Notebook

Present: Kevät, Jesper

Growth plates (LBA, 20 ml)

2x LBA+Amp 100 µg/ml

1x LBA+Kan 500 µg/ml

Streaked ZikV-trigger (Amp) and ZikV-sensor (Amp) to LBA-plates, started incubation at 37℃ at 3 PM. The plates were moved to the fridge at 8:01 AM

Single colonies were inoculated from both plates to culture tubes containing 2xYT-medium with the aforementioned antibiotics. Incubation started at 16.30 and moved to the fridge at 8.47.

875 µl from each of the cultures were used to make a 10 % glyserol preps which are stored at -80℃. The plasmids were purified from the rest of the cultures (3225 µl) with Monarch Plasmid Miniprep Kit. The purified plasmids are stored at -20℃. The concentration of the purified plasmids were measured with biodrop.

The plasmids were confirmed to contain the inserts by restriction analysis. Zikv-Trigger was cut with AvrII and Zikv-Sensor with EcoRI in a 25 µl reaction. They were incubated at 37℃ for 4 hours, followed by 80℃ inactivation for 20 min and then kept at 4℃.

Present: Kristiina, Kevät, Jesper

Cell cultures

Three bottom up tubes were taken from the laboratory. 4 ml of 2 x YT was added into each of the tubes in a laminar flow cabinet. 4 µl of ampicillin (stock c = 100 mg/ml) was added into two of the tubes, 4 µl of kanamycin (stock c = 50 mg/ml) was added into one of the tubes. Streaks were taken separately from the following plates: 1) AMP100 pAD-lyseR 2) AMP100 ZikaV-Trigger 3) KAN50 Zika-V-sensor. Now we have three different cultures: one with a streak from AMP100 pAD-lyseR and ampicillin, AMP100 ZikaV-Trigger and ampicillin and Kan50 ZikaV-Sensor and kanamycin. Cell cultures were grown overnight at 200 rpm at 37 ℃.

Tetracycline stock + LBA plate

Tetracycline stock was created by weighing 13.3 mg of tetracycline to 1.33 ml of UP water. LBA plate was created by adding 25 µl of tetracycline stock to 25 ml of melted LBA, plating 20 ml of it.

LBA + T plate was streaked with bacteria from Addgene stab.

YT + YTP

2 x 1 L bottles of 2xYT were created by weighing 16 g tryptone, 10 g yeast extract and 5 g NaCl to a bottle and filling to 1 L.

4 x 1L bottles of 2xYTP were created as above, with the addition of 3.99 g of potassium phosphate monobasic and 13.93 g of potassium phosphate dibasic.

4 x LBA + AMP100 µg/m

About 90 ml of LBA and 90 µl of ampicillin (stock c = 100 mg/ml) was added into a glass bottle. Shaked delicately. Poured onto four plates.

4 x LBA + KAN100 µg/ml

About 90 ml of LBA and 180 µl of kanamycin (stock c = 50 mg/ml) was added into a glass bottle. Shaked delicately. Poured onto four plates.

1 x LBA + TET10 µg/ml

About 20 ml of LBA and 20 µl of tetracycline (stock c = 10 mg/ml) was added into a glass bottle. Shaked delicately. Poured onto a plate.

Present: Evelina, Kristiina, Jesper

E. coli ZikaV + E. coli ZikaS Plasmid extraction

Plasmids were extracted with the Monarch Plasmid Mini Prep Kit. The pellet was centrifuged down with 2800 x g, 5 min, +4°C. Rest of the steps were centrifuged as described in the protocol (approx. 16 000 x g).

The DNA concentration of the plasmid extract was determined using BioDrop. The results were as follows: ZikaV-Sensor 279.2 ng/µl; ZikaV-Trigger 136.0 ng/µl.

ZikaV-S and ZikaV-T plasmid digestion

Plasmid digestion was made from the extracted ZikaV-S and ZikaV-T plasmids for gel electrophoresis to determine that the extraction was successful. The following reactions were prepared:

Glyserol preps were made of the ZikaV-Sensor and ZikaV-Trigger cell cultures for the cell/plasmid library. The preps were stored in -80℃ in 2B/Patrikainen no.:s 146 and 147.

Cell cultures

Three bottom up tubes were taken from the laboratory. 4 ml of 2 x YT was added into each of the tubes in a laminar flow cabinet. 4 µl of ampicillin (stock c = 100 mg/ml) was added into two of the tubes, 4 µl of kanamycin (stock c = 50 mg/ml) was added into one of the tubes. Streaks were taken separately from the following plates: 1) AMP100 pAD-lyseR 2) AMP100 ZikaV-Trigger 3) KAN50 Zika-V-sensor. Now we have three different cultures: one with a streak from AMP100 pAD-lyseR and ampicillin, AMP100 ZikaV-Trigger and ampicillin and Kan50 ZikaV-Sensor and kanamycin. Cell cultures were grown overnight at 180 rpm at 37°C.

1 x LBA

Approximately 20 ml of LBA was poured onto a plate.

Present: Kristiina, Kevät, Jesper

Gel Electrophoresis of ZikaV-S and ZikaV-T plasmid digestions

The gel was made following the Gel Electrophoresis Protocol in our Drive: 0.6 g Agarose, 60 ml 1XTAE, and 6 µl SYBR Safe. The digestion samples were split in two so in total four samples were made: 2X ZikaV-S digested by EcoRI-HF and 2X ZikaV-T digested by AvrII. The samples were prepared as follows: 25 µl DNA from the digestion and 5 µl Loading Dye Purple 6X (NEB). The ladder used was Purple 1 kb Plus DNA Ladder (NEB).

The gel was run for 37 min on 100 V.

Figure 1. Agarose gel electrophoresis for digested plasmids. Gel diagram: 1: Ladder 2: ZikaV-S-1 3: ZikaV-S-2 4: ZikaV-T-1 5: ZikaV-T-2.

The samples seem to be inverted, as the ZikaV-S plasmid is supposed to be 6455 bp and ZikaV-T plasmid 5675 bp but on the gel, ZikaV-T seems to be bigger than ZikaV-S. New digestion & gel will be made tomorrow to determine that the fault is due to misnaming the samples while digestion and that the extracted plasmids are named correctly and good to use.

2XYTPG preparation

400 ml of 2xYTPG was prepared by dissolving 7.2 g of glucose to 100 ml of 2xYTP and filter sterilizing it. Then, 300 ml of sterile 2xYTP was added.

Present: Evelina, Kristiina, Kevät, Jesper

ZikaV S and ZikaV T Plasmid digestion

Following reactions were prepared:

* Accidentally might have added 7.14 µl instead of 7.4 µl.

Reactions were incubated at 37°C for 4 hours, then 20 minutes at 80°C and kept at 4°C for ∞.

Gel Electrophoresis of ZikaV-S and ZikaV-T plasmid digestions

The gel was made following the Gel Electrophoresis Protocol in our Drive: 0.6 g Agarose, 60 ml 1XTAE, and 6 µl SYBR Safe. The digestion samples were split in two so in total four samples were made: 2X ZikaV-S digested by EcoRI-HF and 2X ZikaV-T digested by AvrII. The samples were prepared as follows: 25 µl DNA from the digestion and 5 µl Loading Dye Purple 6X (NEB). The ladder used was Purple 1 kb Plus DNA Ladder (NEB).

The gel was run for 40 min on 100 V.

Figure 2. Agarose gel electrophoresis for digested plasmids. Gel diagram: 1: Ladder 2: ZikaV-S-1 3: ZikaV-S-2 4: ZikaV-T-1 5: ZikaV-T-2 6: Negative control H₂O.

Now the bands are about the right size (both about 6 kb and the ZikaV-S is bigger than ZikaV-T) and also neg control doesn't show any band --> everything should be alright.

Plasmid extraction

Extracted plasmids from yesterday's cultures with Monarch Plasmid miniprep (NEB). This time the last resuspension was made with 50 µl of the elution buffer instead of 30 µl.

Determined the DNA concentration with BioDrop. ZikaV-T: 87.24 ng/µl; ZikaV-S: 85.28 ng/µl.

Starter culture

4 ml of 2xYT was supplemented with 4 µl of tetracycline and a single BL21-Gold-dLac colony from plate was inoculated o/n.

Present: Evelina, Kristiina, Jesper

Cell lysis by sonication

Sonicated the samples from yesterday. 4 cycles of 30 s sonication and 40 s rest. Peak value 12 and samples were on ice for the whole time. Centrifuged at 20 000 g, 4°C for 60 minutes to pellet the cell debris.

Determined the amount of protein with Bradford assay.

Made the standard curve with a stock solution of 1 mg/ml of BSA. Made the following dilutions: 100 µg/ml, 10-1 µg/ml. Only used 2, 4, 6, 8, 10 µg/ml dilutions for the standard curve. Measured the absorption at 595 nm.

Autolysis production culture

Starter culture was inoculated into 400 ml of 2xYTPG with 100 µg/ml concentration of Ampicillin (Amp). Incubation at 37℃, 220 rpm was started at 8.40

BL21-Gold-dLac

1L flask containing 400 ml of 2xYTPG was supplemented with 100 µg/ml Amp and 4 ml of starter culture. Incubation was started at 8.40.

OD600 was measured at 1 hr intervals, using 2xYTPG as a blank. OD-value of 6.08 was reached at 14.40. Culture was supplemented with 2.2 ml of 0.2 M IPTG and incubated at RT until 17.41.

The culture was transferred to 10 50 ml falcons, each having 40 ml of culture. They were centrifuged at 1800 g, RT for 15 min. Supernatant was discarded and each pellet was resuspended in 4.5 ml of cold S30A buffer. Then they were combined into one falcon tube and centrifuged as above. Then, supernatant was discarded and pellet was weighed. Pellet mass: 1.66 g. Pellet was resuspended in 3320 µl of cold S30A and was divided into 3 eppendorf tubes before freezing at -80℃.

Present: Kristiina, Kevät, Otto, Jesper

Suspension was thawed at RT water bath. Then it was vortexed, transferred to a culture tube, vortexed for 3 min and incubated at 37℃, 300 rpm, starting at 9.45. At 10.34 the suspension was vortexed for 3 min, before starting incubation again at 10.39. At 11.27 the tubes were put on ice, transferred to eppendorfs and centrifuged at 20 000 g, 4℃ for 90 min.

