Labbook

Compartments

Starterculture of BL21: Pick 1 colony from plate from Saturday and incubate 10ml LB no resistance

CaCl2 Solution: Prepared 1 M CaCl2 an 100mM Cacl2. Autoclave didn’t finish and a salt was preticipated after 3+ hours.

CaCl2 1M  and 100mM preparation:

 -->Autoclaved

Making competent BL21 cells

300ml LB no res was inoculated with ~3ml starterculture from yesterday and placed at 37C 200rpm until OD600 = 0.5. Cells (2x50ml) were spun down 4 °C 4000 rpm for 10 min. Resuspended in 20 ml CaCl2 (100 mM) per pellet. Kept on ice 30 min. Centrifuge 4 °C 4000 rpm 10 min. Resuspend in 5 ml 100 mM CaCl2 15 % glycerol. Aliquited 50 µl/eppi total 84 eppis --> See protocol #17

Heat shock trafo of pBAD33 into newly prepared competent BL21: 50µl Bacteria, 10ng pBAD33. See protocol #003. The cells were plated on 2 LB+amp plates (20 µl and 200 µl) and left in incubator 37 °C.

Heat shock trafo of pBAD33 into newly prepared competent BL21: Yesterday we took the wrong plates (with ampicillin res.). However, pBAD33 has an Chloramphinicol resistance. This was also the case for the Gibson assembly. We used 50 µl BL21 and 10ng pBAD33. See protocol #003. The cells were plated on 2 LB+Chl plates (20 µl and 200 µl) and left at incubator 37 °C.

Overnight Cultures: There were colonies on all plates for the pBAD33 into the Bl-21 competent cells. They were almost completely full, so we thought maybe the resistence on the plate is not working. To verify that the cells actually had the pBAD33, we prepared an overnight culture of 2 colonies in LB-Chloramphinicol medium at 30 °C 200 rpm overnight.

There were also colonies on the trafold with the pBAD_SPD5_sfGFP and from these we picked 12 colonies and prepared overnight culture in 5ml LB-Chl medium. These were grown at 30 °C 200 rpm overnight.

Encapsulins

Encapsulins are nanocompartments found natively in bacteria and some archaea. For our project we want to engineer them to encapsulate our target proteins. Firstly, we clone the needed constructs. Secondly, we test the encapsulation with GFP and use fluorescent microscopy and native page verify the results. Responsible person for this subpart: Anne.

PCR of EncA fragment from IDT: in order to amplify via T7 primers, which are incoorporated in the sequence. We make 2 x 25 µl total volume 

Primers: T7 standard primers 

Expected length 900 bp  

Annealing temperature 56°C  

Extension time: 30 sec 

Gel showed bands at the right size, which were cut out and purified via gel extraction. Eluted in 20 µl water. 

Conc.: 26.9 ng/µl

As the concentration of the EncA is not so high we do another PCR with the same conditions but 2 x 50 µl total volume. 

Gelextraction: Gel from yesterdays PCR of EncA was made and bands at the right size (900 bp) were cut out and cleaned: 

Conc.: 55ng/ µl and 39,1 ng/µl

For the EncA the second step is to digest the fragment with restriction enzymes. This we do so we can ligate it into the backbone. This way we can create the same sticky overhangs on the fragment and the backbone.

Digest of pBAD33

Digest of EncA

1. Calculate volume of DNA and water
2. Pipet water, CutSmart buffer and DNA into eppi on ice
3. Incubate at 37°C for 60 min
4. Cool down to room temperature before using, spin down any possible condensed water

Run on agarose gel, we expect a band at 900 kb and one at 5300 bp

Cut out and purify via Monarch gel extraction kit

EncA digest (conc.: 15 ng/µl)

PBAD digest (conc.: 8 ng/µl)

These digests were used to make a ligation with the T4 DNA ligase and T4 ligation buffer 10x. According to protocol #028.

Vector: 0.02 pmol (25.5 ng)

Insert: 0.06 pmol (50 ng)

This was used for a transformation of 1 µl into NEB5-alpha (50 µl), everything was plated on Chloramphinicol plates.

Ligation of EncA and pBAD (ratio 1:3), calculated with NEB calculator.

 Transformation of ligation into top10, 2,5 µl of ligation (EncA)

 all was plated on LB+Cm plates

Plates from yesterday:

Encapsulin: 20 colonies

Miniprep of EncA colonies C1,  C3,  C4,  C7.

Send C1 for sequencing using eurofins and with 2 standard primers

->result: it did not have the insert

Pellets of 3ml from other overnight cultures of different colonies, froze at -20°C

As I saw from the sequencing that the EncA was not inserted I tried to send another 3 colonies to sequencing. This time it was C3, C4, C7. They were sent using Genewiz and standard primers.

->Result: none of them have the insert, at this point I didn’t know why since on the gel it looked correct

To test which bands are produced from the colony PCR of just pBAD I run 9 PCRs with different temperatures to also test if the annealing temperature is ok. Besides I ran a PCR with just the top10 cells to see if the primers bind somewhere in the genome.

Dreamtaq Mastermix

Annealing temp: 55°C and testing between 52°C – 58°C, extension time 2 min, 30 cycles

Overnight cultures of colonies from the pBAD_EncA ligation (C6 + C11)

Overnight culture of #1924 which is pET302_sfGFP plasmid. It contains already sfGFP in a backbone compatible with the pBAD and I will use this for further cloning.

Overnight cultures of pBAD_SPD5 C1, C2, C3: even though they showed no bands at the colony PCR, I will just try to make a miniprep as the primers I used might have not bound properly.

Sequencing using Genewiz and standard primers for pBAD. I send two colonies from the EncA C6 and C11.

 Result: The encapsulins had a region missing at the C-terminal.

PCR for cloning of TP onto sfGFP using #1924 as a template. Amplify sGFP with sfGFP_fwd and sfGFP_TP_rev_1 (primer 31 + 29)

Anneal: 63-65 °C, Extension: 30 sec (600bp), 30 cycles, total 8 samples 2 at every temperature (63, 64,64.5, 65°C).

Gel of the PCR with sfGFP_TP: expected band size: 600bp. 1 kb ladder from Jena bioscience

                      Result: bands were present for all conditions

Band from the gel with sfGFP were cut out and purified using gel extraction.

Since the sequencing from yesterday showed a problem with the IDT fragment, I become the pJET_encA from our supervisor who cloned it in the meantime. An overnight culture is prepared.

PCR for cloning of TP onto sfGFP using PCR above as a template. Amplify sGFP with sfGFP_fwd and sfGFP_TP_rev_2 (primer 30 + 31)

Anneal: 65 °C, Extension: 30 sec (600bp), 30 cycles, total 5 samples.

Digest of pBAD

Digest of EncA using pJET_EncA

All digests were incubated at 37°C for 1 h.

After 1 h digests were run on gels:

Gel 1:

Gel 2:

All the digested backbones and the inserts were cut out and purified using gel-extraction.

From the digests from yesterday I prepare ligation reactions for the final plasmids: pBAD_EncA and pET_sfGFP_TP respectively

pBAD_encA

pET_sfGFP

*for each digest a relegation test was also made, with the respective backbone only.

Both digests were ligated at RT for 10 min and heat-inactivated 65 °C for 10 min. Then 5 µl from each ligation mix was used for a heat shock trafo into top10 competent cells and the total volume was plated on LB + CmR/AmpR plates and incubated overnight.

From this the TA of our supervisor have cloned it into pJET and I get the plate to do the colony PCR of 20 colonies. This was done using the Dreamtaq polymerase green MM and the standard forward and reverse primers for pJET. Anneal at 52°C, Extention for 4 min, 30 cycles.

The colony PCR of pJET_SPD5_1 was run on a gel (OBS C1 is missing) with the ladder from Zymo ZR 1 kb.

This gel showed that all colonies had an insert at the right size about 2000 bp.

Plates from yesterday: There were no colonies on the plates with sfGFP_TP. However, there were nice colonies on the plates with pBAD_EncA and fewer for the relegation with pBAD.

Colony PCR of 20 colonies from pBAD_EncA ligation. This was perfomed using Dreamtaq green MM and “T7 promoter” and “T7 terminal primers”. Annealing at 58°C and extension of 2 min, 30 cycles.

Following a gel was run using the ladder: Jena bioscience 1kb plus. OBS: C8 is missing. There were several colonies with an insert: C1, 3, 4, 5, 6, 9, 10, 11, 13, 15, 16, 17, 18, 20.

Following overnight cultures were prepared: 10 ml of 1924 (AmR), 10 ml of pBAD33 (CmR).

Another heat shock trafo of the ligation with sfGFP_TP and the religation was perfomed using 5 µl of the ligation and 10 µl bought DH5alpha competent cells. The total volume was plated on two LB +AmR plates and incubated overnight.

From the overnight cultures from yesterday miniprep was performed using the Sigma Aldrich kit.

Plates from yesterday: on the pET_sfGFP_TP plate there were 4 colonies and on the relegation 0.

I use these 4 colonies for a colony PCR using the Dreamtaq green MM and the primers: sfGFP_fwd and sfGFP_TP_rev with 64°C as annealing and 1 min as extension for 30 cycles.

Gel of colony PCR with pETsfGFP there were bands at the correct size for all colonies, but because of how the primers were made this could still just be a carryover of undigested whole 1924 without the target peptide. An overnight culture was prepeared from all 4 colonies. Ladder ZR 1kb from Zymo.

Digest of #1924 with BlpI and KplnI:

This was repeated for 5 samples and incubated 2-3 h at 37 °C

Afterwards, the 5 digests were run on an agarose gel and the band from the digested backbone was cut out and purified using gel-extraction. Ladder ZR 1kb from Zymo.

 Concentration after clean-up: 8,9 ng/µl.

From the new digest and the already digested insert (sfGFP_TP) a new ligation was prepared. This time I diluted the sfGFP 2x to avoid the pipetting of small volumes:

As previously, a relegation test without the insert was prepared. The samples were ligated for 10 min at RT and afterwards heat-inactivated at 65°C for 10 min. Following ligation 5 µl of each ligation mix was transformed into 10 µl DH5alpa competent cells and the final volume was plated on two LB + Amp plates and incubated O/N.

Overnight cultures were prepared 4 colonies from the pBAD_encA which all seemed to have an insert at the right size from the colony PCR.

Miniprep of the overnight cultures from yesterday was prepared 4 from pBAD_EncA

--> The colonies from the minipreps were all send for sequencing using eurofins and standard primers.

Colony PCR of the pET_sfGFP_TP plates from yesterdays ligation. Using Thermo fischer Green Taq pol. Master mix.

 The colony PCR was run on a gel with the RZ 1kb ladder.

-->The gel showed that there was a band for multiple colonies, but one colony had a stronger band at the right size.

Overnight cultures were prepared for the colonies of pET_sfGFP_TP which showed bands. (c3,c4,c5,c6).

Sequencing results:

For pBAD_EncA there is also one colony with the right sequence, no mutations, c3.

For pET_sfGFP_TP there is also the correct sfGFP, but it looks like it has only been amplified with the first PCR, so it is missing some part of the target peptide.

Colony PCR of the pET_sfGFP_TP plates from yesterdays ligation. Using Thermo fischer Green Taq pol. Master mix.

The colony PCR was run on a gel with the RZ 1kb ladder.

The gel showed that there was a band for multiple colonies, but one colony had a stronger band at the right size.

Overnight cultures were prepared for the colonies of pET_sfGFP_TP which showed bands. (c3,c4,c5,c6).

From the pBAD_encA c3 a transformation is made to have a clean colony to pick for a glycerol stock.

PCR was prepared using pET_sfGFP_TP c1 as a template to amplify the sfGFP_TP, this time with the full target peptide. Annealing temperature: 65°C, extension time: 30 sec, 30 cycles. Prepared with Q5 polymerase.

An agarose gel was prepared of the above PCR, RZ 1kb ladder.

The bands for the respective fragments were cut out and purified with gel extraction.

                      sfGFP_TP fragment: 97 ng/µl and 207 ng/µl

Miniprep of overnight cultures of pET_sfGFP_TP from the 01/08-22 c3-c6.

Those sequences were send for sequencing using eurofins and standard primers
04/08-22

Sequencing results from yesterday of pET_sfGFP_TP showed that c3 has the entire target peptide incorporated. Therefor I don’t need the extra fragments which I made fresh.

 Overnight cultures of the pBAD_EncA c3 for making glycerol stocks.

A double trafo was made in order to create BL21 (DE3) cells with both the pBAD_EncA and the pET_sfGFP (#1924) and another strain with pBAD_EncA and the pET_sfGFP_TP. Those I plan to use for fluorescent microscopy. Both were plated on LB-plates + Amp + Chl.

Glycerol stocks were prepared for the pBAD_EncA

Overnight cultures were prepared from one colony from the double trafo with pBAD_EncA and pET_sfGFP.

Glycerol stocks were prepared of the two strains of bacteria containing pBAD_encA and either pET_sfGFP with or without target peptide.

Aim: observe wether the GFP with target peptide and the encapsulins come together to form fluorescent “dots” as has been observed for the microcompartments.

To do this I have prepared two strains of BL21 (DE3):

• Carry pBAD_encA and pET_sfGFP
• Carry pBAD_encA and pET_sfGFP_TP

Strain 1 in this experiment is the control as the GFP has no targeting peptide. Strain 2 should show dots.

From these two strains overnight cultures are prepared and later a larger culture which grows until OD around 0.6. Then 3 ml for every condition will be induced or not induced. The plasmid pBAD_EncA is induced by arabinose and pET_sfGFP is induced with IPTG.

After induction the cultures they produce the proteins for 24 h at 18 °C. Then they are subjected to fluorescence microscopy. 1 ml will be spun down and is lysed and both pellet and supernatant will be used for westernblot for detection of a his-tag present on the encapsulins.

For the experiment 2 overnight cultures were prepared from the glycerol stocks containing 1. pBAD_EncA and pET_sfGFP and 2. pBAD_EncA and pET_sfGFP_TP. Both were grown in LB+amp+CmR

LB for tomorrow 200 ml total was prepared with both resistences and left at RT.

Today 2 larger cultures, 100 ml each, were prepared from the overnight cultures and they were grown until OD around 0.6 and then they were induced according to the scheme:

For pipetting a stock solution of IPTG of 100µM was used and for arabinose a 5% solution was used.

The cultures were shaking at 18°C for 24 h

After 24 h has passed 1 ml of each sample is centrifuged at 3000 x g for 10 min and thrown supernatent away and the pellet is frozen at -20°C. The rest of the sample is used for fluorescence microscopy and agarose pads protocol #025 are prepared with each sample for imaging. 3 pictures pr. Slide are taken.

The spun-down samples from 11/08-22 are thawed and resuspeded in 1 ml lysis buffer (20 mM Tris + 50 mM NaCl). Sonication of all the samples. Centrifuge all the samples at 3000 rpm for 10 min

Prepare samples for SDS: Pipette 10 µl supernatent of each sample together with 10 µl loading buffer (Laemmli buffer) and a sample from the pellet is made.

SDS-gels are prepared according to protocol #031. And 1 gel is run pr 12 samples.

Gel 1: samples 1-6 (supernatant) and samples 1-6 (pellet)

Gel 2: samples 7-12 (supernatant) and samples 7-12 (pellet)

Both SDS-gels are then used for transfer for westernblot. For this a semi-dry system is used.

Protocol #030.

Membranes were incubated overnight with the primary antibody (anti-his).

Agarose gel of yesterday’s PCR of different PCR amplified fragments of EncA.

All bands were cut out and purified using gel-extraction

EncA_micro: 96 ng/µl

SPD5_micro: 148 ng/µl

Overnight culture of pBAD_encA + pET_sfGFP_TP and pBAD_EncA + pET_sfGFP for the EncA #02 experiment. This was done by starting from the glycerol stocks, which was prepared last time.

 Digest of EncA

 Induction of EncA and sfGFP_TP according to scheme. This is a part of the experiment EncA #02

Gel with digest from yesterday’s digest of the backbone and the inserts for pMIC_SPD5 and pMIC_EncA

Clean up of digest from gel was performed for all bands using gel-extraction

EncA_micro: 24.9 ng/ µl

Ligation of pMIC_encA and pMIC_SPD5: Ratio was 1:2, 3 ligations were performed as described:

All ligations were incubated 10 min at RT at heat inactivated for 10 min at 65°C.

Fluorescence microscopy of the samples prepared for EncA #02 was performed.

Colony PCR of the constructs; pMIC_SPD5 and pMIC_EncA using the Green Dreamtaq polymerase Mastermix. Total samples 8.

Gel of colony PCR of the 8 colonies showed that 1 of each construct had a band at the right size.

Overnight cultures were prepared of the pMIC_SPD5 c1 and pMIC_EncA c1, which showed bands at the agarose gel.

I send the pMIC_EncA c3 a for sequencing using standard primers for the encapsulins. Result: The pMIC_EncA looks good and has no mutations.

Miniprep of the overnight cultures from yesterday of pMIC_encA, eluted in 50 µl using Sigma Aldrich kit.

Overnight cultures were prepared of pMIC_EncA c3 in 5 ml LB + Amp. They were incubated 25°C for 24 h.

Miniprep of overnight cultures of  pMIC_EncA was prepared and they were subsequently sent for sequencing.

Encapsulins #03

Aim: To test whether a difference in the media composition can influence the induction of encapsulins via arabinose. The two mediums which are compared are TB and LB. Besides, a higher concentration of arabinose is tested, 0.01%.

2 strains in BL21:

Strain 1: (pBAB_EncA + pET_sfGFP)

Strain 2: (pBAD_EncA + pETsfGFP_TP)

Concentrations tested in LB

Strain 1: samples 1-9

Strain 2: samples 10-18

Concentrations tested in TB

Strain 1: samples 1-4

Strain 2: samples 5-8

Overnight cultures from glycerol stock were prepared for the experiments Encapsulins #03. Cultures are grown in LB or TB as described.

pBAD_EncA #03:

Diluted overnight cultures for the different strains 1:50 in 100 ml and grew the larger cultures at 30°C until OD 0.4 (TB medium samples) or OD 0.6 (LB medium samples). This was done with a 1 h delay between the first samples (pBAD_EncA in LB) and last samples (pBAD_encA in TB). Induction was performed at the given OD with the given concentrations stated earlier.

PCRs for cloning of the sfGFP_TP constructs into the mVENUS backbone to have it in a backbone without the T7 promoter. Four x 25 µl was prepared for each fragment.

PCR for fragment sfGFP_TP for the mVEN

Primer 38 + 40, annealing: 63°C, extension time: 30 sec, template pET_sfGFP

PCR for the fragment sfGFP for the mVEN

Primer 38 + 39, annealing: 63°C, extension time: 30 sec, template pET_sfGFP

The PCR samples were run on an agarose gel with the GeneRuler 1 kb ladder:

All PCRs showed bands at the correct size and were cut out for purification using gel-extraction.

Digests of multiple fragments:

• Incubate all at 37°C for 1 h

All Digests were loaded on a gel and subsequently cut out an purified using gel-extraction:

Microscopy of the Encapsulins #03  was performed.

Overnight ligation of pVEN_sfGFP(_TP) ratio 1:3

• Leave at 4°C overnight

Heat shoch transformation of the pVEN_sfGFP(_TP) into DH5-alpha using 2 µl of the overnight ligation. The bacteria were plated on LB + CmR plates overnight.

Preparation of samples from the pBAD_EncA for westernblot. Pellets were stored at -20°C and were first resuspended in Lysis-buffer and sonicated for 30 sec. The samples were centrifuged for 10 min at 4000xg and 15 µl was mixed with 15 µl SDS sample buffer. Then samples were stored at -20°C until SDS-PAGE was prepared.

EncA_#03

Aim: To test whether a difference in the media composition can influence the induction of encapsulins via arabinose. The two mediums which are compared are TB and LB. Besides, a higher concentration of arabinose is tested, 0.01%.

Concentrations tested in LB

Strain 1: samples 1-9

Strain 2: samples 10-18

Concentrations tested in TB

Strain 1: samples 1-4

Strain 2: samples 5-8

Transformations via heat-shock:

pBAD33 (empty) + pET_sfGFP into BL21

pBAD33 (empty) + pET_sfGFP_TP into BL21

Colony PCR of pVEN_sfGFP and pVEN_sfGFP_TP was prepared, screening 8 colonies for each construct. This was done using Green Dream-tag polymerase MM using primers 38 + 39 to amplify sfGFP and 38 + 40 to amplify sfGFP_TP.

Agarose gel of colony PCRs:

Gel 1: marker, sfGFP_TP (primer 38 + 40) (x8)

Gel 2: marker sfGFP (primer 38 + 39) (x8)

Miniprep of mVENUS in TOP10, 3 ml were used for the miniprep with the sigma Aldrich kit.

Overnight cultures were prepared of the colonies which seemed to have a band in the colony PCR of the pVEN_sfGFP_(TP) construct. 3 colonies were picked and cultured overnight.

Miniprep of 3 colonies from each construct of pVEN_sfGFP_(TP) was performed, for sequencing on Monday.

Glycerol stock was prepared from the overnight cultures of pBAD33 + pETsfGFP_TP, pBAD33 + pET_sfGFP.

Heat shock transformation of pBAD33+pET_sfGFP/pBAD33 + pET_sfGFP_TP all in BL21.

Result of sequencing showed that the pVEN_sfGFP_TP_C1 had worked out, for pET_sfGFP there were unfortunately no match.

Overnight cultures from yesterday of pBAD33+pET_sfGFP/pBAD33 + pET_sfGFP_TP were dilutes 1:50 in larger cultures and grown at 30°C until they reach OD: 0.6

Colony PCR of pVEN_sfGFP of the same plate, but with other colonies.Using Dreamtaq Green MasterMix. Primer 38 + 39. Annealing 55°C, Extension 4 min.

Control experiment for the Encapsulins #01

Aim: The aim is to observe whether the observed fluorescent dots for the experiments with Encapsulins are due to the Encapsulins being expressed or if they form even in the absence of the plasmids.

Strain 1: pBAD33 + pET_sfGFP

Strain 2: pBAD33 + pET_sfGFP_TP

Experimental: BL21 was transformed with pBAD33, same backbone as for the Encapsulins, and a plasmid containing either sfGFP or sfGFP with the encapsulation tag C-terminally.  The induction and incubation follow the same procedure as for the previous experiment.

Colony PCR of pVEN_sfGFP using Green Mastermix taq Polymerase and primers 38 +39 at 55°C and extension time 1 min.

Colony PCR was run on an agarose gel with the GeneRuler 1 kb ladder. However, no samples showed an amplified insert.

Digest of pVEN backbone with EcoRI and BamHI.

Incubate 30 min at 37°C and then add 1 µl Fast Alkaline Phosphatase and continue incubation 30 min.

Digest was run on an agarose gel with the GeneRuler 1 kb ladder, there was a nice band for the cut-out and the backbone was cut out and cleaned up using gel extraction according to the kit.

                      Conc.: 4.7 ng/µl

Heat shock transformation was performed with the MG1655 and two double trafos with either: pVEN_sfGFP_TP + 1939 or pVEN_sfGFP + pMIC_EncA. After the 1 h incubation everything was plated on 2 LB + CmR +AmR plates.

Overnight culture of MG1655 with either 1939 + pVEN_sfGFP_TP or pMIC_EncA + pVEN_sfGFP_TP in CmR and AmR.

Colony PCR of MG1655 with either 1939 + pVEN_sfGFP_TP or pMIC_EncA + pVEN_sfGFP_TP checking 2 colonies from both strains for both plasmids. This is done using Green Master mix Taq polymerase and primer 38 + 40 for sfGFP_TP and 32 +33 at annealing temp: 55 or 60°C respectively.

Trafo of overnight ligation from yesterday of pVEN_sfGFP into DH5-alpha.

Plate with trafo of overnight ligation from yesterday of pVEN_sfGFP showed no colonies.

Trafo heat shock of either (pMIC_EncA + pVEN_sfGFP_TP) or (1939 + pVEN_sfGFP_TP) in BL21 and plated on LB + AmR + CmR plates

Glycerol stock of the two MG1655 strains: pMIC_EncA + pVEN_sfGFP_TP or pMIC_EncA + 1939 was prepared

Larger culture and induction of pMIC_EncA/pVEN_sfGFP in MG1655 was induced according to Emcapsulin #04. The cultures were induced 2 hours earlier than planned.

Heat-shock transformation of

pMIC_EncA + pVEN:sfGFP_TP in ME5119                     (AmR + CmR)

1939 + pVEN_sfGFP_TP in ME5119                               (AmR + CmR)

Microscopy of Encapsulin #04

Encapsulins #05

Aim: To observe if the results produced in BL21 can be reproduced in MG1655 and if a strain not devoted to protein production might be better suited for forming the Encapsulins. Both strains are in MG1655. Since last time the expression of sfGFP was too low it had to be repeated.