During centrifuging, 0-10 µg/ml BSA standard was prepared and stardard curve was created. The concentration of the lysate was determined from 1/10000 dilution, it was 13.42 mg/ml. Concentration was < 27 mg/ml, so Amicon Ultra-4 10K concentrator was used to concentrate the solution. New concentration was determined from 1/10000 and 1/20000 dilutions and it was 83.2 mg/ml. The solution was diluted to 30.7 mg/ml by diluting 182 µl of the solution with 311.8 µl of water. Then, solution was aliquoted into 30 µl aliquots and stored at -80℃.

S30A

Prepared 1 L of S30A, pH 7.69 to be autoclaved tomorrow.

Present: Kristiina, Kevät, Otto, Jesper

S30B pH 8.20

Prepared 2 L of S30B, pH 8.20.

2 X YTPG

Prepared 660 ml of 2xYTPG with 11.88g of glucose and filtered it.

1 M DTT

Prepared 6 ml of 1M DTT and filtered it, but due to filtering there're only 5 mls of it anymore....

BL21 Gold deLac starter cultures

Made 2x 4.5ml BL21-Gold-dLac starter cultures with 4.5 µl 10 mg/ml tetracycline. 37°C in a shaker from 16:05 onwards.

Present: Kevät, Otto, Jesper

Energy mix - components

HEPES 2M

4.766 g of HEPES was diluted in approximately 7 ml of MQ-water. After that solution was titrated to reach pH 8.02 with KOH and filled until volume V= 10 ml. Stored at -20°C.

GTP 75mM

ATP 75mM

tRNA 50 mg/ml

5 mg of tRNA was weighed to an Eppendorf tube. 100 µl of MQ-water was added. Mixed gently with a pipette. Stored at -80°C.

NAD 175mM

0.0117 g NAD was weighted to a PCR tube and diluted in 91 µl of MQ-water. After dilution 9 µl of 2M Tris was added to adjust pH around 8 (from Arce et al.). Mixed gently and stored at -20°C.

CoA 65mM

0.0049 g CoA was weighted to a PCR tube and diluted in 100 µl of MQ-water. MIxed gently and stored at -20°C.

cAMP 650mM

0.0118 g cAMP was weighted to a PCR tube and diluted in 31.7 µl of MQ-water. After dilution 18.3 μl of 2M Tris was added to adjust pH around 7.5-8 (from Arce et al.). Mixed gently and stored at -20°C.

Folinic acid 33.9mM

Spermidine 1M

Maltodextrin 650 mg/ml

2.6 g of maltodextrin was diluted in 2.5 ml of MQ-water in a 15 ml Falcon tube. After dilution 0.65 g of maltodextrin was added and filled until V = 5 ml. Stored at 4°C.

HMP 0.03 g/ml

0.12 g of HMP was diluted in 2 ml of MQ-water in a 15 ml Falcon tube and filled until V = 4 ml. Incubated at +95°C for 5 minutes. Stored at -80°C.

Production culture

2 L flasks with 330 ml 2xYTPG each were inoculated with 3.3 ml of the overnight culture and 330 µl of Tet was added to each flask. Incubation at 37°C, 220 rpm was started at 9.00. Cultures reached 0.606 and 0.634 OD₆₀₀ at 12.20. 1.65 ml 0.2 M IPTG was added to each flask. Incubation was continued at RT, 220 rpm until 15.25.

After the IPTG incubation, culture was divided equally to 2 500 ml centrifuge bottles and centrifuged at 5000 g, 4℃ for 12 min. Supernatant was discarded and pellet was suspended in 100 ml of S30A and centrifuged as above. This step was repeated. Pellets were resuspended with 20 ml of S30A per bottle and then transferred to a falcon tube, which was centrifuged at 2000 g, 4℃ for 8 min. Supernatant was discarded and wash repeated with 40 ml of S30A. Then the pellet was centrifuged for 2 min and resuspended in 4 ml of S30A. The suspension was stored at -20℃.

Tris

Approximately 36.342 g of Tris base was diluted in 150 ml of MQ-water. The concentration of the solution is 2 M. The solution was filtrated with a syringe and a filter.

Glycerol prep of E. coli lyseR

0.875 µl of E. coli lyseR was added into a microcentrifuge tube and 0.125 µl of 80 % glycerol was added. Mixed with a pipette. Stored at -80°C.

Present: Kevät, Otto, Jesper

5 X LBA+KAN50 Plates

Approximately 120 ml of melted LBA was into a flask. 120 µl of kanamycin (stock concentration 50 mg/ml) was added. Mixed. Poured onto six plates. One plate failed.

LacZ culture

BL21 (DE3) strain containing the pET-15b-LacZ-plasmid was streaked to an LBA + Amp 100 plate. Incubation at 37℃ started at 10.03. Single colonies from the plate were inoculated into a 5 ml 2xYT + Amp 100 culture tube and incubation was started at 20.20.

2 x Liquid cultures of E. coli BL21 pET-15 lacZ

5 ml of 2 x YT was transferred into a tube in a laminar flow cabinet. 5 µl of ampicillin (stock concentration = 100 mg/ml) was added. Mixed. A streak of a plate containing E. coli BL21 pET-15 lacZ was added. Two liquid cultures were prepared. Grown overnight at 37°C at 250 rpm.

mScarlet transformation

2 preps of competent DH5a cells were thawed on ice for 15 min. 1 µl of Purified pEAS001 (mScarlet) and 1 µl of pEAS002 (mScarlet-I) were added to the tubes. They were incubated on ice for 30 min. Cells were heat-shocked on 42℃ water bath for 45 s and kept on ice for 5 min. Then, 500 µl of LB was added to each of the tubes and they were placed on 37℃, 200 rpm for 1 hr. From each of the tubes, 100 µl was plated on a LBA + Kan 50 plate and the remaining 400 µl on a second plate. They were incubated at 37℃, starting at 16.05.

Primer dilutions for mScarlet and lacZ

100 µM dilutions:

mScarlet F

n = 30.8 nmol

V = n/c => 30.8 ⋅ 10 ^-9 / 100 ⋅ 10 ^-6 M = 0.000 308 l = 308 µl

308 µl of MQ-water was added into the tube containing 31.6 nmol mScarlet F. Mixed. Stored at -20°C.

mScarlet R

n = 31.6 nmol

V = 31.6 ⋅ 10 ^-9 / 100 ⋅ 10 ^-6 M = 0.000 316 l = 316 µl

Prepared the same way as mScarlet F except for the volume of MQ-water.

lacZ F

n = 25.2 nmol

V = 25.2 ⋅ 10 ^-9 mol/ 100 ⋅ 10 ^-6 M = 0.000 252 l = 252 µl

Prepared the same way as mScarlet F except for the volume of MQ-water.

lacZ R

n = 35.2 nmol

V = 35.2 ⋅ 10 ^-9 mol/ 100 ⋅ 10 ^-6 M = 0.000 352 l = 352 µl

Prepared the same way as mScarlet F except for the volume of MQ-water.

10 µM dilutions:

180 µl of MQ-water was added into four microcentrifuge tubes. 20 µl of each primer (c = 100 µM) was added into separate tubes. Stored at -20°C. Total amount: four different tubes for mScarlet F and R, lacZ F and R

Present: Evelina, Kevät, Otto, Jesper

Sonication

Pellet suspension was thawed on ice and then sonicated for 4x30 s. Suspension was centrifuged at 48 000 g, 4℃ for 30 min. Supernatant was transferred to 2 15 ml falcons and incubated at 37℃, 220 rpm for 60 min.

Solution was transferred to eppendorfs and centrifuged at 12 000 g, 4℃ for 10 min. 10 µl of the supernatant was saved for Bradford assay. The rest was loaded into a 10 ml 10 MWCO dialysis cassette. Dialysis was completed in a 1 L beaker at 4℃ for 3 hours.

Bradford assay

BSA standards were created at 0, 2, 4, 6, 8 and 10 µg/ml concentrations, 800 µl of each were created and 200 µl of Coomassie solution was added to each. Their absorbance at 595 nm was measured and a standard plot was created. Sample was diluted to 1/10000 and 1/20000 for the assay. The concentration of the sample undiluted sample was 41,01 mg/ml.

LacZ PCR

lacZ PCR amplification from plasmid

Following reaction was prepared:

Following PCR cycle was run:

Steps 1)-3) repeated 40 X. Stored at -20°C.

Present: Anni, Kevät, Jesper

One-day competent DH5a cells

Competent cells were made using the modified protocol for competent cells (fast) from Hari's (SBC group) protocol. DH5α cells from LBA plate inoculated into 2 x 8 ml SOC in 25 ml Erlenmeyers. Incubation at 37 ℃ and 220 rpm until cells reached OD600 value of 0.4-0.5. The cells were then centrifuged at 10000 x g, 4 ℃, 2min.

lacZ cleanup and concentration

lacZ was cleaned up with Monarch PCR & DNA Cleanup Kit (5 µg) Protocol Card according to the DNA Cleanup and Concentration (because lacZ gene size is 3075 bp > 50 bp). Sample type of lacZ is dsDNA < 2 kb (some amplicons, fragments) so 5:1 was chosen as the ratio of binding buffer:sample (125 µl:25 µl). The concentration of the purified lacZ sample was 62.97 ng/µl.

Transformation to DH5α cells with lacZ-plasmid

Transformation was made using the heat-shock transformation protocol for E. coli cells. 10 µl DNA (lacZ plasmids) on the cells. Cells were incubated on ice little over than half an hour (maybe 35 min) → heat shock 45 s 42 ℃ → kept 6 min on ice. 1 hour 10 min incubation at 37 ℃. We used kanamycin as antibiotics. We plated 100µl on LBA plate and the remaining cell culture on another LBA plate.

Agarose gel electrophoresis for lacZ

60 ml of 1 x TAE buffer was heated with 0,60 g agarose. 6 µl of SYBR safe color was added and mixed. Left to cool. Poured onto gel tray. Approximately 3.75 µl of Gel Loading Dye 6X was added to the sample (sample volume 18 µl). 10 µl of 1 kb Quick-Load DNA Plus Ladder Purple was loaded onto the gel. Approximately 22 µl (all of the sample) was loaded onto the gel. Gel was run at 100 V for 35 mins.

Figure 3. Gel electrophoresis for LacZ.

Gel extraction lacZ

m(lacZ gel slice) = 0.09 g = 90 mg (~ 90 µl)

lacZ gel slice was extracted with Monarch DNA Gel Extraction Kit. The concentration of the purified lacZ was 8.039 ng/µl (quite low!!). Stored at -20°C.