Strain A: in MG1655

Strain B: in BL21

Strain A1: 1934 + pVEN_sfGFP_TP

Strain A2: pMIC_EncA + pVEN_sfGFP_TP

Strain B1: 1934 + pVEN_sfGFP_TP

Strain B2: pMIC_EncA + pVEN_sfGFP_TP

Pipetting was done using a 100mM stock of IPTG and 50ng/µl Doxycyclin stock.

The samples of which I did microscopy were:

Strain A: none showed the results seen on Friday so I didn’t spend more time on them

Strain B: sample 1, 2, 4, 8

Induction of Encapsulin #05 according to previously described

Microscopy of Encapsulins #05

ENCAPSULINS #06

Aim: To detect via Native PAGE and microscopy whether the sfGFP is encapsulated correctly and whether sfGFP alone forms aggregates.

Experimental setup: Overnight cultures of the BL21 strains were prepared from glycerol stocks and on the next day grown at 30 °C to OD: 0.6-0.8 and induced accordingly. Then samples were incubated for 24 h at 18 °C.

Strain 1: pBAD_EncA + pET_sfGFP (sample 1-6)

Strain 2: pBAD_EncA + pET_sfGFP_TP (sample 7-12)

After induction the samples are incubated at 18 °C 24 h.

Overnight culture of Encapsulins #06 for the last experiment of Encapsulins from glycerol stocks.

Strain 1: pBAD_EncA + pET_sfGFP (sample 1-6)

Strain 2: pBAD_EncA + pET_sfGFP_TP (sample 7-12)

Induction of Encapsulins as described above for the #06 experiment

Microscopy of Encapsulins #06

Spun down 3ml of the samples for the Native PAGE, see protocol. Froze pellets down at -20 °C.

Buffers according to the Native PAGE protocol from Invitrogen (). I prepared the Running buffer 1X and the Cathode buffer additive 20X, and Sample buffer 4X.

Sample preparation for Native PAGE, see protocol. This was done for the samples (2,3,5,6,8,9,11,12)

OBS: the sample buffer was prepared with too less Glycerol and a new one was prepared for the next native PAGE.

Native PAGE of the Encapsulins samples.

                      Run at RT at 150 V for 90-115 min.

Detect gel using Typhoon in the Cy2 (488nm) channel for GFP signal.

Sample preparation for Native PAGE. This was done for the samples -.

Native PAGE of the Encapsulins samples. Detect gel using Typhoon in the Cy2 (488nm) channel for GFP signal.

Liquid Droplets

SPD-5 is a protein capable of self-assembling into liquid droplets in vivo and in vitro. They are natively expressed in C.elegans where their liquid droplets allow for nucleation of microtubule. For our project we fuse SPD-5 to spy- and snoop-tag and thereby allow targeting of cargo to liquid droplets. Firstly, we clone the SPD-5 constructs and secondly we test its capability of specifically catching sfGFP. Responsible person for this subpart: Anne.

PCRs of pBAD33 backbone, SPD5_1, SPD5_2 and sfGFP_linker using Q5 polymerase 2X Mastermix.

Program for pBAD33 Backbone

 Program for SPD5_1, SPD5_2, sfGFP_linker

Pippeting scheme:

For the fragments 1µl was used as the concentration was unknown, and 10ng for the Backbone.

Gel for PCR fragments SPD5_1, SPD5_2, linkeR_sfGFP (1% agarose gel, 1kb plus quick load ladder)

 --> All fragments were amplified!

Gel for PCR fragment pBAD33 backbone (0,7% agarose gel, 1kb plus quick load ladder)

--> All fragments were amplified!

HiFi Gibson Assembly of SPD5_sfGFP (pIG22_007)

 See protocol #020. Because the concentrations from the cleanup of the PCR fragments were too low to do an assembly with 0.2 pmol we did:

Total amount of DNA= 0.05 pmol

Ratio of vector: insert 1:2

Backbone: 0.017pmol

Per fragment: 0.017pmol/3 = 0.00567 pmol

Example of calculations:

Pmol*(basepairs x 650 daltons) / 1000 = __ ng

Heat shock trafo of SPD5_sfGFP_pBAD33 in competent Top10: 50µl Bacteria, 1.5µl Plasmid. See protocol #003. Cells were plated on 2 LB + amp plates (200 µl and 800 µl) + left at incubator  37 °C.

Clean-up of PCR fragments from gel

Fragments that were cut out and purified: SPD5_1, SPD5_2, sfGFP, pBAD33. Protocol #021

Heat shock trafo of SPD5_sfGFP_pBAD33 in competent Top10: We used 50 µl Bacteria, 1.5 µl assembled pBAD33_SPD5_sfGFP. See protocol #003. Cells were plated on 2 LB + Chl plates (200µl and 800µl) + left at incubator 37 °C.

Colony PCR of the pIG22_007 (pBAD_SPD5_sfGFP) (colony 1- 12)

In order to check if the overnight cultures worked we prepared a colony PCR of all 12 colonies by amplifying the insert between primer (9) and (14). This should be in total 4,3kb.

Here we use the Q5 polymerase as this was what we had in the lab

We added 5 ml to the overnight cultures and left them in the fridge as we won’t do the miniprep today.

 Gel of the colony PCRs on 0,7% agarose, 1kb plus Quick load ladder

None of the colonies had a band at 4,3 kb instead they all had a band at 0,5 kb and 1 lower.

Colony PCR of the pIG22_007 (c13-c24): We decide to do another colony PCR from 12 other colonies. Similar to the one from yesterday. However this time we ran out of Q5 polymerase, so it wasn't enough. We ran the PCR anyway.

HiFi Gibson Assembly of SPD5_sfGFP (pIG22_007):  We also decided to repeat the assembly of the plasmid with extractions of the fragments with higher concentrations. This time we did following:

Total amount of DNA= 0.2pmol

Ratio of vector: insert 1:1

0.2pmol / 2 parts = 0.1 pmol

Backbone: 0.1pmol

Per fragment: 0.1pmol/ 3 = 0.033 pmol

Note: The total volume of DNA was a bit more than 10 µl.

Heat shock trafo of pIG22_007 in competent Top10. We used 50 µl bacteria and 1.5 µl of assembled plasmid. See protocol #003

Cells were plated on 3 LB + Chl plates (20 µl, 200 µl and 780 µl) + left at incubator 37 °C

Colony PCR of the pIG22_007 (c1-c12) 

Same protocol as last week. But this time we test the new Gibson assembly (0,2pmol 1:1 ratio). 

Gel of colony PCRs: The bands are again at 0,5 kb and not at 4,3 kb. 

Miniprep of c1 of pIG22_007 

We spin down 5 ml. The concentration is very low. Conc: 29,7 ng/µl. See protocol #008 

Restriction digest of pIG22_007 : In order to check if the reason we get wierd bands is because something with the PCR/primers we make a digestion of the plasmid to compare with the undigested. Protocol #022 

This was incubated 15 min at 37C. 

Gel for comparison of digested and undigested pIG22_007: We prepared a 0,7% agarose gel and used the 1kb+ Quick load ladder. 

 The band on the gel looked like it was about 5 kb, which would fit with just the backbone. We decide to stop working with the old primers from the 2019 Freiburg team and instead design our own new primers.

We did another apmlification of the SPD5_1 fragment with the new 2022 primers. This is done to test different annealing temperatures of the new primers as we have had difficulties to amplify via the new primers. 12 PCRs were prepared (25 µl) and temperatures between 60 - 66°C were tested.

Result: Annealing temperature at 64°C gives bands and we continue with this temperature for future PCRs. Bands at the right size (2000bp), were cut out and purified via gel extraction.

Conc.: 8 ng/µl

PCR amplification of SPD5_1: As we used all the PCR amplified SPD5_1, we first need to amplify it with the 2019 primers, before we can amplify it with optimized conditions and new 2022 primers.

 50 µl HIFI master mix (Primers: 9 and 10)

Annealing: 64,5 °C, Extension time 1 min 10 sec

Gel extraction of agarose bands:

SPD5_1: 29 ng/µl

For the SPD5_1 we did another PCR with the 2022 primers to get the right overlaps for the Gibson assembly.

-> Same conditions as for the previous PCRs

Gel extraction: Bands with the right size for SPD5_1 (2000 bp) were cut out and purified:

SPD5_1: 30 ng/µl and 20.4 ng/µl

Gibson of pBAD_sfGFP, SPD5_1 and SPD5_2

According to protocol 020#. Total amount of DNA: 0,2 pmol (Ratio 1:1)

Vector: 0,067 pmol

        PBAD33_sfGFP: 269,5 ng

Inserts : 0,067 pmol

       SPD5_1: 87,5 ng

      SPD5_2: 69,4 ng

Add water to 10 µl and 10 µl HIFI assembly mix.

--> This was to low a ratio and too much DNA as 50-100 ng of the backbone is recommended.

A heat shock transformation of the Gibson was performed with 1µl into 50 µl Top10 cells

Plates from yesterday: the Gibson of SPD5 did not seem to work.

• Next time higher ratio, lower amount of vector

Plates from trafo from yesterday: 5 colonies

PCR of SPD5_2 to amplify it as I am running out of the fragment

2x 25µl using 2 X Mastermix, annealing at 64°C extend for 1min 10sec

-->Gel showed a small band which is cut out and stored at 4 °C overnight.

Gibson of SPD5 and pBAD with fresh assemblymix (ratio1:3)

incubate 1 h at 50 °C

Transformation of 2.5 µl SPD5 gibson into top10

 all was plated on LB+Cm plates

                      SPD5 gibson: nothing

Clean up from gel piece of SPD5_2 from yesterday:

Kit: protocol #. Eluted in water.

                      Conc: 37 ng/µl

PCR of SPD5_whole (both SPD5_1 and SPD5_2)

Using primer 17 and 18, and both fragments as templates.

Expected size: 3.6 kb, annealing temperature 62.6 °C, 63.9°C, 64.9 °C. Extension time 2 min.

Using Q5 HIFI mastermix (NEB) 25µl/rxn, 15 cycles.

Gel showed only smeared band around 1600 bp. Probably, the conditions were not optimal for amplifying SPD5_1.

PCR of SPD5_whole (both SPD5_1 and SPD5_2)

Using primer 17 and 18, and both fragments as templates.

Expected size: 3.6 kb, annealing temperature 64.5 °C. Extension time 2 min.

Using Q5 with GC enhancer (NEB) 25µl/rxn, 15 cycles.

Gel showed a bigger band, which could be that SPD5_1 was also amplified, but maybe the number of cycles was not high enough.

Electoporation of SPD5 gibson 1:3 ratio from previous was via electroporation

1µl gibson in 50 µl bacteria.

Plate with electroporation of SPD5: no colonies

PCR of both SPD5_1 and SPD5_2

Using primer:

SPD5_1: 10 and 17

SPD5_2: 11 and 18

Expected size: 2 kb and 1.6 kb respectively, annealing temperature 64 °C. Extension time 1min 10sec.

Using Q5 with GC enhancer (NEB) 50µl/rxn, 30 cycles. For each 2 x 50 µl was prepared.

Miniprep of SPD5_2 in pJET colony 18 + colony 20

Send both colonies for sequencing with eurofins and primer 11 + 18

->result: C18 had the SPD5 and did not have any problems with the sequence

Gel of PCR of 3 colonies of pBAD_SPD5 using primer 21 + 22 (binding at the promoter an the terminal)

• Gel showed band for all samples with backbone but no band for samples using top10 cells.

I realized that there is a blurry band underneath the marker and that what I thought was 2000 bp was really the 1000 bp mark.

 I continue with C 6 and C11 as they have a band at actually 1000bp, which should be correct.

Electoporation of SPD5_gibson ratio 1:3

I try again an old Gibson to see if another electroporation would give any results.

1 µl of Gibson + 50 µl electrocompetent cells + 950 µl after 1 h at 37°C it was plated on a LB + CmR plate.

Overnight culture of pJET_SPD5_2 from plate C18

Colony PCR of the 3 colonies pBAD_SPD5 with primer 17 + 18

Dreamtaq Mastermix, Annealing temp: 65 °C, extension time 4 min, 30 cycles

PCR of SPD5_2 using pJET_SPD5_2 as template. I made 5 x 25 µl amplifying it with primer 11+18, annealing temp: 64°C, extension time: 1 min 10 sec

PCR of SPD5_1 using different gel extractions from different days to test if any of them works for amplifying the fragment. I made 4 x 25 µl and amplified using primer 9 + 10. Annealing: 64,5°C, extension time 1min 10 sec.

Gel of PCRs

The SPD5_2 bands were cutout for purification, 3 together on one column.

The SPD5_1 band was cut out and extracted using gel extraction kit.

Another PCR with SPD5_1 using the template which gave bands on the previous PCR. I made 1 x 25 µl. Annealing: 64,5 °C and extension: 1 min 10 sec, 30 cycles.