Minipreps for mScarlets

Minipreps for mScarlets were made using single colonies of DH5α pEAS001 and DH5α pEAS002 plates. Colonies were inoculated in 5 ml 2xYT + 50 µg/ml Kan.

Present: Evelina, Jonna, Anni, Kristiina, Kevät, Jesper

Energy mix

The reagents were added in order to microcentrifuge tube and Vortex after each addition. The tube was kept on ice. The mix was then dived in tubes, 4 x 150 µl and 1 x 100 µl and put to -80 ℃.

Reagents for energy mix:

RNA synthesis of Zika V trigger

To the reaction tube, the reagents were added in the following order: 1.5 µl 10 X RNAP reaction buffer; 1.5 µl each of the NTPs: ATP (100 mM), GTP (100 mM), CTP (100 mM), UTP (100 mM); 1 µg, 11 µl, Zika V trigger DNA (87.24 ng/µl), 1.5 µl T7 RNAP. 20 µl reaction. The reaction was incubated at 37℃ for 3 hours. Note: After 3 hours of incubation, it was heated to 42℃ for 1 min and incubated at 37℃ for 1 hour.

RNA pufication

RNA was purified with Monarch total RNA miniprep kit. 2 volumes (20 µl) of RNA lysis buffer was added to the 20 µl reaction and mixed with pipette. 60 µl of 95 % ethanol was added and mixed with pipette. The mixture was transferred to an RNA purification column and spinned for 30 seconds, the flow-through was discarded. 500 µl of RNA Priming Buffer was added and spinned for 30 seconds, the flow-trough was discarded. 500 µl of RNA Wash Buffer was added and spinned for 30 seconds, the flow-through was discarded. Another 500 µl of RNA was added and spinned for 2 minutes. The column was transferred to a new microcentrifuge tube. The RNA was eluted with 50 µl of water and spinned for 30 seconds. The tube was put to -80℃.

After purification, the RNA concentration was 148.5 ng/µl or about 5.25 mM (calculated without terminator).

Minipreps for mScarlets and lacZ

Minipreps for mScarlets were made using single colonies of DH5α pEAS001 and DH5α pEAS002 plates. Colonies were inoculated in 5 ml 2xYT + 50 µg/ml Kan. These mScarlet minipreps are for preparing glycerol preps. Miniprep for lacZ was made using a single colony of DH5α pET-15b lacZ plate. Colony was inoculated in 5 ml 2 x YT + 100 µg/ml Amp. Grown overnight at 220 rpm at 37 ℃.

Purify mScarlet plasmids

mScarlet and mScarlet-I plasmids, pEA5001 and pEA5002 respectively, were purified using the NEB plasmid miniprep kit. The first centrifuge step was done as follows: 5 mins, 3000 rcf. This worked well. The plasmids were eluted to 50 µl of the elution buffer.

The concentrations of the DNA in the samples were determined by using BioDrop. pEA5001 61,28 ng/µl; pEA5002 74,68 ng/µl. 2 µl of both samples were used for this, and the rest (48 µl) are in the freezer.

PCR for both mScarlets

First PCR for mScarlet and mScarlet-I.

Second PCR (different temperature) for both mScarlets

Same amounts as previous one, different temperatures in PCR.

--> failed, PCR machine error (W27), we will try again tomorrow.

Agarose gel

Agarose gel was made for mScarlets PCR products. 60 ml of 1 x TAE buffer was heated with 0.60 g agarose. 6 µl of SYBR safe color was added and mixed. Left to cool. Later stored in plastic wrap in the fridge.

Present: Evelina, Anni, Kristiina, Kevät, Otto, Jesper

Third PCR (different temperature) for mScarlets

PCR for pEA5001 and pEA5002. Same amounts as yesterday, same temperature (annealing at 72℃) as yesterday's second PCR. The PCR cycle was adjusted!! 2 x 72℃ 30 s was changed to 1 x 72℃ 1 min. The PCR worked just fine but we need to monitor the PCR in the future if there are some problems again.

Electrophoresis for the mScarlets PCR products

Agarose gel from yesterday was taken out of the fridge and used in gel electrophoresis.

Samples: 25 µl of each PCR product was mixed with 5 µl of Purple Dye and injected to the wells. Also 7 µl of ladder was injected to two wells. 100 V was used to run the gel for 42 minutes first and then 15 minutes to be sure.

Below is the picture of the gel electrophoresis after 42 min of running.

Figure 4. Agarose gel electrophoresis for mScarlet and mScarlet-I products. 1: Ladder 2: pEA5001, annealing 62 ℃ 3: pEA5002, annealing 62 ℃ 4: H2O, annealing 62 ℃ 5: empty 6: ladder 7: pEA5001, annealing 72 ℃ 8: pEA5002, annealing 72 ℃ 9: H2O, annealing 72 ℃

Gel analysis did show that there is no visible difference between the PCR products (annealing temperature on the left 62℃ and on the right 72℃) so all of the samples were taken to the next step.

Gel extraction

Gel extraction was made using Monarch® DNA Gel Extraction Kit protocol from NEB. Wanted DNA fragments were excised out from the gel with sterile knife (remember to use flame and paper to clean the knife) and transferred to 1.5 ml microfuge tubes and weighted (empty microfuge tube to tare):

pEA5001 62°C --> 200 mg --> 800 µl gel dissolving buffer

pEA5002 62°C --> 160 mg --> 640 µl gel dissolving buffer

pEA5001 72°C --> 150 mg --> 600 µl gel dissolving buffer

pEA5002 72°C --> 170 mg --> 680 µl gel dissolving buffer

concentrations of the samples, biodrop ng/µl:

pEA5001 62°C : 58.54 ng/µl

pEA5002 62°C : 35.75 ng/µl

pEA5001 72°C : 56.16 ng/µl

pEA5002 72°C : 44.77 ng/µl

--> put in the freezer.

Glycerolpreps

Three glycerolpreps were made: HD5α pET 15b-lacZ, HD5α pEA 5001 and HD5α pEA 5002

--> 875 µl of each sample was mixed with 125 µl glycerol and stored in the freezer -80°C.

pJUMP sfGFP transformation (acceptor plasmid backbone)

Transformation was made using the heat-shock transformation protocol for E. coli cells.

5 µl DNA (pJUMP sfGFP plasmids) on the cells. Cells were incubated on ice for half an hour, then heat shock 45 s 42℃ and kept 4 min on ice. 500 µl of LB was added and incubated for 1 hour at 37℃. With 200 rpm shaking. We used kanamycin as antibiotics. We plated 100 µl on LBA plate and 400 µl cell culture on another LBA plate.

Present: Evelina, Anni, Kristiina, Kevät, Jesper

Agarose gel and electrophoresis for RNA

Gel electrophoresis for ZikaV Trigger RNA pure. Before gel electrophoresis the RNA concentration was measured with biodrop (148.5 ng/µl).

Agarose gel was made for the purified Zika V trigger RNA. 60 ml of 1 x TAE buffer was heated with 0.72 g agarose. 6 µl of SYBR safe color was added and mixed. Left to cool.

Sample: 18 µl formamide, 5 µl loading dye, 7 µl Zika V trigger RNA sample = 30 µl. Also 7 µl of ladder was loaded.

100 V was used to run the gel for 10 minutes. Then the voltage was raised by ten after every ten minutes. 110 V after 10 min, 120 V after 20 min, 130 V after 30 min, 140 V after 40 min, 150 V after 50 min. The gel was run for 70 minutes.

Figure 5. Agarose gel electrophoresis for ZikaV trigger RNA. Wells: 1. Ladder 2. RNA sample

--> The gel run failed, next time incubation after formamide needs to be performed before loading the sample to the well.

13 x LBA+KAN50 plates

Approximately 250 ml of melted LBA was into a flask. 250 µl of kanamycin (stock concentration 50 mg/ml) was added. Mixed. Poured onto 13 plates. LBA might have been too hot when the antibiotic was applied.

Plasmid extraction for the remaining cultures of mScarlet, mScarlet-I and LacZ.

Plasmid extraction was done following the instructions of the kit (NEB Monarch Plasmid miniprep). In the first step the culture tubes were centrifuged in 3000 x g for 5 min. In the last step plasmids were eluted to 50 µl of the elution buffer.

The concentrations of the plasmids were: pET-15b 209.8 ng/µl; pEA5001 81.47 ng/µl; pEA5002 80.62 ng/µl.

PCR reactions for LacZ, mScarlet and mScarlet-I

3 reactions were made of each sequence (3x LacZ, 3x mScarlet, 3x mScarlet-I); in total 9 reactions + 2 controls with MQ H₂O. One of the H₂O controls was made using lacZ primers and the other one with mScarlet primers. Assembled as follows:

The amount of DNA was 25 µl more than the initial amount, because of a mistake in pipetting. This shouldn’t affect the reaction too much, it might slightly increase the amount of the product.

The PCR was run with the following protocol:

Gel electrophoresis for lacZ, mScarlet and mScarlet-i PCR products

25 µl of each PCR product was mixed with 5 µl of 6 X Purple Dye and injected to the wells. Notion: the volume of PCR products seemed to differ from each other, which might affect the results. 7 µl of both ladders were injected into two separate wells. The gel was run at 100 V for 35 min.

Figure 6. Agarose gel electrophoresis for LacZ, mScarlet and mScarlet-I PCR products. Wells: Quick-Load Purple 1 kb DNA Ladder, lacZ 1, lacZ 2, lacZ 3, H2O with lacZ primers, 100 bp Ladder, 001 1 (mScarlet 1), 001 2 (mScarlet 2), 001 3 (mScarlet 3), H₂O with mScarlet primers, 002 1 (mScarlet-I 1), 002 2 (mScarlet-I 2), 002 3 (mScarlet-I 3)

Gel extraction for lacZ, mScarlet and mScarlet-I

Despite the apparent problems with the PCR, the products were extracted with NEB Monarch DNA Gel extraction kit. The products from the three parallel PCR reactions of each sequence were combined to reduce the amount of the reagents used. LacZ tube 1 and lacZ tube 2 were combined into one lacZ tube (the third was ignored because it didn't produce the correct product); mScarlet 1, 2 and 3 were combined to one mScarlet tube and mScarlet-I 1, 2 and 3 were combined to one mScarlet-I tube.

The weights of the gel pieces in the final tubes: lacZ 80 mg → 320 µl buffer; mScarlet 200 mg → 800 µl buffer; mScarlet-I 220 mg → 880 µl buffer. The samples were eluted to 20 µl in the final step.

The concentrations were:

lacZ 20.60 ng/µl

mScarlet 104.0 ng/µl

mScarlet-I 83.51 ng/µl

Cell-free reaction 1 for ZikaV

A Mastermix was prepared using the excel sheet to calculate the amounts to pipette for 90 µl mastermix. Sonication 2/41 was used as the extract. The pipetted amounts are here:

Mastermix was created in tube 1. From the mastermix, 19.5 µl was transferred to tube 3 (water control) and 0.5 µl of water was added to complete the sample.

0.33 µl of Zika-S plasmid was added to tube 1. 34.1 µl of this mixture was transferred to tube 2 (negative sample). The sample was completed with 0.57 µl of water.

0,57 µl of Zika-T plasmid was added to tube 1 (positive sample)

From tube 3, one 5 µl and one 10 µl sample was loaded to a clear V-bottom 96 well-plate. From tubes 1 and 2, two 20 µl and two 10 µl samples were loaded into the plate as follows:

The reaction was incubated at 29℃ for 2 hrs. And additional 1 hour at RT in the beginning, we did not program the reader correctly. Absorbance at 420 nm was measured at 1 min intervals.

Figure 7. β-galactosidase activity in cell-free reaction.

Present: Evelina, Anni, Kristiina, Jesper

Agarose gel and electrophoresis

Second gel electrophoresis for ZikaV Trigger RNA pure.

Agarose gel was made for the purified Zika V trigger RNA. 60 ml of 1 x TAE buffer was heated with 0.72 g agarose. 6 µl of SYBR safe color was added and mixed. Left to cool.

Sample 1: 18 µl formamide, 5 µl loading dye with SDS, 7 µl Zika V trigger RNA

Sample 2: 18 µl formamide, 5 µl loading dye without SDS, 7 µl Zika V trigger RNA

sample = 30 µl. Also 7 µl of ladder was loaded.

Before loading the samples to the wells samples were incubated for 5 min at 65°C and left on ice for 5 min.

100 V was used to run the gel for 10 minutes. Then the voltage was raised by ten after every ten minutes. 110 V after 10 min, 120 V after 20 min, 130 V after 30 min, 140 V after 40 min, 150 V after 50 min and 160 V after 60 min. The gel was run for 70 min.

Figure 8. Agarose gel electrophoresis for ZikaV trigger RNA. Wells: 2. ladder 3. sample 1 (with SDS) 4. sample 2 (without SDS)

--> Gel run still did not work.

Level one acceptor plasmid pOdd-1 transformation

Transformation was made using the heat-shock transformation protocol for E. coli cells.

5 µl DNA (pOdd-1 plasmids) on the cells. Cells were incubated on ice for almost 50 min, then heat shock 45 s 42℃ and kept 3 min on ice. 500 µl of LB was added and incubated for 1 hour at 37℃ with 200 rpm shaking. We plated 100 µl on LBA plate and 500 µl cell culture on another LBA plate. We used kanamycin as antibiotics.

Present: Kristiina, Jonna

10X PBS

Tried to prepare 10X PBS, but it didn't work.

The pOdd-1 plates were collected from the incubator at 12:30. They didn't seem too bad even though they were incubated too long and were likely overgrown.

Present: Evelina, Kristiina, Kevät

10X PBS

Recipe: 100 ml of 10X PBS, 8 g NaCl, 0.2 g KCl, 1.44 g Na ₂ HPO ₄ , 0.24 g KH ₂ PO ₄ -> to 100 ml MQ H ₂ O

The salts were added to 80 ml of MQ water one at a time while stirring with a magnetic stirrer. Then the pH was adjusted to 7.2 with NaOH and the rest of the water was added using a volumetric flask. Then the buffer was filtered it with .2 µm filter to two 50 ml falcons and stored in +4℃.

Prepared 100 µl of 1X PBS by diluting the 10X buffer and then used the 1X PBS for the ONPG solution.

Competent DH5α cells

Cold DH5α cells were inoculated in 5 ml of LB medium in two separate tubes. Grown overnight at 220 rpm at 37℃.

Cell free reaction for ZikaV with sonication cell extract

A mastermix was prepared in tube 1. Everything was pipetted according to the table below in that order. Sonication 2/41 was used as the extract.

Reagents for mastermix:

Mix was created in tube 1. From the mix, 19.5 µl was transferred to tube 3 (water control) and 0.5 µl of water was added to complete the sample.

0.33 µl of Zika-Sensor plasmid was added to tube 1. 34.1 µl of this mixture was transferred to tube 2 (negative sample). The sample was completed with 0.57 µl of water.

0.57 µl of Zika-Trigger RNA was added to tube 1 (positive sample).

From tube 3, one 5 µl and one 10 µl sample was loaded to a clear V-bottom 96 well-plate. From tubes 1 and 2, two 5 µl and two 10 µl samples were loaded into the plate as follows:

The reaction was incubated at 29℃ for 3 hrs. Absorbance at 420 nm was measured at 1 min intervals.

Figure 9. β-galactosidase activity in cell-free reaction.

Present: Evelina, Jonna, Kristiina, Kevät, Jesper

Some of our growing cells were transferred to new plates.

Figure 10. A. pET15b-LacZ in E.coli DH5 α. B. pET15b-LacZ in E.coli DH5 α, pAD-lyseR, ZikaV trigger and BL21 pET15b-LacZ. C. pEAs002 in E.coli DH5 α, pEAs001 in E.coli DH5 α and ZikaV sensor. D. pOdd1 in E.coli DH5 α., pEAs002 in E.coli DH5 α and pEAs001 in E.coli DH5 α

Competent DH5α cells

About 800 µl from the O/N culture was inoculated into 160 ml of LB. Unfortunately, the competent cells failed because we contaminated the new culture accidentally twice when measuring the OD. Two new cultures were made of the O/N culture by inoculating 20 µl of the old O/N culture into 5 ml of LB. Grown overnight.

Our old DH5a plate had gone bad, so we moved the transformation to tomorrow. We should do a new one tomorrow (12.7.) and always keep two plates of them in the fridge so that if something happens to one of them, we'd have a backup.

LacZ PCR

lacZ PCR amplification from plasmid

Following 4 X MasterMix was prepared:

Following PCR cycle was run 40 cycles:

Steps 1)-3) repeated 40 X. Stored at -20 ℃.

OBS! There were problems again with the PCR programme stopping. This time it said something about the lids being wrong.

The needed amounts of reagents were calculated in our sheet, using 50 µl as the final volume. Sonication 2/41 and autolysis 1 batches were used as the extract in the samples.  To tubes numbered 1 (samples), we pipetted the following amounts:

After pipetting, 24.4 µl of this mastermix was transferred to tubes labeled 2 (water control), followed by 0.6 µl of water. 0.6 µl of pEAS002 (78.4 ng/µl) was added to the samples.

After that, two 10 µl samples from each tube were pipetted to a flat-bottom. black 96-well plate as follows:

The reaction was incubated for 3 hrs in 29℃ and the fluorescence (569/593 nm) was measured at 1 min intervals.

Figure 11. mScarlet-I produced in cell-free reaction.

Present: Evelina, Anni, Kristiina, Kevät, Jesper, Sofia

Cell-free reaction testing different Potassium Glutamate concentrations

Using the mastermix calculator, the following mastermix was created into tube labeled 56, using sonication 2/41 batch as the extract. Pipetted as follows:

11.5 µl of the mastermix was transferred to tube labelled 0 (water control). 0.5 µl of water was added to complete the control.

4.4 µl of pEAS002 plasmid was added to tube 56.

The mastermix was aliquotted by 21.89 µl to tubes 6, 8, 10, 15, 20, 30, 56.

The following amounts of K-G and water were added to each tube:

10 µl of water sample and 2 x 10 µl of the samples were loaded onto a black flat-bottom 96 well-plate. The reaction was run at 29 ℃ for 3 hrs. The fluorescence at 569/593 nm was measured at 1 min intervals.

No signal was detected from the reaction, likely due to not vortexing the samples.

Figure 12. mScarlet-I produced in cell-free reaction.

Glycerol prep of E. coli pOdd-1

0.875 µl of E. coli pOdd-1 was added into a microcentrifuge tube and 0.125 µl of 80 % glycerol was added. Mixed with a pipette. Stored at -80℃.

Plasmid pOdd-1 extraction

Plasmids were extracted from two cultures, the one was pink as it should but the other one wasn't. Plasmid extraction was done using NEB Monarch Plasmid miniprep following the manufacturer’s protocol. In the first step the culture tubes were centrifuged in 3000 x g for 5 min. In the last step plasmids were eluted to 50 µl of the elution buffer.

The DNA concentrations were measured with BioDrop. The pink culture concentration was 73.92 ng/µl and the other one was 64.88 ng/µl.

Competent DH5α cells

From the O/N culture, 0.1 - 0.5% was inoculated into 160ml of LB and grew the cells to OD600= ~0.4-0.5. During the procedure the cells were kept on ice as much as possible. Incubated the culture at 4℃ for 30 minutes. Centrifuged at 5000rpm – 5 mins at 4℃ in 4 x 50 ml Falcon tubes. The centrifuge was maintained at 4℃. The supernatant was discarded. The cells were resuspended in each tube in 20 ml of ice-cold sterile 0.1 M MgCl2 and incubated in ice for 15 mins. The cells were centrifuged at 5000 rpm 4℃ for 5 mins. The supernatant was discarded and resuspended the cells in 25 ml of ice-cold sterile 0.1 M CaCl2 and incubated in ice for 15 mins. The cells were centrifuged (5000 rpm - 5mins - 4℃) and the supernatant was discarded. The cells were resuspended in each tube in 1ml of ice-cold sterile 0.1 M CaCl2 supplemented with 15-20% glycerol and aliquoted 100 µl into each 1.5 ml tube. Stored immediately at -80℃.

Zika-T RNA production

To a 200 µl PCR tube, the following reagents were added in the following order. 1.5 µl 10 X RNAP reaction buffer; 1.5 µl each of the NTPs: ATP (100 mM), GTP (100 mM), CTP (100 mM), UTP (100 mM); 1 µg, 11 µl, Zika V trigger DNA (87.24 ng/µl), 1,5 µl T7 RNAP. The final volume was 20 µl. Reaction was incubated in a 37℃ water bath for 5 hrs and the reaction tube was stored at +4℃.