The SPD5_1 band was cut out and cleaned up with the piece from yesterday using gel extraction.

Conc: 24 ng/ul eluted in 10 µl

This was enough to make a cloning into pJET using #__ and a 1:3 ratio

This was ligated at RT 5min and 5 µl was directly used for heat shock trafo into Top10 cells. The complete volume was plated on a LB + Amp plate O/N.

Since there was a band for the c2 from the pBAD_SPD5 at 10 kb I also make an overnight culture of this to check the plasmid with sequencing. 10 ml LB +CmR.

Miniprep of c2 from the pBAD_SPD5 overnight culture: Conc: ~60 ng/µl.

Colony PCR of 20 colonies from pJET_SPD5_1. Using standard primers for pJET and the Green Dreamtag Mastermix. Annealing at 52°C and extension was 2 min, 30 cycles.

Gel 1% agarose, Jena Bioscience 1 kb ladder. The gel showed multiple colonies with a band at 2000 bp.

Three were chosen for following overnight culture: C2,5,9 in 10 ml LB + Amp.

Sequencing using Genewiz and standard primers for pBAD I send the C2 from pBAD_SPD5 using standard primers.

Result: The pBAD_SPD5 only had the SPD5_2 inserted

Results from sequencing from the pJET_SPD5_1 colonies: all had some kind of mutation.

As we ordered the SPD5_1 again from fresh (the other was from 2019) we will continue with the new fragment.

Gel of sfGFP_TP from 2^nd PCR, 100 bp plus thermos fischer.

Bands were all at the right size and were cutout out and purified using gel-extraction.

                      Conc: 260 ng/µl

Digest of #1924 with BlpI and KplnI:

Digest of sfGFP_TP from PCR 2

All digests were incubated at 37°C for 1 h.

After 1 h digests were run on gels:

Gel 1:

All the digested backbones and the inserts were cut out and purified using gel-extraction.

Overnight cultures were prepared of 4 of the colonies from the pJET_SPD5_1 (new fragment)

Miniprep of the overnight cultures from yesterday was prepared 4 from pJET_SPD5_1.

-->The colonies from the minipreps were all send for sequencing using eurofins and standard primers.

Sequencing results from pJET_SPD51: There are an insert for all colonies and colony nr. 4 also has the right sequence with no mutations.

.

For pJET_SPD5_1 c4 a transformation is made to have a clean colony to pick for a glycerol stock.

PCR was prepared using the pJET_SPD5_1 c4 as a template for amplifying the SPD5_1 using the primers 10 + 17. Annealing temperature: 64,5°C, extension time 1 min 10 sec, 30 cycles. Prepared with Q5 polymerase. This is done to achieve the correct overhangs for the following Gibson.

An agarose gel was prepared of the two above PCR, RZ 1kb ladder.

The bands for the respective fragments were cut out and purified with gel extraction.

                      SPD5_1: 139 ng/µl

Gibson was prepared for inserting the SPD5_1 and SPD5_2 fragments into pBAD33.

Ratio 1:2

I will use 100ng of the backbone, which is pBAD33 amplified with two primers. As the backbone is 5,3 kb this is 30.5 fmol. As the ratio is 1:2 the amount of insert will be 61 fmol. From the concentrations and lengths of my fragments I get:

This incubate 1 h at 50 °C and then 1.5 µl of the Gibson mix was used for a trafo with 10 µl competent bought Dh5-alpha cells. And plated on Chloramphenicol plates.

 Overnight cultures of the  pJET_SPD5_1 for making glycerol stocks.

Colony PCR was made of colonies from the 6 colonies I had an the pBAD_SPD5 plate, using Thermo fischer green mastermix. The primers were the T7-primers. Annealing was at 52°C and extension tim was 5 min. 30 cycles.

The colony PCR was run on an agarose gel with the RZ 1 kb ladder. The colonies c1 and c2 showed a band at the expected size ~4,3 kb.

Glycerol stocks were prepared for  pJET_SPD5_1

Overnight cultures were prepared of the two colonies C1 and C2 of pBAD_SPD5 which showed to have an insert in the colony PCR.

Minipreps were prepare of the SPD5 c1 and c2, eluted in 30 µl:

PCR of SPD5 to produce different fragments with and without the Spy-tag and the Snoop-tag.

First PCRs:

SPD5_snoop: 34 + 26 anneal at 59°C

SPD5_spy: 35 + 24 anneal at 59°C

SPD5_both: 26 + 24 anneal at 59°C

Agarose gel of yesterday’s PCR of different PCR amplified fragments of SPD5.

All bands were cut out and purified using gel-extraction

SPD5_micro: 148 ng/µl

Trafo of BAD_SPD5 into Bl21 and 2332 into Bl21 using heat-shock. 30 µl was plated on LB + CmR plates.

Digest of pHT1_spy_snoopT2T3

Digest of SPD5

Overnight culture of BL21 with either pBAD_SPD5_sfGFP or pBAD_sfGFP were prepared.

Miniprep pMIC_SPD5_c1 and pHT1_spy/snoop_T2T3

I ran the second PCR for cloning of the pMIC_SPD5_both, which contains both the spy and the snoop catcher. This was done using Q5 polymerase and the pimers 36 + 37 annealing at 64 °C.

An agarose gel was made to confirm the PCR fragments of SPD5_both. The band was at the expected size and was cut out and purified using gel-extraction.

Gel with digest from yesterday’s digest of the backbone and the inserts for pMIC_SPD5 and pMIC_EncA

Clean up of digest from gel was performed for all bands using gel-extraction

SPD5_snoop: 99 ng/µl

SPD5_micro: 41.5 ng/µl

pHT1spy_snoopT2T3: 45 ng/µl

Clean up from gel of the SPD5_both from the first and the second PCR. This was done using gel-extraction.

                      SPD5_both 2°: 136 ng/µl

Ligation of pMIC_SPD5: Ratio was 1:2, 3 ligations were performed as described:

All ligations were incubated 10 min at RT at heat inactivated for 10 min at 65°C.

Digest of SPD5_tags

Incubate 1 h at 37°C

An agarose gel was run with the digests of the 3 digests and all had bands at the correct size. These were cut out and purified using gel extraction.

                      SPD5_both: 43 ng/µl

                      SPD5_snoop: 60 ng/µl

                      SPD5_spy: 50 ng/µl

Glycerol stocks were prepared of pBAD_SPD5_sfGFP and #2332 (pBAD_sfGFP) in BL21(DE3) for microscopy at a later timepoint. 1 ml of the overnight cultures were centrifuged and resuspended in 1:1 glycerol and LB.

Ligation of pMIC_SPD5 with tags: 3 ligations were prepared with ratio 1:2 of vector:insert. For each ligation 50 ng of the backbone was used, corresponding to 22 fmol.

pHT1(snoop/spy)T2T3 digest: 1,1 µl

SPD5_snoop/spy: 44 fmol

T4 ligase: 1µl

T4 ligase buffer: 2µl

Ligation time was 10 min at RT and followed by heat inactivation 10 min at 65°C

Heat shock: Trafo was performed using 10 µl of bought competent DH5-alpha and 2 µl ligation mix. The total volume was plated og LB+Amp plates.

Electoporation of the SPD5 with tags constructs. As the last trafo yesterday did not give many colonies I try the trafo again using electroporation to get more colonies.

I send the pMIC_SPD5 c1 for sequencing using standard primers for the encapsulins and self-sent primers for SPD5

Result: The primers for the SPD5 were wrong.

Miniprep of the overnight cultures from yesterday of pMIC_SPD5, eluted in 50 µl using Sigma Aldrich kit.

Plates from yesterday’s electroporation: there were 1 colony of the two SPD5_spy/snoop respectively, no colonies of the SPD5_snoop.

Colony PCR of the 2 colonies from SPD5_snoop/spy as well as the colonies from last trafo of the same ligation, total samples 9. Colony PCR was done using Green Dreamtaq Mastermix and primer 35+34.

Gel of colony PCR

The gel showed no bands for any of the colonies.

Overnight ligation: As none of the colonies showed bands at the expected size, I decide to try to make an overnight ligation instead at 16°C. 2 ligations were prepared with ratio 1:3 of vector:insert. For each ligation 50 ng of the backbone was used, corresponding to 22 fmol.

pHT1(snoop/spy)T2T3 digest: 1,1 µl

SPD5_snoop/spy: 66 fmol

T4 ligase: 1µl

T4 ligase buffer: 2µl

Both ligations were incubated at 16°C overnight.

From the ligation from yesterday of pMIC_SPD5_Spy/snoop a heat-shock was performed with DH5-alpha and plated on LB + Amp plates.

Overnight cultures were prepared of pMIC_SPD5 c1 in 5 ml LB + Amp. They were incubated 25°C for 24 h.

PCRs were prepared to amplify different parts of the SPD5 with and without tags. Primers and annealing temperatures were as described. This time I amplify the SPD5 in two parts, split at the SpeI restriction site. For fragments amplifying the tags 2 PCRs are needed:

SPD5_spy: 23+24, 60°C

SPD5_snoop: 26 +28, 65°C

SPD5_c-term: 28+35, 59°C

SPD5_n-term 34+23, 59°C

All PCRs were run on an agarose gel and all samples took up 2 lanes. The bands were cut out and purified with gel-extraction

For the second PCRs of the fragments SPD5_tags different primers were used to amplify it, with specified annealing temperatures:

SPD5_spy: 23+36, 64°C

SPD5_snoop: 37 +28, 60°C

The PCRs were run on an agarose gel:

The gel was thrown out as there were multiple unspecific bands. The PCR would need optimization.

Overnight cultures of SPD5_snoop/spy were prepared

Miniprep of overnight cultures of pMIC_SPD5 c1 was prepared and they were subsequently sent for sequencing.

Transformation via heat-shock of SPD5_snoop/snpy into Dh5 alpha

Colony PCR of the SPD5_spy/snoop in DH5-alpha from yesterday.

From the overnight culture of SPD5_snoop/spy c2, 3 ml were spun down and stored at -20°C.

Trafo of already done ligations, to screen more colonies: trafo of SPD5_snoop/spy and SPD5_both. 2 µl were transformed into highly competent DH5-alpha. Afterwards, plated on LB+amp. 

SPD5 #01

Aim: to check via fluorescent microscopy if there are fluorescent “dots” shown for the SPD5 like was shown in 2019. Different concentrations of arabinose are tested.

2 strains in BL21:

                      Strain 1: pBAD_sfGFP

                      Strain 2: pBAD_SPD5_sfGFP

Overnight cultures from glycerol stock were prepared for the experiments SPD5 #01. Cultures are grown in LB.

Colony PCR of SPD5_both tags and SPD5_snoop/spy from yesterdays transformation. For this green dream-tag polymerase MM was used and the primers 34 and 35. Annealing temp: 55°C and Extension time: 4min.

Colony PCR was run on an agarose gel with the ladder GeneRuler 1kb

For 2 colonies a band at the correct size, 3.6 kb, was seen and those colonies came from the plate with SPD5_both tags.

Overnight culture of the SPD5_snoop C1 was spun down and the pellet was stored in -20°C.

Overnight cultures of two colonies of SPD5_both were prepared and grown at 30°C, C9 and C10.

Miniprep of the two colonies of SPD5_both C9 and C10 as well as the stored pellet of SPD5_snoop C1.

Send SPD5_snoop c1 for sequencing using primers amplifying the ends.

Digests of multiple fragments:

• Incubate all at 37°C for 1 h

All Digests were loaded on a gel and subsequently cut out an purified using gel-extraction:

Microscopy of the SPD5 #01 was performed.

However, the SPD5 showed no fluorescence what so ever, so there might be a problem with the glycerol stock.

Sequencing results: from the SPD5_both tag: the two colonies I sent for sequencing looks good. Two of the primers had no result, I will resend those for another sequencing.

Small culture of SPD5_snoop c1 in LB + amp. After 6 hours 3 ml of the small culture was spun down and miniprep was prepared. The SPD5_snoop c1 was send for sequencing using 4x primers.

New 2° PCR of SPD5_snoop using primer 34 + 37, using Q5 polymerase. Annealing temp: 59°C and Extension: 4 min. This amplifies the whole SPD5, I choose this  as the other method gave unspecific bands.

Digest of BMC backbone

2000ng = 15µl

NdeI 2µl

XhoI 2µl

Cutsmart 10 µl

Water 71 µl

Digest of SPD5_snoop fragment

2000ng = 17.3 µl

NdeI 2µl

XhoI 2µl

Cutsmart 10 µl

Water 66.4 µl

All digests were incubated 1h at 37 °C and then loaded on a gel. The digests were cut out and purified using gel-extraction

Ligation of SPD5_snoop 1:1 ratio

Ligation mix was kept at 4°C overnight

_______________________SPD5 #02_____________________

Aim: to repeat the SPD5_sfGFP microscopy results, which were done in 2019 by the Freiburg iGEM team with our newly cloned construct. This time from freshly prepared strains, which I make sure contain the plasmids.

Strains are prepared in BL21 and are transformed with either pBAD_sfGFP or pBAD_SPD5_sfGFP

Sequencing results of SPD5_both showed that both C9 and C10 looked good and had no mutations. I will continue with C9 for future experiments.

Transformations via heat-shock:

Ligation of pMIC_SPD5_snoop into DH5-alpha

pMIC_SPD5 + pBbA2c_mVenus_ SpyCatcher (mVENUS)

pMIC_SPD5_both + mVENUS

pMIC_SPD5 + mVENUS + mTurquoise

pMIC_SPD5_both + mVENUS + pBbA2k_mTurquoise2-SnoopCatcher (mTurquoise)

Colony PCR of new colonies of the trafo of pBAD_sfGFP or pBAD_SPD5_sfGFP. This was done using Green Dream-tag polymerase MM using primers 14 +16 to amplify sfGFP.

3 colonies of pBAD_sfGFP was screened and 5 colonies of pBAD_SPD5_sfGFP as well as a positive control using the pBAD_SPD5_sfGFP plasmid. Besides, the current not-working glycerol stock were screened.

Colony PCR of SPD5_snoop C1 re-trafo colonies. This was done using Green Dream-tag polymerase MM using primers  34 + 37 to amplify SPD5_snoop.

Agarose gel of colony PCRs:

Gel 2: marker, pBAD_sfGFP (x3), pBAD_SPD5_sfGFP (x5), positive control, Glycerol stock (x2)

Gel 3: marker sfGFP (primer 38 + 39) (x8)

Miniprep of mVENUS in TOP10, 3 ml were used for the miniprep with the sigma Aldrich kit.

Overnight culture of one the screened pBAD_sfGFP/pBAD_SPD5_sfGFP colonies was prepared for microscopy on Sunday and for glycerol stock.

SPD5_#02: Preparation of 50 ml larger culture from the overnight culture (1:50) and grown to OD 0.6 at 30°C and shortly at 37°C. Induction was performed according to previous scheme from a 20% arabinose stock:

0,1%: 15 µl

0,005%: 0.75µl

Then samples were incubated at 18°C overnight. 2 additional samples were prepared with 0,1% arabinose but incubated at 37°C overnight.

Heat shock transformation was performed of; pMIC_SPD5_both into TOP10, pMIC_SPD5 + mTURQ, pMIC_SPD5_both + mTURQ.

Glycerol stock was prepared from the overnight cultures of pMIC_SPD5 + mVEN, pMIC_SPD5_both + mVEN, pMIC_SPD5 + mVEN + mTUR, pMIC_SPD5_both + mVEN +mTUR.

Overnight cultures were prepared of the trafos from yesterday of pMIC_SPD5_both into TOP10, pMIC_SPD5 + mTURQ, pMIC_SPD5_both + mTURQ.

Colony PCR of colonies from the overnight ligation of PMIC_SPD5_snoop, this was done using Dream-taq green MM and primers 34 + 37 at annealing 55°C and extension 4 min.

Glycerol stocks of yesterdays overnight cultures and pMIC_SPD5 + mTURQ, pMIC_SPD5_both + mTURQ

Miniprep of the colonies for pMIC_SPD5_both was prepared an afterwards send for sequencing.

Induction of the pMIC_SPD5 + mTURQ, pMIC_SPD5_both + mTURQ was prepared according to previous description

SPD5 #03

Aim: to repeat the SPD5_sfGFP microscopy results, which were done in 2019 by the Freiburg iGEM team with our newly cloned construct. Repeat of SPD5 #02 because the last time I thought I made a mistake by induction.

Strains are prepared in BL21 and are transformed with either pBAD_sfGFP or pBAD_SPD5_sfGFP

Overnight cultures from yesterday of pBAD_SPD5_sfGFP/pBAD_sfGFP and pBAD33+pET_sfGFP/pBAD33 + pET_sfGFP_TP were dilutes 1:50 in larger cultures and grown at 30°C until they reach OD: 0.6

Microscopy of pMIC_SPD5 + fluorescent catcher

Heat shock transformations of the following was performed:

SPD5 #04

Aim: To see if there is a difference in the dots which are formed in BL21 and in MG1655.

Experimental setup: MG1655 is transformed with either pBAD_SPD5_sfGFP (Strain 2) or pBAD_sfGFP (Strain 1) and from an overnight culture a larger culture is grown to OD: 0.6 and induced according to the scheme.

Strain 1: samples 1-4

Strain 2: samples 5-8

Glycerol stocks of all overnight cultures was prepared.

Grew a larger culture (50 ml) of the 1:50 diluted overnight culture pBAD_SPD5_sfGFP or pBAD_sfGFP in MG1655 and induced at OD: 0.6 according to SPD5 #04 scheme. Then they were incubated at 18 °C.

Microscopy of the prepared samples of pBAD_SPD5_sfGFP and pBAD_sfGFP, this time in MG1655.

 This time the drops could move!

Send pBAD_SPD5_sfGFP for sequencing the promoter region to look for mutations in the region causing it to be constitutively expressed.

Sequencing result of SPD5_sfGFP from yesterday showed for the PETUP primer that it could be a mix of two different plasmids, but the primer I send showed a cleaner result. However, there seems to be a deletion in front of the AraC, which could influence how well the AraC is translated.

Heat-shock transformation of

pMIC_EncA + pVEN:sfGFP_TP in ME5119                     (AmR + CmR)

1939 + pVEN_sfGFP_TP in ME5119                               (AmR + CmR)

pMIC_SPD5 + mVEN in C321∆exp                                 (AmR + CmR)

pMIC_SPD5_both + mVEN in C321∆exp                       (AmR + CmR)

pMIC_SPD5 + mVEN in Genome Reduced                    (AmR + CmR)

pMIC_SPD5_both + mVEN in Genome Reduced           (AmR + CmR)

Microscopy of Encapsulin #04

Prepare overnight cultures for the microscopy on Sunday from the MG1655

pMIC_SPD5 + mVEN

pMIC_SPD5_both + mVEN

pMIC_SPD5 + mTURQ

pMIC_SPD5_both + mTURQ

pMIC_SPD5_spy/snoop #02

Aim: to check whether the pMIC_SPD5 with and without tags, look like the SPD5_sfGFP in regards of being dynamics and if the catching seems to work.

Experimental setup: all strains in MG1655

Strain A1: SPD5 + mVEN

Strain A2: SPD5_both + mVEN

Strain B1: SPD5 + mTURQ

Strain B2: SPD5_both + mTURQ

Not all samples were microscoped, only the following based on initial microscopy.

Strain A: 10, 4, 3, 1

Strain B: 8, 5, 2, 1

SPD5 #05

Aim: To see if there is a difference in the dots which are formed in BL21 and in MG1655. Experimental setup: MG1655 is transformed with either pBAD_SPD5_sfGFP (Strain 2) or pBAD_sfGFP (Strain 1) and from an overnight culture a larger culture is grown to OD: 0.6 and induced according to the scheme. This time one concentration lower than previous was also performed.

Strain 1: samples 1-3

Strain 2: samples 4-6

Microscopy of pMIC_SPD5_spy/snoop #02

Induction of samples for FRAP pBAD_sfGFP and pBAD_SPD5_sfGFP accordingly

FRAP + fluorescent microscopy of pBAD_sfGFP and pBAD_SPD5_sfGFP

SDS of the pMIC_SPD5_spy/snoop #02 (12% SDS gel prestained protein ladder plus, thermos fischer)

Transfer via semidry (Run for 1 h at constant voltage 10V)

Blocking with 10% milk powder PBS-T 1X

Primary antibody anti-GFP (Rabbit)

• Lost focus almost immediately

pMIC_SPD5 + fluoro #03

Aim: To repeat the experiments with the SPD5 tag construct, which showed fluorescent dots that did not move. This time repeat again in MG1655, but microscope multiple timepoints before 24 h. I expect that the lang incubation times cause the droplets to turn into gel-like state.

Experimental setup: Liquid cultures from glycerol stocks of the strains were grown until OD 0.6-0.8 and induced accordingly. Then the cultures incubated further at 18 °C for 2-2-5 hours until agarpads were prepared for microscopy. These were then microscoped with intervals of 1-2 hours and observed for dynamic droplets.

Strain 1: pMIC_SPD5 + mVen

Strain 2: pMIC_SPD5_both + mVen

Overnight culture for pMIC_SPD5 + fluoro #03 were prepared and incubated at 37 °C

Overnight experiment of pMIC_SPD5_both + mVENUS #03

• Lost focus immediately

Trafo of new Site-directed Mutagenesis of SPD-5 from Jeromy + mVenus or + mTurquoise into MG1655 and plated on LB +amp + (cmR/kan)

Overday culture of pMIC_SPD5_both + mVENUS #03, same culture later used for induction.

                      Strain 2: induced at 16:55

                      Strain 1: induced at 17:25

• The agar pad was measured every 2h until 02:30, however when I changed to new agar pad, the dots had already formed and did not move.

Overnight culture of trafos from yesterday, SPD-5_sdm_both + mVenus or + mTurquoise in MG1655 were incubated at 37 °C.

pMIC_SPD5 + fluoro #04

Aim: To repeat the experiments with the SPD5 tag construct, this time with the site-directed mutagenesis version, from Jeremy.

Experimental setup: Liquid cultures from single colonies of the strains were grown until OD 0.6-0.8 and induced accordingly. Then the cultures incubated further at 18 °C for 24 hours until agarpads were prepared for microscopy.

Strain 1: pMIC_SPD5_both + mVen

Strain 2: pMIC_SPD5_both + mTur

Strain 1:

Strain 2:

Microscopy pMIC_SPD5_sdm_both + mVenus or + mTurquoise #04

SDS-gel of samples were prepared by measuring OD and spinning down 100 µl of culture and then resuspending of SDS-sample buffer.

Run 110 V for 1 hour 10 min.

Westernblot transfer, wet blot according to protocol. Run at 0.002A for 2 hour at RT

Blocking with 10% Milk in PBST for 1 h

Wash 4x, incubation with primary antibody (anti-gfp) wash 4x, incubation with secondary antibody, wash 4x, detect. Incubation with primary antibody anti-RNA-pol, wash 4x, Incubation with secondary antibody HRP-peroxidase, detect.

Wiffleballs

Noncanonical amino acids are amino acids which are not found naturally in proteins. The incorporation can lead to novel characteristics of the protein of interest. In this part of our project we seek to characterize the incorporation of ncAA into sfGFP. Furthermore, we incorporate ncAA into the main proteins of the bacterial microcompartments. This is done using the amber stop-codon and incorporation will be tested through FACS, western-blot and crosslinking. Responsible for this part were: Fabian, Johanna, Leon, Jeromy.

Preparing 250 ml LB medium following the protocol #001 and adding 250µl K and 250µl Sm 

Preparing 250 ml LB agar following the protocol #004 and adding 250µl K and 250µl Sm

Picking 2 colonies from the E.coli strain with the plasmid pHT1 and 2 from the one with the plasmid pHT1T2T3 --> overnight culture in LB-medium + Amp

Glycerol stocks of all overnight cultures using protocol #008 

Miniprep of all overnight cultures using protocol #007 

wiffleballs:

1. SnpCat-mTurquoise2  KanR
2. SpyCat-mVenus2  CmR
3. pHT1SnpISpyTagT2T3  AmpR
4. pHT1SnpISpyTag  AmpR

Transformation in BL21 of:

wiffleballs:   a + b + c

              a + b + d

Transformation in BL21 of:

wiffleballs:   a + b + c

              a + b + d

Overnight culture of :

• wiffleballs:   a + b + c and a + b + d (in +K+Cm+Amp medium)

Glycerol stocks of the overnight cultures of the previous day (2 each):

• wiffleballs:   a + b + c and a + b + d (in +K+Cm+Amp medium)

wiffleballs: we want to prove that they can form and we also want to make them visible

We made 4 transformations with BL21 competent cells from Nicole with following plasmids, following the protocol #003:

• pHT1 + mVenus2-spyCat
• pHT1T2T3 + mVenus2-spyCat
• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat

We let the bacteria grow on CmR + AmpR plates at 37°C.

Overnight cultures of all transformations and let them grow at 30°C.

Prepare medium for the induction next day and also let it at 30°C to have a constant temperature.