Present: Evelina, Jonna, Anni, Kristiina, Kevät, Jesper, Sofia

Testing the cell-free system with 2/41 sonication batch and variable Potassium Glutamate concentrations

Using the master mix calculator (excluding the water from the calculator), the following master mix was created taking care to vortex each stock and the master mix after each addition. The extract was not vortexed, and neither was the master mix after adding extract. Sonication 2/41 batch was used as the extract. Pipetted as follows:

10.68 μl of the mastermix was transferred to tube labelled 0 (water control). 1.32 μl of water was added.

17.36 μl if the mastermix was aliquotted to tubes 4, 6, 8 10, 15, 20, 30 and 56. Then they were filled to 22.25 μl with the following amounts of water and Potassium Glutamate:

10 μl samples from each tube were loaded onto a black flat-bottom 96-well plate as follows (the numbers represent the tube labels / K-G concentration in mM):

The reaction was incubated at 37℃ for 3 hrs. The fluorescence intensity at 569/593 nm was measured at 1 min intervals.

No signal was detected.

Figure 13. mScarlet-I produced in cell-free reaction.

14.07.2022 Transformations for DH5α competent cells

Transformations were made using Heat-shock transformation protocol for E. coli cells. Competent DH5α cells were made 13.7.

5 µl DNA was added on the cells (B 46 -> 3 µl). 4 transformation rounds (3 at a time altogether 12 transformations)

round 1 DNA: T 7 term, T7 max, T7prom

round 2 DNA: Tr 3, A 95, B 69

round 3 DNA: B 46, A 92, Tr 2

round 4 DNA: B 39, Tr 1, A 70

picture 220714 plates

Figure 14. A. T7 max in E.coli DH5 α. B. Trigger 3 in E.coli DH5 α. C. A95 in E.coli DH5 α. D. B69 in E.coli DH5 α. E. T7 promoter in E.coli DH5 α. F. T7 terminator in E.coli DH5 α.

Figure 15. A. A92 in E.coli DH5 α. B. A70 in E.coli DH5 α. C. B46 in E.coli DH5 α. D. Trigger 1 in E.coli DH5 α. E. Trigger 2 in E.coli DH5 α. F. B39 in E.coli DH5 α.

Gel electrophoresis for lacZ fragments

60 ml of 1 x TAE buffer was heated with 0.60 g agarose. 6 µl of SYBR safe color was added and mixed. Left to cool. Poured onto a gel tray. 6 µl of Gel Loading Dye 6X was added to the sample (sample volume 25 µl). 7 µl of 1 kb Quick-Load DNA Plus Ladder Purple was loaded onto the gel. All of the sample was loaded onto the gel. Gel was run at 100 V for 35 mins.

14.07.2022 ZikaV trigger RNA purification attempt

RNA was attempted to purify with Monarch total RNA miniprep kit. 2 volumes (20 µl) of RNA lysis buffer was added to the 20 µl reaction and mixed with pipette. 60 µl RNA wash buffer (mistake) was added and mixed with pipette. The mixture was transferred to an RNA purification column and spinned for 30 seconds, the flow-through was discarded. 500 µl of RNA Priming Buffer was added and spinned for 30 seconds. Then the mistake was noticed, so the purification was not continued.

Zika V trigger RNA synthesis

Two reactions were made. To the reaction tubes, the reagents were added in the following order: 1.5 µl 10 X RNAP reaction buffer; 1.5 µl each of the NTPs: ATP (100 mM), GTP (100 mM), CTP (100 mM), UTP (100 mM); 1 ug, 11 µl, Zika V trigger DNA (87.24 ng/µl), 1.5 µl T7 RNAP. 20 µl reaction. After final addition, mixed with pipette. The reactions were incubated at 37℃.

Zika-T RNA purification

Both ZikaV-T RNA tubes were purified using Monarch total RNA miniprep kit (according to our protocol). Concentrations for tubes (measured with nanodrop) were 160 ng/µl and 423.5 ng/µl. Stored in -20℃.

Present: Jonna, Anni, Kristiina, Kevät, Jesper, Sofia

Golden gate minipreps

12 Golden gate minipreps (DH5a + T7 term, T7 max, T7prom, Tr 3, A 95, B 69, B 46, A 92, Tr 2, B 39, Tr 1, A 70) were made 5ml + kanamycin, overnight 37℃ in shaker.

15.07.2022 RNA gel electrophoresis

Agarose gel was made. 60 ml of 1 x TAE buffer was heated with 0.60 g agarose. 6 µl of SYBR safe color was added and mixed. Left to cool. Loaded and run.

Figure 16. Agarose gel electrophoresis for ZikaV trigger RNA.

Present: Anni, Kevät

Glycerol preps

Glycerol preps were made using 875 µl of each sample and 125 µl glycerol.

samples: DH5a + T7 term, T7 max, T7prom, Tr 3, A 95, B 69, B 46, A 92, Tr 2, B 39, Tr 1, A 70

stored in -80℃.

Present: Jonna, Anni, Kevät

Plasmid minipreps

Plasmid minipreps were prepared from the cultures stored in the fridge on Saturday, with Monarch Miniprep Kit (according to manufacturer's protocol). In the last step, the elution was done in 21 µl water. Stored in -20℃, in the GoldenGate box.

Concentrations: shown in the image below.

Golden Gate Assembly of the triggers and the toeholds

A total of 9 reactions were made. Tubes 1-3 contained pODD1 plasmid, T7 promoter and a trigger, and tubes 4-9 contained pODD1, T7 promoter, a toehold switch, LacZ and terminator, according to the tables below. The concentrations needed are calculated below.

Figure 17. Calculations of needed concentrations for golden gate reactions.

Golden gate reaction protocol (according to instructions on NEB site): (1 min 37℃, 1 min 16℃) X 30, 5 min 60℃, keep on 4℃ for infinity.

Stored at -20℃.

Growth cultures BL21 Gold deLac and BL21 pET15 lacZ

A streak of BL21 Gold deLac freeze stock was inoculated in 5 ml of YT + 5 µl tetracycline (stock concentration 10 mg/ml). A streak of BL21 pET15 lacZ containing plate was inoculated in 5 ml of YT + 5 µl ampicillin (stock concentration 100 mg/ml). Kept overnight in a shaker at 37℃.

Present: Anni, Kristiina, Kevät, Sofia

Preparing 0,1 M MgCl₂

4.062 g of MgCl₂ x H₂O was diluted into ~ 100 ml of MQ water. Filled up to 200 ml of MQ water. Autoclaved.

Growth/starter culture BL21 Gold deLac

A streak of BL21 Gold deLac freeze stock was inoculated in 5 ml of YT + 5 µl tetracycline (stock concentration 10 mg/ml) same as yesterday. Kept overnight in a shaker at 37℃.

Transformation for Golden Gate assembled plasmids (3+6)

Transformations were made using the Heat-shock transformation protocol for E. coli cells.

Figure 18. A. Golden Gate assembled pOdd1 T7+A92+LacZ+terminator in E.coli DH5 α. B. Golden Gate assembled pOdd1 T7+A92+LacZ+terminator in E.coli DH5 α. C. Golden Gate assembled pOdd1 T7+A95+LacZ+terminator in E.coli DH5 α. D. Golden Gate assembled pOdd1 T7+A95+LacZ+terminator in E.coli DH5 α. E. Golden Gate assembled pOdd1 T7+A70+LacZ+terminator in E.coli DH5 α. F. Golden Gate assembled pOdd1 T7+A70+LacZ+terminator in E.coli DH5 α.

Figure 19. A. Golden Gate assembled pOdd1 T7+B46+LacZ+terminator in E.coli DH5 α. B. Golden Gate assembled pOdd1 T7+B46+LacZ+terminator in E.coli DH5 α. C. Golden Gate assembled pOdd1 T7+B69+LacZ+terminator in E.coli DH5 α. D. Golden Gate assembled pOdd1 T7+B69+LacZ+terminator in E.coli DH5 α. E. Golden Gate assembled pOdd1 T7+B39+LacZ+terminator in E.coli DH5 α. F. Golden Gate assembled pOdd1 T7+B39+LacZ+terminator in E.coli DH5 α.

Figure 20. A. Golden Gate assembled pOdd1 T7+trigger 3 in E.coli DH5 α. B. Golden Gate assembled pOdd1 T7+trigger 3 in E.coli DH5 α. C. Golden Gate assembled pOdd1 T7+trigger 1 in E.coli DH5 α. D. Golden Gate assembled pOdd1 T7+trigger 1 in E.coli DH5 α. E. Golden Gate assembled pOdd1 T7+trigger 2 in E.coli DH5 α. F. Golden Gate assembled pOdd1 T7+trigger 2 in E.coli DH5 α.

Present: Evelina, Anni, Kristiina, Kevät, Sofia

BL21 Gold deLac Competent cells

Competent cells were made using protocol for competent cells. 60 ml production culture was made instead of 100 ml (we made half of what the protocol said). When OD600 were 0.4-0.5 the cultures were divided into falcon tubes. 2 falcons, each 25 ml of the culture. Stored at -80℃.

RNA gel electrophoresis

Gel electrophoresis for ZikaV Trigger RNA.

Agarose gel was made for the purified Zika V trigger RNA. 50 ml of 1 x TAE buffer was heated with 0.60 g agarose. 5 µl of SYBR safe color was added and mixed. Left to cool.

Sample 1: 18 µl formamide, 5 µl loading dye, 7 µl Zika V trigger RNA (160 ng/µl)

Sample 2: 18 µl formamide, 5 µl loading dye, 7 µl  Zika V trigger RNA (423.5 ng/µl)

sample = 30 µl.

Also 7 µl of 1 kb ladder and 7 µl 100 bp ladder was loaded.

Before loading the samples to the wells, samples were incubated for 50 min at 60°C and left on ice for about 5 min.

100 V was used to run the gel for 10 minutes. Then the voltage was raised by ten after every ten minutes. 110 V after 10 min, 120 V after 20 min, 130 V after 30 min, 140 V after 40 min, 150 V after 50 min and 160 V after 60 min. The gel was run for 70 min.

Figure 21. Agarose gel electrophoresis for ZikaV trigger RNA.

Gel run failed again :)

Zika V trigger overnight miniprep

5 ml LB, 5 µl amp 100 and Zika V trigger from plate. Grown overnight in shaker 37℃.

BL21 pET15 lacZ growth culture for the measurement test

A streak of a plate containing BL21 pET15 lacZ was inoculated in 5 ml of 2 x YT and 5 µl of ampicillin. Grown overnight at ~ 200 rpm at 37°C. Stored in the refrigerator for ~ 5 hours before induction.