--> 5 ml LB + 5 µl Cm + 5 µl Amp pro tube

-->100 ml LB + 100 µl Cm + 100 µl Amp pro Erlenmayer glas

We induced the bacteria of all trafos. First we prepared the bacteria as following:

• Centrifuge 2 ml at 3000 g for 100 min in an eppi in order to get a 1:50 culture in a 100 ml LB
• Throw supernatent away and resuspend bacteria in little amount of the LB that has been prepared on the previous day, then put all the bacteria into the 100 ml culture
• Prepare a cuvette with just LB as blank and measure the OD600 of the bacteria until they have reached 0,6-0,8
• Distribute the 100 ml culture into 9 tubes for the 9 induction conditions

Induction conditions: we used a 100 mM stock solution of IPTG for the wiffleballs and a 50 µg/ml stock solution of doxycycline for Venus.

Samples:

• pHT1 + mVenus2-spyCat: samples n.1-9
• pHT1T2T3 + mVenus2-spyCat: samples n.10-18
• pHt1snpspyTag + mVenus2-spyCat: samples n.19-27
• pHT1snpspyTagT2T3 + mVenus2-spyCat: samples n.28-36

24h incubation at 18°C

We prepared the cells for fluorescence microscopy as following:

• Take 1 ml bacteria from induced cultures
• Centrifuge them at 8000 rpm for 1 min and throw supernatent away
• Resuspend in 10-20 µl PBS (wash)
• Centrifuge again and resuspend again
• Prepare the agar pads for each sample --> fluorescence microscopy

We also prepared the cells for SDS as following:

• Take 1 ml bacteria from induced cultures
• Centrifuge them at 3000g for 10 min and throw supernatent away
• Freeze the pellet for the next day

We prepared the cells for sonication by resuspending the pellet in 1 ml lysis buffer (20 mM Tris + 50 mM NaCl)

--> Sonication of all the samples

Centrifuge all the samples at 3000 rpm for 10 min

Prepare samples for SDS:

• Pipette 10 µl supernatent of each sample together with 10 µl loading buffer (Laemmli buffer)
• Prepare gel by mixing and polymerizing separating gel and stacking gel

Separating gel (12,5%)

1,33 ml H2O

1,5 ml Tris/HCl 1,88 M pH 8,8

1,5 ml SDS 0,5% w/v (1 g per 100 ml)

3,13 ml Acrylamide (37, 5:1)

7,5 µl TEMED

37,5 µl APS (10% w/v)

Invert the tube before pouring the gel in the chamber

Add 500 µl isopropanol on top and let it polymerize

Stacking gel (5%)

1,06 ml H2O

0,5 ml Tris/HCl pH 6,8

0,5 ml SDS 0,5% w/v

0,42 ml Acrylamide

2,5 µl TEMED

12,5 µl APS

 Invert the tube

Pour the isopropanol away and give the stacking gel on top of the polymerized separating gel

We loaded all the samples in the chambers as in the table below, poured the running buffer and let the SDS PAGE run at 120V with 0,08 Amper

We stained the gels with Roti blue overnight

We ran a SDS PAGE again with the samples 1-9, then we transferred the proteins on nitrocellulose membrane with the semidry blotting technique

--> We ran the western blot at 10 V with 1 Amper, for 1 h at 4°C

We blocked the membrane with 10% milk in PBS-Tween20 solution for 1 h, then washed it 4 times for 5 min with PBS-Tween20 buffer (PBS 1x + Tween20 0,1%)

1st Ab incubation:

We prepared a solution of BSA 3%, 0,1% sodium azide, PBS-Tween20 + antiHis6xTag-Ab 1:1000

Incubate overnight at 4°C

Wash the membrane 4 times for 5 min with PBS-Tween20

2nd Ab incubation:

We prepared a solution of 5% milk in PBS-Tween20 + 1:10000 HRP(anti-mouse) and incubated the membrane for 2 h at room temperature

We washed again the membrane 4 times for 5 min with PBS-Tween20 and then detected the proteins with the imager

--> for the detection we mixed 500 µl luminol enhancer with 500 µl peroxide solution and incubated for 1 min before taking the picture with the imager

We ran SDS gels of the following samples:

• 11, 13, 15, 16, 17,18 (pHT1T2T3 + mVenus)
• 19, 21, 22, 25, 26, 27, 28 (pHT1snpspyTag + mVenus)
• 29, 30, 31, 32, 33, 34, 35, 36 (pHT1snpspyTagT2T3 + mVenus)

--> We had to repeat the last row since the marker went into all the wells; for this we made a 8% separating gel and a 3% stacking gel

We ran a Western blot for the first two rows following the same procedure as the days before, until the 1st ab incubation

We ran the Western blot for the pHT1snpspyTagT2T3 + mVenus samples until the 1st ab incubation and finished the other ones for pHT1T2T3 + mVenus and for pHT1snpspyTag + mVenus)

We finished the Western blot procedure for pHT1snpspyTagT2T3 + mVenus samples

The detection did not work, so we incubated the membranes again with the 1st ab

Run SDS gels at 120 V, 0,4 Ampere

The samples have been pipetted in the wells as following:

• Gel 1: marker, 1, 2, 3, 4, 5, 6 7, 8, 9
• Gel 2: 36, marker, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35

Western Blot process until 1st Ab incubation (overnight)

Finish Western Blot + detection on imager

We made 4 transformations with BL21, ME5000 and MG1655 competent cells  with following plasmids, following the protocol #003:

• pHT1 + mVenus2-spyCat
• pHT1T2T3 + mVenus2-spyCat
• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat
• pBAD33 (empty backbone)

We let the first 4 transformed cultures grow on CmR + AmpR plates, pBAD33 on CmR plates, at 37°C

Overnight cultures of:

• pHT1
• pHT1T2T3
• pHt1snpspyTag
• pHT1snpspyTagT2T3
• mVenus2-spyCat
• mTurquoise-spyCat

Overnight cultures in BL21 of following transformations;

• pHT1 + mVenus2-spyCat
• pHT1T2T3 + mVenus2-spyCat
• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat
• pBAD33 (empty backbone)

Induction of all overnight cultures as following:

Samples:

• pHT1 + mVenus2-spyCat: 1-4
• pHT1T2T3 + mVenus2-spyCat: 5-8
• pHt1snpspyTag + mVenus2-spyCat: 9-12
• pHT1snpspyTagT2T3 + mVenus2-spyCat: 13-16
• pBAD33 (empty backbone): 17-20

Same procedure as the 1st of July but we induced 5 ml cultures with 1:1000 inducers

Growth curve assay in a 96-well plate with 180 µl of each sample, as in the table below (indicated are the sample numbers and the conditions of the samples). We pipetted each sample 3 times in order to get the mean of the values and so get more precise results. We let the plate shake at medium intesity continuosly, at 37°C, measuring the values every 10 min.

Fluorescence microscopy

Transformation of:

ME5000 with

• pHT1 + mVenus2-spyCat
• pHT1T2T3 + mVenus2-spyCat
• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat
• pBAD33 (empty backbone)
• mVenus2-spyCat

NEBDH5a with

• pHT1
• pHT1T2T3
• pHt1snpspyTag
• pHT1snpspyTagT2T3
• mVenus2-spyCat
• mTurquoise-spyCat

BL21 with

• pHT1snpspyTagT2T3 + mVenus2-spyCat + mTurquoise-spyCat

Sonication

SDS of all induced samples 1-20 (separating gel: 8%, stacking gel: 3%)

Gel A: marker, 1, 2, 3, 4, 5, 6, 7, 8

Gel B: marker, 9, 10, 11, 12, 13, 14, 15, 16

Western Blot --> transfer did not work properly

• Overnight cultures of all the NEBDH5a transformations and pHT1snpspyTagT2T3 + mVenus2-spyCat + mTurquoise-spyCat

We repeated the SDS and made the Western blot again until the 1st Ab incubation

(Western blot at 9-10V, 0,4 ampere, 4W, 1h)

Roti-Blue staining of the gels overnight

Miniprep of all the NEBDH5a overnight cultures

Transformations of ME5000 with new DNA (minipreps):

• pHT1 + mVenus2-spyCat
• pHT1T2T3 + mVenus2-spyCat
• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat
• 1939(AmpR) + pBAD33(CmR) (empty backbones)
• 1939

Finish Western blot + detection --> not enough bands and bands at wrong places

Transformations of ME5000 with new DNA (minipreps):

• pHT1 + mVenus2-spyCat
• pHT1T2T3 + mVenus2-spyCat
• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat
• 1939(AmpR) + pBAD33(CmR) (empty backbones)
• 1939

Induction (protcol #040) of 20 samples of BL21 (1-20) and 20 samples of MG1655 (21-40) like on the 19th of July

In MG1655 and BL21:

• pHT1 + mVenus2-spyCat: 1-4 
• pHT1T2T3 + mVenus2-spyCat: 5-8
• pHt1snpspyTag + mVenus2-spyCat: 9-12
• pHT1snpspyTagT2T3 + mVenus2-spyCat: 13-16
• p1939(AmpR) + pBAD33(CmR) (empty backbones): 17-20

Condition 0-3:

(addendum 28/07 -> Doxycyclin stock was prepared wrong -> 1000 fold concentration used)

Growth curve assay of MG1655 (20h)

Growth curve assay in a 96-well plate with 200 µl of each sample, as in the table below (indicated are the sample numbers and the conditions of the samples). We pipetted each sample 3 times in order to get the mean of the values and so get more precise results. We let the plate shake at medium intesity continuosly, at 30°C, measuring the values every 10 min for 20h.

96 well-plate schema:

Incubator was turned of by someone … no further experiments --> repeat

Trafo of BL21 w. pBAD33 & 1939 for WT control

Overnight cultures of all following transformed of BL21 and MG1655 

• pHT1 + mVenus2-spyCat
• pHT1T2T3 + mVenus2-spyCat
• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat
• p1939(AmpR) + pBAD33(CmR) (empty backbones)

Induction of BL21 and MG1655 like Monday 25/07 

… but -> Doxycyclin stock was prepared wrong the past-> 1000 fold concentration used, also on 25/07 and 19/07 

Induction of BL21 and MG1655 like Monday 25/07

Fluorescence Microscopy w. Agarose Pads (protocol #037) of induced BL21 & MG1655 and 

their sample preparation (protocol #038): Sonication, fractioning freeze -20°C for later SDS-PAGe and WB

Transformations of ME5000 (SmR) and ME5119(SmR +TetR):

• pHT1 + mVenus2-spyCat
• pHT1T2T3 + mVenus2-spyCat
• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat
• p1939(AmpR) + pBAD33(CmR) (empty backbones)
• P1939 (empty backbone)

Protein Miniprep w. Nickel/IDA spin column (Zymno) 87µg/ml, but 260/280n ratio ~.097

SDS-PAGE (8% seperating gel; protocol #31) of induced BL21 (from 29/07) with following constructs

• pHT1 + mVenus2-spyCat
• pHT1T2T3 + mVenus2-spyCat
• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat

Condition 0-3: 

One construct per gel; 4 gels in total  
loading schema (M = marker) :

Well:          1  2  3  4  5  6  7  8  9 

Condition: M  0  1  2  3  0  1  2  3

                  Supernatent    Pellet

Western Blot (protocol #030) of all 4 Gels until anti-His Antibody -> overnight

Western Blot (protocol #030) part 2: 

Incubate anti-mouse antibody and detection --> worked well! J 

SDS-PAGE like Monday but with MG1655 

SDS-PAGE (8% seperating gel; protocol #31) of induced MG1655 (from 29/07) with following constructs 

• pHT1 + mVenus2-spyCat 
• pHT1T2T3 + mVenus2-spyCat 
• pHt1snpspyTag + mVenus2-spyCat 
• pHT1snpspyTagT2T3 + mVenus2-spyCat 

Condition 0-3:  

One construct per gel; 4 gels in total  
loading schema (M = marker) : 

Well:          1  2  3  4  5  6  7  8  9  

Condition: M  0  1  2  3  0  1  2  3 

                  Supernatent    Pellet 

Western Blot (protocol #030) of all 4 Gels, anti-His Antibody; then anti RNAPol antibody -> overnight 

Antibiotic resistance test of ME5119 

Western Blot:

Also anti-RNAPol on BL21 Western Blot membranes

Sec Anti mouse antibody on all 8 membranes (Bl21 and MG1655) 

Detection:

BL21: nice 
MG1655: RNA pol = nice;  
wiffleball expression no detectible, only pHt1snpspyTag + mVenus2-spyCat and pHT1T2T3 + mVenus2-spyCat

ME5119 also grew on Streptomycin--> maybe wrong strain? Or not working antibiotic

Meeting for transmission electron microscopy + new strategy to enhance the wiffleball-Protein expression/ formation

Also test different concentration of IPTG for higher expression

Induction (protcol #040) of BL21 and MG1655 

Constructs in MG1655 and BL21: 

• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat
• p1939(AmpR) + pBAD33(CmR) (empty backbones)

Conditions: 

Incubation for 24h, 18°C, 200rpm

Glycerol stocks of MG1655 

• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat
• p1939(AmpR) + pBAD33(CmR) (empty backbones, WT control)

and and BL21: p1939(AmpR) + pBAD33(CmR) (empty backbones, WT control)

Check competent ME5125 for antibiotic resistance:

ME5125:

AmpR: no 

KanR: yes 

TetR: yes  
-> maybe wrong strain was given? Maybe ME5010?