Present: Evelina, Kristiina, Kevät, Jesper

RNA gel electrophoresis

Gel electrophoresis for ZikaV Trigger RNA.

Agarose gel was made for the purified Zika V trigger RNA. 50 ml of 1 x TAE buffer was heated with 0.60 g agarose. 5 µl of SYBR safe color was added and mixed. Left to cool.

Sample 1: 18 µl formamide, 5 µl loading dye, 7 µl Zika V trigger RNA (160 ng/µl)

Sample 2: 18 µl formamide, 5 µl loading dye, 7 µl Zika V trigger RNA (423.5 ng/µl)

sample = 30 µl.

Also 7 µl of 1 kb ladder and 7 µl 100 bp ladder was loaded.

Before loading the samples to the wells samples were incubated for 50 min at 60°C and left on ice for about 5 min.

100 V was used to run the gel for 10 minutes. Then the voltage was raised by ten volts after every ten minutes. 110 V after 10 min, 120 V after 20 min, 130 V after 30 min, 140 V after 40 min, 150 V after 50 min. The gel was run for 60 min.

Figure 22. Agarose gel electrophoresis for ZikaV trigger RNA.

Gel run failed.

Plasmid extraction Zika V trigger

Plasmids were purified using the NEB plasmid miniprep kit. The first centrifuge step was done as follows: 5 mins, 3000 rcf. The plasmids were elued to 50 µl elution buffer.

The concentrations of the DNA in the samples were determined by using BioDrop. Concentration 117.0 ng/µl, A260/A230=1.158, A260/A280=1.857. Stored in the freezer.

IPTG induction for BL21 pET15 lacZ

100 µl of BL21 pET15 lacZ cell culture was inoculated in 10 ml of 2 x YT + 10 µl ampicillin in Erlenmeyer. Grown until OD600 was about 0.52. The total volume of the culture was 7 ml after taking 3 ml for measuring OD. 0.35 µl of IPTG (stock concentration 0.2 M) was added into the culture. IPTG concentration was 1 mM in the culture. Grown at room temperature overnight.

Present: Evelina, Anni, Kristiina, Kevät, Jesper, Sofia

Measurement test with IPTG induced cells

7 ml of E. coli BL21 DE3 pET15b-LacZ cells from overnight induction were supplemented with 63.25 μl of 10 mg/ml ONPG in DMSO.

10, 20, 40 and 60 μl samples were loaded onto a clear, V-bottom 96-well plate as follows:

Reactions were incubated without agitation at 29℃ for 3 hrs and absorbance at 420 nm was measured at 1 min intervals.

Figure 23. β-galactosidase activity in cell-free reaction.

22.07.2022 Transformation for BL21-Gold-dLac comp.

Transformation for BL21-Gold-dLac comp. and mScarlet-I (pEAS002) were made using the Heat-shock transformation protocol for E. coli cells.

The transformation was made using 50 µl of competent BL21-Gold-dLac cells. 10 µl DNA was added to the cells.

Zika V trigger plasmid linearisation

4 50 µl reactions were made. To the tubes were pipetted the following reagents: 35.4 µl autoclaved MQ-water, 8.6 µl Zika V trigger plasmid DNA (1 µg, 117.0 ng/µl), 5 µl NEBuffer 3, 1 µl Pst1 restriction enzyme. The reactions were incubated at 37℃ for 15 min.

Zika V trigger DNA purification

Linearised plasmid were purified with NEB Monarch PCR & DNA cleanup kit, according to the kit instructions. The elution was made to 12 µl elution buffer. Concentrations and purity were measured with BioDrop:

Zika V trigger RNA synthesis

2 RNA synthesis reactions were made. Samples 1 and 2 were combined (20 µl, total 590, 3 ng DNA) and samples 3 and 4 were combined (20 µl, total 689.7 ng DNA). The reagents were pipetted in the tubes in following order: 2 µl of RNAP reaction buffer; 2 µl of each of the NTPs; 20 µl of linearised DNA; 2 µl of T7 RNAP. (32 µl total) The reactions were incubated at 37℃ for 8 hours. The tubes were stored at -20℃.

Present: Evelina, Anni, Kristiina, Kevät, Jesper

O/N cultures of Toeholds A92, A95, A70, B39, B46, B69 and Triggers 1, 2, 3 and pOdd1

5 ml LB overnight cultures of Toeholds A92, A95, A70, B39, B46, B69 and Triggers 1, 2, 3 were made with KAN50. Two 5 ml LB O/N cultures of pOdd1 were made with KAN50.

Transformation for BL21-Gold-dLac comp.

Transformation for BL21-Gold-dLac comp. and mScarlet-I (pEAS002) were made again using the Heat-shock transformation protocol for E. coli cells.

The transformation was made using 50 µl of competent BL21-Gold-dLac cells. 10 µl DNA was added to the cells. Cells were plated on a double antibiotic (kanamycin and tetracycline) plate.

Linear plasmid was used by accident and the tubes were discarded.

Starter culture for LacZ purification

Single colony from the LBA plate containing BL21 Gold-dLac pET15b-LacZ was inoculated into 5 ml LB + 100 μg/ml ampicillin in a culture tube. Culture was incubated in 37℃, 220 rpm for 16 hours.

25.07.2022 Zika-T RNA purification

Both ZikaV-T RNA tubes were purified using Monarch total RNA miniprep kit (according to our protocol). Concentrations for tubes (measured with nanodrop) were 209.7 ng/µl and 257.9 ng/µl. Stored in -20℃.

RNA gel electrophoresis

Gel electrophoresis for ZikaV Trigger RNA.

Agarose gel was made for the purified Zika V trigger RNA. 50 ml of 1 x TAE buffer was heated with 0,60 g agarose. 5 µl of SYBR safe color was added and mixed. Left to cool.

Sample 1: 18 µl formamide, 5 µl loading dye, 7 µl Zika V trigger RNA (160 ng/µl)

Sample 2: 18 µl formamide, 5 µl loading dye, 7 µl Zika V trigger RNA (423.5 ng/µl)

sample = 30 µl.

Also 7 µl of 1 kb ladder and 7 µl 100 bp ladder was loaded.

Before loading the samples to the wells, samples were incubated for 50 min at 60°C and left on ice for about 5 min.

100 V was used to run the gel for 20 minutes. Then the voltage was raised to 120 V for 30 min.

Figure 24. Agarose gel electrophoresis for ZikaV trigger RNA.

Present: Anni, Kristiina, Kevät, Jesper, Sofia

Cell-free reaction with pET15b-LacZ

Using the master mix calculator with 230 μl as the final volume, the following mastermix was created into tube 1 (positive sample):

28.71 μl of the master mix was transferred to tube 2 along with 1.28 μl of water.

8.58 μl of pET15b-LacZ DNA (62.66 ng/μl) was added to tube 1.

A 30 μl sample from tube 2 (water control) and 2 x 20 μl, 2 x 30 μl and 2 x 40 μl samples from tube 1 were transferred to a clear v-bottom 96 well-plate as follows:

The reactions were incubated at 29℃ for 3 hrs and absorbance at 420 nm was measured at 1 min intervals.

Figure 25. β-galactosidase activity in cell-free reaction.

Glycerol preps

Glycerol preps were made using 875 µl of each sample and 125 µl glycerol.

samples: DH5α + pOdd1 + Tr 1 or Tr 2 or Tr 3, or A 95 or B 69 or B 46 or A 92 or B 39 or A 70

--> stored in -80℃

Plasmid extractions Toeholds A92, A95, A70, B39, B46, B69 and Triggers 1, 2, 3

Extraction was made using the NEB Monarch Plasmid Miniprep Kit, according to the manufacturer's protocol. First spin was done in 3000 x g 5 min. Concentrations measured with BioDrop:

Transformation for BL21-Gold-dLac comp

THIRD TIME :) Transformation for BL21-Gold-dLac comp. and mScarlet-I (pEAS002) were made again using the Heat-shock transformation protocol for E. coli cells.

The transformations were made using 50 µl of competent BL21-Gold-dLac cells. 10 µl DNA was added to the cells.

--> Plated on a double antibiotic (kanamycin and tetracycline) plate.

Tris A buffer, 200ml, pH 7.5

20 mM Tris-HCl = 4 ml 1M Tris-HCl stock

150 mM NaCl = 1.753 g

20 mM imidazole = 0.272 g

to 200 ml MQ H₂O

pH was adjusted to 7.5 with HCl. Stored in 4℃.

Phosphate buffer 0.08 M 100 ml, pH 7,7

KH2PO4 0.369 g

K2HPO4 0.921 g

pH was adjusted to 7.7 with KOH. Stored in 4℃.

10 mg/ml ONPG in DMSO

10.6 mg ONPG

1060 ml DMSO

Stored in -20℃.

ONPG in PBS

10.58 µl 10 mg/ml ONPG in DMSO

89.42 µl 1X PBS

O/N cultures of Trigger 1, 2, 3

5 ml overnight cultures of DH5ɑ pOdd1 Trigger 1, 2, 3 in LB with KAN50.

Production culture for LacZ purification

4 ml of overnight culture was added to 200 ml of 2xYT with about 70 μg/ml ampicillin. When OD600 reached 0.613, 1 ml of 0.2 M IPTG was added and incubation was continued at 220 rpm, 23℃ for 3 hrs.

Pellet suspension was thawed on ice and then sonicated for 4x30 s. Suspension was centrifuged at 48 000 g, 4℃ for 30 min.

Protein was purified according to the following protocol:

1 ml of Ni2+ sepharose fast flow was pipetted into a filter tube

Ethanol was washed away with 4 ml of water

The column was balanced with 4 ml of Tris A buffer

The supernatant was filtered through column

Column was washed with 4 ml of Tris A buffer

5 fractions were collected, each with 1 ml of Tris B buffer pipetted into the column

FInally the Ni2+ was collected with 10 ml of water

Purification was unsuccessful, as we got very little protein.

Present: Anni, Jesper, Sofia

Restriction analysis of toehold plasmids & linearisation of trigger plasmids

51 µl reactions digestion reactions for each of the toehold (EcoRI-HF) and trigger (PstI-HF) plasmids were composed of the following reagents:

Normally the reaction volume is not 51 µl, but the volume of the enzyme was forgotten about the calculations.

The reaction was incubated was 37℃ for 4 hours, followed by 20 minutes at 80℃.

Purification of linearised toehold and trigger plasmids.

Purifications were made using Monarch PCR & DNA Cleanup Kit.

After purification the triggers DNA concentrations were measured with BioDrop:

Trigger 1: 0.125 μg/ul

Trigger 2: 0.105 μg/ul

Trigger 3: 0.103 μg/ul

After measure there were left 5 µl each trigger sample.

RNA synthesis of triggers

RNA synthesis of triggers 1, 2 and 3 were made using the RNA production protocol.

Because the concentration of the each DNA were too low, all of the DNA were used in the synthesis reaction and the reaction volumes were reduced to 15 µl.

Was left to incubate at 37℃ overnight.

Present: Anni, Jesper, Sofia

Gel electrophoresis for toehold plasmids

10 µl of 6X Purple loading dye was added to digested 51 µl samples. 20 µl of these samples were loaded to the gel, and electrophoresis was run at 100 V for 40 minutes.

Samples were pipetted to the gel in the following order:

Figure 26. Agarose gel electrophoresis for digested plasmids.

There should be two bands, 2.4 kb and 3.2 kb. Those bands cannot be seen on the gel, the plasmid is most likely undigested.

Restriction analysis of toehold plasmids again

Since last time the plasmids didn't digest properly, this time more enzyme is added

20 µl reactions as follows:

pos control: pET-15b lacZ

Negative control: A92 toehold, no enzyme.

Incubation in 37℃

Figure 27. Agarose gel electrophoresis for digested plasmids

RNA purification of the triggers 1, 2 and 3

RNA purification to the triggers were made using RNA production protocol.

After purification the concentrations of the triggers RNA were measured with biodrop:

Trigger 1: 416.1 ng/μl

Trigger 2: 673.5 ng/μl

Trigger 3: 385.6 ng/μl

Stored in the freezer (-20℃).

Present: Jesper

A single colony of BL21 pET15b-LacZ was inoculated into a culture tube containing 5 ml of 2xYT + ampicillin at 100 μg/ml.

A single colony of BL21 Gold-dLac pEAS002 was inoculated into a culture tube containing 5 ml of 2xYT + tetracycline at 10 μg/ml and kanamycin at 50 μg/ml.

Cultures were incubated at 37°C, 220 rpm for 12 hrs.

Present: Kevät, Jesper

Cell-free reaction test with mScarlet-I

This time, we have a couple of modifications. We changed the amount of cell extract so the final concentration is 10 mg/ml (before was 15 mg/ml) of protein. We also changed the plate (black PCR plate with clear wells) and we are testing bigger concentrations, as we did last week.

Mastermix amounts were calculated using 230 μl as the total volume. The following amounts were to tube labeled 1 (positive sample master mix). Each stock tube was and the mastermix were vortexed before each addition until cell extract, which we did not vortex. Pipetted as follows:

29.26 μl of the mix was transferred to a tube labelled 2 (water control). 0.74 μl of water was added to it.

4.95 μl of DNA was added to tube 1.

A single 30 μl water control sample was pipetted onto the plate. 2 x 20 μl, 2 x 30 μl and 2 x 40 μl samples were pipetted onto the plate and reactions were incubated at 29℃ for 3 hrs. Fluorescence at 569/593 nm was measured at 1 min intervals.

Figure 28. mScarlet-I produced in cell-free reaction.

Production cultures for protein purification

Overnight cultures were each added to 400 ml of 2xYT with their appropriate antibiotics. Incubation at 37°C, 220 until cells reached OD600 0.6. 2 ml of 0.2 M IPTG was added and incubation started at 220 rpm, 23°C.

pEAS002 reached 0.059 OD600 at 12.55. IPTG was added as above.

Present: Evelina, Kristiina, Kevät, Jesper

10 x LBA+KAN50 plates

Approximately 200 ml of melted LBA was put into a flask. 200 µl of kanamycin (stock concentration 50 mg/ml) was added. Mixed. Poured onto 10 plates.

Trigger plasmid linearization

50 µl reactions were made to all 3 triggers. To the tubes were pipetted the following reagents: 35.4 µl autoclaved MQ-water, 1 µg trigger plasmid DNA, 5 µl buffer, 1 µl Pst1 restriction enzyme. The reactions were incubated at 37°C for 2 h and 80°C for 20 min.

Linearized trigger plasmid purification

Linearized plasmids were purified with NEB Monarch PCR & DNA cleanup kit, according to the kit instructions. The elution was made to 11.5 µl elution buffer. Concentrations and purity were measured with BioDrop:

Trigger RNA synthesis

RNA synthesis reactions were made to all 3 samples. The reagents were pipetted in the tubes in following order: 1.5 µl of RNAP reaction buffer; 1.5 µl of each of the NTPs; 5 µl of linearised DNA; 1.5 µl of T7 RNAP. (14 µl total) The reactions were incubated at 37℃ overnight.

Gel electrophoresis for linearized trigger plasmids

60 ml of 1 x TAE buffer was heated with 0.60 g agarose. 6 µl of SYBR safe color was added and mixed. Left to cool. Poured onto gel tray. Approximately 1 µl of Gel Loading Dye 6X was added to the sample (sample volume 5 µl). 7 µl of 1 kb Quick-Load DNA Plus Ladder Purple was loaded onto the gel. Approximately 6 µl (all of the sample) was loaded onto the gel. Unfortunately, Trigger Plasmid 2 vanished from the well. Gel was run at 100 V for 35 mins.

Figure 29. Agarose gel electrophoresis for ZikaV trigger, triggers 1, 2 and 3 RNA. Wells: 1 kb DNA Plus Ladder, Trigger Plasmid 1, Trigger Plasmid 2, Trigger Plasmid 3.

Protein purification for mScarlet-I (pEAS002) and β-galactosidase

Both cell suspensions were thawed on ice and sonicated at peak value of 12 for 4 x 30 s, with 30 s intervals. The sonicated liquids were then centrifuged at 48 434 G, 4°C for 3 minutes. The supernatant was collected and the pellet discarded.

Both proteins were purified using the following protocol:

1 ml of Ni²⁺ sepharose fast flow was pipetted into a filter tube

Ethanol was washed away with 4 ml of water

The column was balanced with 4 ml of Tris A buffer

The supernatant was filtered through column

Column was washed with 4 ml of Tris A buffer

5 fractions were collected, each with 1 ml of Tris B buffer pipetted into the column

FInally the Ni2+ was collected with 10 ml of water

From each of the samples, the first two fractions were combined (to a volume of 2 ml). They were mixed with 3.2 ml of 80 % glycerol and stored at -20°C for later use.

The fractions did not seem to contain any protein based on a modified Bradford assay, but the first two mScarlet-I fractions clearly contained protein, so we combined the first two from each sample.

ZikaV Trigger Growth Cultures

A streak of a plate containing E. coli ZikaV Trigger was inoculated in 5 ml of 2 x YT and 5 µl of ampicillin. Two growth cultures were prepared. Grown overnight at 37°C at 200 rpm in Falcon tubes.

2 M Imidazole 5 ml

0.6808 g imidazole (M= 68.08 g/mol)

to 5 ml MQ H2O

Tris B buffer, 15 ml

12.75 ml Tris A

2.25 ml 2 M imidazole (total conc. 300 mM)

Present: Evelina, Jonna, Kevät, Jesper

Golden gate assembly for T7 + mScarlet-I

6 reactions were made, one for each toehold. All the concentrations were as before (18.7.), but the concentration of the reporter PCR product is 44.77 ng/µl. The components for each of the reactions are:

Golden gate reaction protocol (according to instructions on NEB site): (1 min 37℃, 1 min 16℃) X 30, 5 min 60℃, keep on 4℃ for ∞. Stored at -20℃.

ZikaV Trigger Growth Cultures

A streak of a plate containing E. coli ZikaV Trigger was inoculated in 5 ml of 2 x YT and 5 µl of ampicillin. Two growth cultures were prepared. Grown overnight at 37°C at 200 rpm in Falcon tubes.

Trigger RNA purification

Trigger RNA tubes were purified using Monarch total RNA miniprep kit (according to our protocol). Concentrations for tubes (measured with nanodrop):

Agarose gel electrophoresis for trigger RNAs

0.72 g of agarose was dissolved in 60 ml of 1 x TAE + 25 µl of ethidium bromide. Left to solidify. 15 µl of glyoxylate mix was added to 7.5 µl sample and denatured at 60℃ for 50 min. 2.5 µl of DNA Loading Dye and was added just before loading the gel. Run at 100 V for 1h 15 min.

Wells:

1 kb DNA Ladder

Trigger 1 29.7.

Trigger 2 29.7.

Trigger 3. 29.7.

Trigger 1 3.8.

Trigger 2 3.8.

Trigger 3 3.8.

ZikaV Trigger

=> RNAs were once again invisible on the gel. Juha proposed that we could have a meeting in which we go through the whole protocol and talk generally about how to handle RNAs. We also discussed the possibility of using RNA specific stains/dyes instead of SYBR Safe and ethidium bromide.

ZikaV Trigger Plasmid Linearization

17.6 µl of ZikaV Trigger Plasmid (c = 56 smth ng/µl), 24.6 µl of MQ Water, 5 µl of rCutSmart and 1 µl of AvrII was added into a PCR tube. Incubated for two hours at 37°C and then for 20 minutes at 80°C.

Present: Evelina, Kevät, Jesper

Cell-free reaction with mScarlet-I

This reaction was meant to be done with LacZ, but we made a mistake and used mScarlet-I instead so we changed the measurement to fluorescence.

The reactions were performed on a black 384-well PCR plate with transparent wells.

We used sonication 2/41 and autolysis batches. The following mastermixes were made into tube labeled 1 (sample):

From both mastermixes, 10.94 μl were transferred into tubes labeled 2 (water control) along with 1.06 μl of water.

6 μl of pEAS002 solution was added to both samples.

From each of the water controls, 10 μl was loaded into the plate. From both of the sample tubes, 2 x 10 μl and 2 x 20 μl reactions were loaded onto the plate. The reactions were incubated at 29°C for 3 hrs, with fluorescence at 569/593 nm measured at 1 min intervals.

Figure 30. mScarlet-I produced in cell-free reaction.

Heat-shock transformation for A70+MS, A92+MS, A95+MS and pEAS002

Four preps of competent DH5a cells were thawed on ice for 15 min. 5 µl of A70MS, A92+MS, A95+MS and pEAS002 were added to separate tubes. They were incubated on ice for 30 min. Cells were heat-shocked in a 42℃ water bath for 45 s and kept on ice for 7 min. Then, 500 µl of LB was added to each of the tubes and they were placed on 37℃, 200 rpm for 1 hr. From each of the tubes, 50 µl was plated on a LBA + Kan 50 plate and 300 µl on a second plate. They were incubated at 37℃ overnight.