Fluorescence Microscopy w. Agarose Pads (protocol #037) of induced BL21 & MG1655 from 05/08

+ their sample preparation (protocol #038): Sonication, fractioning freeze -20°C for later SDS-PAGEand WB 

SDS-PAGE (8% seperating gel; protocol #31) of induced MG1655 (from 05/08) with following constructs in MG1655 and BL21: 

• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat

Conditions: 

4 Gels:

MG1655

• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat

BL21

• BL21 pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat

loading schema (M = marker) :

Well:          1  2  3  4  5     6    7       8     9  10  11  12   13    14     15

Condition: M  0  1  2  3  400 700  1000  0   1    2    3   400   700  1000  

                                Supernatent                               Pellet

Western Blot (protocol #030) of all Gels till blocking,

• Lab mate messed membranes up… all again… 

Again: SDS-PAGE (8% seperating gel; protocol #31) of induced MG1655 (from 05/08) with following constructs in MG1655 and BL21: 

• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat

Conditions: 

4 Gels:

MG1655

• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat

BL21

• BL21 pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat

loading schema (M = marker) :

Well:          1  2  3  4  5     6    7       8     9  10  11  12   13    14     15

Condition: M  0  1  2  3  400 700  1000  0   1    2    3   400   700  1000  

Western Blot (protocol #030) till anti-mous antibody -> overnight

Preparation for immunofluoresce to see the distribution and formation of the BMCs at the cell poles. 

Prepare Lid coation with Triton X-100 for better growth curve assays in the microplate reader 
Coating solution: 20%EtOH, 0.05% Triton X-100

Incubate 96 Well plate lid for 15sec, at RT, then air dry

Detection of Western Blot membranes from 10/08

BL21: both constructs nice

MG1655

• pHt1snpspyTag + mVenus2-spyCat

--> only RNAPol detectable

• pHT1snpspyTagT2T3 + mVenus2-spyCat

--> little bit of expression

Conclusio: wiffleball expression in MG1655 not optimal, but repeat 

New Antibodies from Barbaras Lab:

- Anti-GFP-Antibody (polyclonal) (rabbit) for mVenus or mTurquoise detection 

- anti-rabbit-Antibody-Cy5 conjugate 

Incubate same membranes again with Anti-GFP-Antibody and anti-rabbit antibody

• only BL21 BMC-C worked --> maybe because of anti rabbit CY5 Antibody ? 

Making competent ME5119 bacteria (protocol #017), because wrong strain was given last time

Replicate Experiment

Induction (protcol #040) of MG1655 

Constructs in MG1655 and BL21: 

• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat
• p1939(AmpR) + pBAD33(CmR) (empty backbones)

Conditions: 

Incubation for 24h, 18°C, 200rpm

Also Growth assay with coated lid from10/08 

Dilute bacteria after induction from OD600= 0.6 to 0.1

Addendum 31.08 also inducer concentration diluted… not the right inducer concentration anymore

Fluorescence Microscopy w. Agarose Pads (protocol #037) of induced MG1655 from 13/08 with a Zeiss Axiovert with Kolibri LEDs

Induced bacteria with pHT1snpspyTagT2T3 + mVenus2-spyCat have dots at their poles. 

Sample preparation (protocol #038) from induced MG1655 from 13/08: samples spinned down and freezed -20°C for later SDS-PAGE and WB 

Sample preparation (protocol #038): Sonication, fractioning freeze -20°C for later SDS-PAGEand WB (samples from 14/08)

SDS-PAGE (8% seperating gel; protocol #31) of induced MG1655 (from 13/08) with following constructs

• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat

Conditions: 

3 Gels:

MG1655

• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat

+ MG1655 sample from 09/08 to check for same results

• pHT1snpspyTagT2T3 + mVenus2-spyCat

loading scheme (M = marker) :

Well:          1  2  3  4  5     6    7       8     9  10  11  12   13    14     15

Condition: M  0  1  2  3  400 700  1000  0   1    2    3   400   700  1000  

                                Supernatent                               Pellet

Transformation of: 

ME5119 (genome reduced E.coli) with 

• pHt1snpspyTag + mVenus2-spyCat 
• pHT1snpspyTagT2T3 + mVenus2-spyCat 
• pBAp1939(AmpR) + pBAD33(CmR) (emptyvectors)

Western Blot (protocol #030) of gels from, 15/08:

After blotting: Ponceau staining showed no proteins, also Comassie staining showed no proteins left on gel

--> again 

Overnight cultures of ME5119 (genome reduced E.coli) 

with 

• pHt1snpspyTag + mVenus2-spyCat 
• pHT1snpspyTagT2T3 + mVenus2-spyCat 
• pBAp1939(AmpR) + pBAD33(CmR) (empty   vectors)

Induction (protcol #040) of ME5119 like 04/08

Constructs in ME5119: 

• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat
• p1939(AmpR) + pBAD33(CmR) (empty backbones)

Conditions: 

Incubation for 24h, 18°C, 200rpm

Also Growth assay with Triton X-100 coated lid like 10/08 

Dilute bacteria after induction from OD600= 0.6 to 0.1

Addendum 31.08 also inducer concentration diluted… not the right inducer concentration anymore

Transformation of new made BL21 with 

• pHt1snpspyTag + mVenus2-spyCat 
• pHT1snpspyTagT2T3 + mVenus2-spyCat 
• pBAp1939(AmpR) + pBAD33(CmR) (empty   vectors)

Fluorescence microscopy of induced ME5119 (17/08) with Agar Pads (protocol #37) with a Zeiss Axiovert with Kolibri LEDs 

• no foci at the poles detectable + weakly expressed fluorescence

Sample preparation (protocol #038) from induced ME5119 spinned down and freezed -20°C for later SDS-PAGEand WB 

Sample preparation (protocol #038): Sonication, fractioning of ME5119 samples from 18/08

SDS-PAGE (8% seperating gel; protocol #31) of induced ME5119 (17/08) and induced MG1655 from 15/08 with following constructs: 

• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat

Conditions: 

4 Gels:

MG1655

• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat

BL21

• BL21 pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat

loading schema (M = marker) :

Well:          1  2  3  4  5     6    7       8     9  10  11  12   13    14     15

Condition: M  0  1  2  3  400 700  1000  0   1    2    3   400   700  1000  

                                Supernatent                               Pellet

Western Blot (protocol #030) till anti-His antibody -> overnight

Induction (protcol #040) of transformed BL21 (from 18/08) like 04/08 

Constructs in BL21: 

• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat
• p1939(AmpR) + pBAD33(CmR) (empty backbones)

Conditions: 

Incubation for 24h, 18°C, 200rpm

Also Growth assay with Triton X-100 coated lid like 10/08 

in a 96-well plate with 200 µl of each sample, as in the table below We pipetted each sample 3 times in order to get the mean of the values and so get more precise results. We let the plate shake at medium intesity continuosly, at 30°C, measuring the values every 10 min for 20h

Western Blot (protocol #030) of Mg1655 and ME5119 samples (19/08)

Anti-RNAPol Antibody ~1.5h

Anti-mouse Antibody ~1h

Detection

Stripping test of these membranes after detection w. 2x 5min incubation  
w. 0.2M NaOH, then 4x 5min wash with PBST

Again detection with ECL solution 

• still signal detectable

Overnight cultures of BL21 

with 

• pHt1snpspyTag + mVenus2-spyCat 
• pHT1snpspyTagT2T3 + mVenus2-spyCat 
• pBAp1939(AmpR) + pBAD33(CmR) (empty vectors)

for third time repetition of the experiments: Fluorescence microscopy, growth curve assay, SDS-PAGE, Western Blot

nduction (protcol #040) of BL21 (overnight cultures from 22/08) like 04/08 for growth assay

Constructs in BL21: 

• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat
• p1939(AmpR) + pBAD33(CmR) (empty backbones)

Conditions: 

Growth assay with Triton X-100 coated lid like 10/08 

In a 96-well plate with 200 µl of each sample, as in the table below We pipetted each sample 3 times in order to get the mean of the values and so get more precise results. We let the plate shake at medium intesity continuosly, at 30°C, measuring the values every 10 min for 20h

Fluorescence microscopy of induced BL21 (23/08) with Agar Pads (protocol #37) with a Zeiss Axiovert with Kolibri LEDs 

Sample preparation (protocol #038) from induced BL21 spinned down and freezed -20°C for later SDS-PAGE and WB 

Sample preparation (protocol #038): Sonication, fractioning of BL21 samples from 24/08

SDS-PAGE (8% seperating gel; protocol #31) of induced BL21 (23/08) with following constructs: 

• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat

Conditions: 

2 Gels:

BL21

• BL21 pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat

loading schema (M = marker) :

Well:          1  2  3  4  5     6    7       8     9  10  11  12   13    14     15

Condition: M  0  1  2  3  400 700  1000  0   1    2    3   400   700  1000  

                                Supernatent                               Pellet

Western Blot (protocol #030) till anti-His antibody -> overnight

Western Blot (protocol #030) of Mg1655 and ME5119 samples (19/08)

Incubated with Anti-RNAPol Antibody, Anti-mouse Antibody

Then detection

Induction(protocol #040) of BL21 for immunofluorescencestaining (Johanna alreadyprepaired the overnight cultures)

Constructs in BL21: 

• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat
• p1939(AmpR) + pBAD33(CmR) (empty backbones)

Conditions: 

• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat

• Wild type control p1939(AmpR) + pBAD33(CmR) (empty backbones)

Incubationfor 16h, 18°C, 200rpm

Immunofluorescence staining of induced BL21 (29/08) based on the paper from Park et al. 2019. “An Improved Method for Bacterial Immunofluorescence Staining To Eliminate Antibody Exclusion from the Fixed Nucleoid“

Fixation 
1) 5ml cells spinned down at 4000g for 5min 
2) Resuspend in 1ml of 4% formaldehyde in 1xPBS  
3) Incubation for 30min at room temperature 
4) Spin down at 1500g for 5 min resuspend in 1xPBS 
5) Repeat step 4)

EtOH treatment 
1) 10µl of 70% EtOH per 108 Cells (OD600=1 --> 8*108 cells 
2) Incubation on nutator for 1h

Cell immobilisation 
1) Precoat in 8well chambered cover glass (ibiTreat from ibidi used) with 0.1% Polylysine in ddH2O and incubate for 1h 
2) 3x washing with ddH2O + airdrying 
3) 5µl of EtOH treated cells + 5µl ddH2O -> centrifuge at 600g for 5min 
4) Resuspend pellet in 130µl 1xPBS  
-> add in single well and incubate for 1h at 4°C 
5) check cell density under microscope until desired cell density is reached then remove unattached cells with PBS replacement

Lysozyme treatment 
1) 2µL of 1000 U/µL Lysozyme (from fisher scientific) per well (130µl -> 2000U) 
2) Incubate 30min at roomtemperature 
3) Wash 3x with PBS

DNase I treatment 
1) 10x DNase reaction buffer w. MgCl2 to 130µl in well (14.6µL) + 2µL of DNase 1U/mL (fisher scientific)  
2) incubate 1h at 37°C 
3) Wash 3x with PBS

Immunostaining 
1) Incubation with blocking buffer (0.1% BSA, 0.05% Tween20® in PBS) 
2) 1st antibody Anti-His (from Biolegend) 1:250 in PBS + incubate 1.5h at roomtemperature 
3) 3x wash with PBS with 5min incubation 
4) 2nd antibody anti-mouse dyelight 600 conjugate (from invitrogen) 1:500 in PBS + incubate for 1h at roomtemperature 
5) 3x wash with PBS at roomtemperature

Storage for later use because SIM microscope was not availible 
store with PBS with 0.1% Sodiumazide at 4°C

Transformation of C321∆exp with 

• pHt1snpspyTag + mVenus2-spyCat 
• pHT1snpspyTagT2T3 + mVenus2-spyCat 
• pBAp1939(AmpR) + pBAD33(CmR) - empty        vectors

Western Blot (protocol #030) again staining of membranes from 26/08:

Anti-GFP, polyclonal, rabbit host, (from cell signaling 255555) for mVenus detection

-Antibody solution with Sodiumazide 0.1% -> Sodiumazide blocks HRP from last secondary antibody. There should be no or nearly less signal.

 
Detection with ECL solution after anti-GFP incubation -> really less signal of RNA-Pol, but also high background noise

• Stripping not necessary

 
Incubate with secondary antibody anti-rabbit-HRP overnight

Detection of Western Blot from 31/08

mVenus is heavily expressed and already degraded when bound to T1, maybe less mVenus induction better

Tranformation results from 31/08: worked, but pHT1snpspyTagT2T3 + mVenus2- spyCat not

Transformation of C321∆exp with 

• pHT1snpspyTagT2T3 + mVenus2-spyCat 
• strain test

Transformation of BL21 with 

• pHT1snpspyTagT2T3 + mTurqouise2-snpCat 
• pHT1snpspyTag + mTurqouise2-snpCat 
• pHT1snpspyTagT2T3 + mTurqouise2-snpCat + mVenus2-spyCat 
• mTurqouise2-snpCat + mVenus2-spyCat 
• for FRET

Transformation of DH5a with 

• pHT1snpspyTag 
• pHT1snpspyTagT2T3 
• mTurqouise2-snpCat 
• mVenus2-spyCat 
• Glycerol stocks and Miniprep

Overnight cultures of BL21 with 

• pHT1snpspyTagT2T3 + mTurqouise2-snpCat 
• pHT1snpspyTag + mTurqouise2-snpCat 
• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat
• p1939(AmpR) + pBAD33(CmR) (empty backbones)

Overnight cultures of MG1655 with 

• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat
• p1939(AmpR) + pBAD33(CmR) (empty backbones)

Transformation of C321∆exp with:

• pHT1snpspyTagT2T3 + mVenus2-spyCat

Glycerol stocks of DH5a with 

• pHT1snpspyTag 
• pHT1snpspyTagT2T3 
• mTurqouise2-snpCat 
• mVenus2-spyCat 

Glycerol stocks of BL21 with 

• pHT1snpspyTagT2T3 + mTurqouise2-snpCat 
• pHT1snpspyTag + mTurqouise2-snpCat 
• pHT1snpspyTagT2T3 + mTurqouise2-snpCat + mVenus2-spyCat 

Induction (protcol #040) of BL21 (overnight cultures from 22/08) like 04/08 for growth assay

Constructs in BL21: 

• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat
• p1939(AmpR) + pBAD33(CmR) (empty backbones)
• pHT1snpspyTagT2T3 + mTurqouise2-snpCat 
• pHT1snpspyTag + mTurqouise2-snpCat 

Conditions for growth curve assay with: 

• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat
• p1939(AmpR) + pBAD33(CmR) (empty backbones)

Growth curve assay in 96 well plate with Triton-X100 coated lid.

After OD600= 0.6 induction: dilution to 0.1, but with right inducer concentration.

30°C, medium shake, OD600 read every 10min for 18h.

Conditions for fluorescence microscopy with: 

• pHT1snpspyTagT2T3 + mTurqouise2-snpCat 
• pHT1snpspyTag + mTurqouise2-snpCat 
• p1939(AmpR) + pBAD33(CmR) (empty backbones)
• pHT1snpspyTagT2T3 + mVenus2-spyCat

Induction at 18°C, 200 rpm for 20h

Fluorescence microscopy of induced BL21 (03/04) with Agar Pads (protocol #37) with a Zeiss Axiovert with Kolibri LEDs 

• 50ng/µl Doxycyclin to much for mTurquoise, seems to aggregate, check with WB
• mVenus induction with 25ng/µL Doxycyclin perfect

Overnight cultures of BL21 with 

• pHt1snpspyTag + mTurqouise2-snpCat + mVenus2-spyCat
• mTurqouise2-snpCat  + mVenus2-spyCat
• p1939(AmpR) + pBAD33(CmR) (empty backbones)

Overnight cultures of MG1655 with 

• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat
• p1939(AmpR) + pBAD33(CmR) (empty backbones)

Overnight cultures of C321∆exp with 

• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat
• p1939(AmpR) + pBAD33(CmR) (empty backbones)

Test newly competent made BL21 for Chloramphenicol contamination -> overnight culture

Induction (protocol #040) of MG1655 and C321∆exp with following constructs:

·      pHt1snpspyTag + mVenus2-spyCat

·      pHT1snpspyTagT2T3 + mVenus2-spyCat

·      p1939(AmpR) + pBAD33(CmR) (empty backbones)

Conditions: 

Incubation at 18°C; 200rpm, for 18h

Growth curve assay of MG1655 in 96 well plate with Triton-X100 coated lid.

After OD₆₀₀= 0.6 induction: dilution to 0.1, but with right inducer concentration.

30°C, medium shake, OD₆₀₀ read every 10min for 18h.

Sample preparation (protocol #038): Sonication, fractioning SDS-PAGE and WB (samples from 03/09)

SDS-PAGE (8% seperating gel; protocol #31) of induced BL21 (from 03/09) with following constructs

·      pHt1snpspyTag + mTurqouise2-snpCat 

·      pHt1snpspyTagT2T3 + mTurqouise2-snpCat 

·      pHT1snpspyTagT2T3 + mVenus2-spyCat

Conditions: 

One construct per gel; 3 gels in total
loading schema (M = PageRuler Plus Thermo scientifc) :

-       pHT1snpspyTagT2T3 + mVenus2-spyCat

Well:          1  2     3       4        5         6            7        8                9-15

Condition: M  0  0/10   0/50  100/0  100/10  100/25 100/50     same as well 2-8

                                              Supernatent                                       Pellet

-       pHt1snpspyTag + mTurqouise2-snpCat 

-       pHt1snpspyTagT2T3 + mTurqouise2-snpCat 

Well:          1  2     3       4        5         6            7        8                9-15

Condition: M  0  0/10   0/25  100/0  100/10  100/25 100/50     same as well 2-8

                                               Supernatent                                       Pellet

Western Blot from 06/09 incubation of anti-rabbit-HRP to detect the mVenus

• all mVenus seem to be catched, some of the mVenus and mTurquoise proteins seem to be degraded

Overnight cultures of BL21 for FRET with 

• mTurqouise2-snpCat + mVenus2-spyCat
• pHt1snpspyTagT2T3 + mTurqouise2-snpCat
• pHt1snpspyTagT2T3 + mVenus2-spyCat
• pHt1 + mTurqouise2-snpCat + mVenus2-spyCat
• pHt1T2T3 + mTurqouise2-snpCat + mVenus2-spyCat
• pHt1snpspyTag + mTurqouise2-snpCat+ mVenus2-spyCat
• pHt1snpspyTagT2T3 + mTurqouise2-snpCat+ mVenus2-spyCat
• p1939(AmpR) + pBAD33(CmR) (empty backbones)

Induction of BL21 for FRET with 

• mTurqouise2-snpCat+ mVenus2-spyCat

• 0 µM IPTG/ 25 ng/ml Doxycycline
• 0 µM IPTG/ 50 ng/ml Doxycycline

• pHt1snpspyTagT2T3 + mTurqouise2-snpCat

• 0 µM IPTG/ 0 ng/ml Doxycycline
• 0 µM IPTG/ 25 ng/ml Doxycycline

• pHt1snpspyTagT2T3 + mVenus2-spyCat

• 0 µM IPTG/ 0 ng/ml Doxycycline
• 0 µM IPTG/ 25 ng/ml Doxycycline

• pHt1 + mTurqouise2-snpCat + mVenus2-spyCat
• 100 µM IPTG/ 25 ng/ml Doxycycline
• 100 µM IPTG/ 50 ng/ml Doxycycline

• pHt1T2T3 + mTurqouise2-snpCat + mVenus2-spyCat

• 100 µM IPTG/ 25 ng/ml Doxycycline
• 100 µM IPTG/ 50 ng/ml Doxycycline

• pHt1snpspyTag + mTurqouise2-snpCat+ mVenus2-spyCat

• 100 µM IPTG/ 25 ng/ml Doxycycline
• 100 µM IPTG/ 50 ng/ml Doxycycline

• pHt1snpspyTagT2T3 + mTurqouise2-snpCat+ mVenus2-spyCat

• 100 µM IPTG/ 25 ng/ml Doxycycline
• 100 µM IPTG/ 50 ng/ml Doxycycline

Incubation at 18°C, 200rpm for 18h

FRET with mVenus2 and mTurquoise2 for encapsulation/ spatial distance proof:

• In FACS Cyan Flowcytometer from Beckman and Coulter samples were diluted 1:100 in PBS

• Fluorescence microscopy ->not possible, not the right filter option at the Epifluorescence and the accesible confocal microscope

• Microplate reader 1:1 dilution with LB;measured the excitation, emission around 400nm till 600 nm range

Colony PCR (protocol #019) for Anne

Run 0.7% Agarose Gel

Transformation for Anne

In MG1655
1) pMic_SPD5 + + mVenus2-spyCat

2) pMic_SPD5_both + mVenus2-spyCat

3) pMic_SPD5 + mTurqouise2-snpCat 

4) pMic_SPD5_both + mTurqouise2-snpCat 

5) pBAD_SPD5_sfGFP

6) pBAD_sfGFP

7) pMic_EncA + pVen_sfGFP

In C321∆exp

8) pMic_EncA + pVen_sfGFP

No more pVen_sfGFP left -> make Overnight cultre for new miniprep

Overnight cultures of BL21

• pVen_sfGFP

Overnight cultures of MG1655

• pHt1snpspyTagT2T3 + mVenus2-spyCat

Overnight cultures of C321∆exp

• pHt1snpspyTagT2T3 + mVenus2-spyCat
• pHt1snpspyTag + mVenus2-spyCat

Overnight cultures of ME5119

• pHt1snpspyTagT2T3 + mVenus2-spyCat
• pHt1snpspyTag + mVenus2-spyCat
• p1939(AmpR) + pBAD33(CmR) (empty backbones)

Miniprep with sigma aldrich Kit Elute of freezed DH5a samples from 03/09 overnight cultures and of BL21 pVen_sfGFP overnight culture 10/09

Transfomation: repetition of 7) from 10/09 (hadn’t shown colonies)

Transformation of BL21 and MG1655

• pMic_SPD5 + pVen_sfGFP
• pMic_SPD5_both + pVen_sfGFP

Induction of MG1655 with pHt1snpspyTagT2T3 + mVenus2-spyCat

with 100µM IPTG and 50ng/ml DOXY as induction control

Induction of C321∆exp and ME5119 with

• pHt1snpspyTagT2T3 + mVenus2-spyCat
• pHt1snpspyTag + mVenus2-spyCat

Incubation at 18°C; 200rpm

Overnight cultures of ME5119

• pHt1snpspyTagT2T3 + mVenus2-spyCat
• pHt1snpspyTag + mVenus2-spyCat
• p1939(AmpR) + pBAD33(CmR) (empty backbones)

 for Growth curve assay

Induction (protcol #040) of MG1655 (overnight cultures from 11/09) like 03/09 for growth assay

Conditions for growth curve assay with: 

• pHt1snpspyTag + mVenus2-spyCat
• pHT1snpspyTagT2T3 + mVenus2-spyCat
• p1939(AmpR) + pBAD33(CmR) (empty backbones)

Growth assay with Triton X-100 coated lid like 10/08

In a 96-well plate with 200 µl of each sample, as in the table below We pipetted each sample 3 times in order to get the mean of the values and so get more precise results. We let the plate shake at medium intesity continuosly, at 30°C, measuring the values every 10 min for 20h

Fluorescence microscopy of induced ced MG1655; C321∆exp and ME5119 (23/08) with Agar Pads (protocol #37) with a Zeiss Axiovert with Kolibri LEDs 

Samples of MG1655 were taken after 19h incubation and after 24h incubation to see difference in foci formation. 19h seem to have litte more foci

C321∆exp only one foci per 200-300 cells
ME5119 no foci formation

• check all again after 48h of induction

Sample preparation (protocol #038) from induced MG1655; C321∆exp and ME5119 were taken after 24h incubation, spinned down and freezed -20°C for later SDS-PAGE and WB 
Additionally samples of MG1655  after 19h incubation were taken

Fluorescence microscopy of same samples from 12/09 only after 48h of induction

Dry lab

Primer arrived for switching the BMC Promotor and Operator from IPTG to Tetracyclin inducible per Gibson Assembly
Forward and reverse primers for BMC backbone amplification.  (Tm = 64°C)