3 x LBA+TET plates

51 ml of melted LBA was added into a flask. 51 µl of tetracycline (stock concentration 10 mg/ml) was added. Mixed. Poured onto three plates.

Transfer into a new plate - lacZ deficient plasmid containing E. coli

Bacteria was streaked from the old plate containing lacZ deficient plasmid containing E. coli into a new one (LBA+TET10). Grown overnight at 37°C.

Linearized trigger plasmid purification

Linearized plasmid were purified with NEB Monarch PCR & DNA cleanup kit, according to the kit instructions. The elution was made to 11 µl elution buffer. Concentrations and purity were measured with BioDrop:

Gel electrophoresis for linearized trigger plasmids

60 ml of 1 x TAE buffer was heated with 0.60 g agarose. 6 µl of SYBR safe color was added and mixed. Left to cool. Poured onto gel tray. Approximately 1 µl of Gel Loading Dye 6X was added to the sample (sample volume 5 µl). 7 µl of 1 kb Quick-Load DNA Plus Ladder Purple was loaded onto the gel. Approximately 6 µl (all of the sample) was loaded onto the gel. Gel was run at 100 V for 35 mins.

Figure 31. Agarose gel electrophoresis for linearised ZikaV triggers. Wells: 1: 1 kb DNA ladder, 2: Zika V trigger plasmid sample 1, 3: Zika V trigger plasmid sample 2.

Zika V trigger RNA synthesis

Two RNA synthesis reactions were made. The reagents were pipetted in the tubes in following order: 6 µl H2O; 1.5 µl of RNAP reaction buffer; 1.5 µl of each of the NTPs; 5 µl of linearised DNA; 1.5 µl of T7 RNAP. (20 µl total) The reactions were incubated at 37℃ overnight.

Present: Evelina, Kevät

Cell-free reactions for A92 and B39 toeholds

The following reagents were pipetted to tube 2 (A92 pos):

7.8 µl was transferred to tube 1 (water control) with 4.2 µl water.

28.76 µl was trasferred to tubes 4 (B39 pos) and 6 (ZikaV pos).

0.57 µl A92 was added to tube 2 and 14.67 µl transferred to tube 3 (A92 neg) with 7.33 µl water. 0.3 µl trigger 1 RNA and 7.04 µl water was added to tube 2.

0.61 µl B39 was added to tube 4 and 14.69 µl transferred to tube 5 (B39 neg) with 7.32 µl water. 0.3 µl trigger 1 RNA and 7.02 µl water was added to tube 4.

0.31 µl Zika sensor was added to tube 6 and 14.54 µl transferred to tube 7 (ZikaV neg) with 7.46 µl water. 0.32 µl trigger RNA and 7.14 µl water was added to tube 6.

10 µl samples were pipetted to 384 plate:

The reactions were incubated at 29°C for 3 hrs, with fluorescence at 569/593 nm measured at 1 min intervals.

Figure 32. β-galactosidase activity in cell-free reaction.

Zika V trigger RNA purification

Trigger RNA tubes were purified using Monarch total RNA miniprep kit (according to our protocol). Concentrations for tubes (measured with nanodrop):

Present: Evelina, Kristiina, Jesper

Figure 33. β-galactosidase activity in cell-free reaction.

15 LBA+KAN50 plates were made and stored in the fridge (+4°C).

Present: Evelina, Kristiina, Jesper

Figure 34. β-galactosidase activity in cell-free reaction.

Linearized trigger plasmid purification

Linearised plasmid were purified with NEB Monarch PCR & DNA cleanup kit, according to the kit instructions. The elution was made to 10 µl elution buffer. Concentrations and purity were measured with BioDrop:

0.020 M IPTG 1 ml

100 µl 2 M IPTG stock solution

900 µl of MQ H₂O

Present: Evelina, Kristiina, Jesper

Trigger Plasmid Linearization

50 µl reactions and 2 for each trigger were made to all triggers, according to our protocol. Incubated for two hours at 37°C.

Linearized trigger plasmid purification

Linearised plasmid were purified with NEB Monarch PCR & DNA cleanup kit, according to the kit instructions. The elution was made to 11 µl elution buffer. Concentrations and purity were measured with BioDrop:

RNA gel electrophoresis

Gel electrophoresis for all the triggers.

Agarose gel was made for the purified Zika V trigger RNA. 50 ml of 1 x TAE buffer was heated with 0.60 g agarose. 10 µl of SYBR safe color was added and mixed. Left to cool.

Samples: 24 µl of glyoxal mix was added to 12 µl of RNA and denatured 50 min in 60℃. 4 µl of loading buffer was added just before loading the gel. The gel was run for 25 min and 15 min after that (Fig. 34). Gel run was successful.

The wells:

Figure 35. Agarose gel electrophoresis for ZikaV trigger, triggers 1, 2 and 3 RNA. A. After 25 min of running. B. After 40 min of running.

Present: Evelina, Jonna, Kristiina, Jesper

Cell-free reaction

The following reagents were pipetted to a master mix tube:

7.8 μl of the mastermix was transferred to water control and positive control tubes. 4.2 μl of water was added to the water control. 0.521 μl pET15b-LacZ, 0.6 μl 0.02 M IPTG and 3.08 μl of water were added to the positive control tube.

To the mastermix tube, 1.26 µl Zika sensor plasmid was added and 14.4 µl transferred to negative control tube and added 7.6 µl water. To the mastermix tube 6.94 µl water was added and 0.66 µl of trigger RNA.

From water and positive control, 10 μl was loaded into the plate. From both of the sample tubes, 2 x 10 μl reactions were loaded onto the plate as follows:

The reactions were incubated at 29°C for 3 hrs, with absorbance measured at 1 min intervals.

Figure 36. β-galactosidase activity in cell-free reaction.

O/N cultures of B69, B46, B39 Toeholds, pEAS002, pET15b-LacZ and ZikaV-Sensor

5 ml overnight cultures were made with 5 ml LB and the following antibiotics: KAN50 for B39, B46, B69, pEAS002, ZikaV-S and AMP100 for pET15b-LacZ.

Trigger RNA purification

Trigger RNA tubes were purified using Monarch total RNA miniprep kit (according to our protocol). Concentrations for tubes (measured with Nanodrop):

RNA gel electrophoresis

RNAs were denatured for 50 minutes in 60℃ with glyoxal mix according to Juha's protocol.

1,2 % agarose gel was run in 1X TAE buffer for 30 min at 100 V.

Figure 37. Agarose gel electrophoresis for ZikaV trigger, triggers 1, 2 and 3 RNA. Legend: ZT ZT T1 T1 T2 T2 T3 T3

Present: Jonna, Kristiina, Jesper

Colony PCR to analyze GG assembled LacZ-toehold plasmids (T7+toehold+LacZ+T7) and MS-toehold A-series plasmids

2 colonies from each plate were picked for colony PCR (inoculated in 20 µl of MQ-water). Reactions for colony PCR were performed as below (a master mix of primers and Q5 was also made).

Forward primer: toehold linker

Reverse primer: T7 terminator

Figure 38. Concentrations for colony PCR reaction.

PCR reaction:

(40 cycles)

Figure 39. PCR cycle for PCR reaction.

Colony PCR was then performed to analyze the results (two colonies from each plasmid).

As can be seen on the gel, none of the LacZ colony PCR reactions show positive results (band size should be 3200 bp), whereas the MS plasmids all have the correct sized band (760 bp).

Figure 40. Agarose gel electrophoresis for colony PCR products. Ladder - LacZ_A70 - LacZ_A92 - LacZ_A95 - ladder - LacZ_B39 - LacZ_B46 - LacZ_B69 - ladder - MS_A70 - MS_A92 - MS_A95 - ladder - Neg. control (no DNA.

O/N cultures for MS_A70 (#1), MS_A92 (#1) and MS_A95 (#2) were started.

Plasmid extractions for B46, B39, B69, pEAS002, pET15b-LacZ, ZikaV-S

Extraction was made using the NEB Monarch Plasmid Miniprep Kit, according to manufactures’ protocol. First spin was done in 5000 x g 5 min. Samples were eluted in 50 µl of MQ H₂O.

Concentrations were as follows:

Present: Jesper

5 ml of LB was supplemented with 5 µl on kanamycin stock, yielding a final concentration of 50 μg/ml. Single colony of DH5α with Zika-S plasmid was inoculated and incubated overnight at 220 rpm, 37℃.

Present: Evelina, Anni, Jesper

Plasmid extraction for ZikaV sensor, MS A70, MS A92, MS A95

Extraction was made using the NEB Monarch Plasmid Miniprep Kit. First spin was done in 5000 x g 5 min.

Concentrations:

PCR reaction for pet15b-LacZ

Four reactions and one water control, where DNA was replaced with MQ H₂O, was made according to following:

Following PCR cycle was run:

Gel electrophoresis for PCR products

60 ml of 1 x TAE buffer was heated with 0.60 g agarose. 6 µl of SYBR safe color was added and mixed. Left to cool. Poured onto a gel tray.

Samples: 25 µl of each PCR product (4+ neg. control) was mixed with 5 µl of loading Dye and injected to the wells. Also 7 µl of ladder was injected to two wells.

Gel extraction for PCR products

Gel extraction was made with the NEB gel extraction kit according to the protocol. The concentrations were measured with Biodrop.

Heat-shock transformation for A70+MS, A92+MS, A95+MS and pEAS002

Two preps of competent DH5a cells were thawed on ice for 15 min. One prep was split in half into two tubes 5 µl of B39_MS, B46_MS, and B69_MS separate tubes. They were incubated on ice for 30 min. Cells were heat-shocked in a 42℃ water bath for 45 s and kept on ice for 7 min. Then, 600 µl of LB was added to each of the tubes and they were placed on 37℃, 200 rpm for 1 hr. From each of the tubes, 100 µl was plated on a LBA + Kan 50 plate. They were incubated at 37℃ overnight.

Present: Evelina, Jonna, Anni, Kristiina, Kevät, Jesper

Golden gate assembly for T7 + mScarlet-I

3 reactions were made, one for each toehold. All the concentrations were as before (18.7.), but the concentration of the reporter PCR product is 20.6 ng/µl. The components for each of the reactions are: