N O T E B O O K
Notebook
20220718
The Synthetic gene synthesized gene is transferred to DH5a
Import the plasmid carrying the fragment of interest into DH5α for preservation
Experimental operation:
1. Plasmid dry powder (transport at room temperature, store at -20 degrees, 90 days shelf life, please be sure to use after converting and extracting plasmid)
2. Centrifuge the plasmid tube, 13000g for 3min, then add 80uL of sterile water to the tube, shake well, dissolve the plasmid dry powder;
3. Add 10μl of plasmid, chemically transform into DH5α strain competent state, named: pUC57-SulAp_eGFP, pUC57-hrpR, pUC57-hrpS_PhrpL, pUC57-SulAp_relE, and coated on resistant plates;
4. Incubate the plate forward for 1h, and then invert at 37 °C for 14h;
（If there are too many colonies, dilute the plasmid before transformation. If there are no colonies, add 10 μl of plasmid for transformation. In addition, do not directly transform to competent cells, but first transform cloned competent cells, re-extract plasmids and then transform into expression competent cells）
20220719
1. SulAp_eGFP、hrpR、hrpS_PhrpL Fragment sequencing
Pick transformed single colonies (host bacterial DH5a) from yesterday's plates (pUC57-SulAp_eGFP, pUC57-hrpR, pUC57-hrpS_PhrpL), design primer amplification SulAp_eGFP, hrpR, hrpS_PhrpL fragments, agarose gel electrophoresis to verify fragment size and send for sequencing, run the program above.
Primer list：
PCR outcome：
PCR outcome：
2. Save bacteria and shake bacteria
- Inoculate the Successfully transformed colonies of pUC57-SulAp_eGFP, pUC57-hrpR, pUC57-hrpS_PhrpL into 4 ml of LB medium (with antibiotics) at 220 rpm overnight;
- Pick a single colony of pSB1C3 (43) from the plate and inoculate into 4 ml of LB medium (with antibiotics) at 220 rpm overnight;
20220720
3. Plasmid extraction and amplification of the fragment of interest
Experimental operation：
1. Plasmid extraction: Extract the plasmids of pUC57-SulAp_eGFP, pUC57-hrpR, pUC57-hrpS_PhrpL, pSB1C3(43) according to the instructions;
2. PCR to obtain the gene and skeleton of interest
Gene of interest: from pUC57-SulAp_eGFP
Skeleton: From pSB1C3（43）
Configure the PCR system: (refer to the instructions for using high-fidelity polymerases):
Procedure (refer to instructions for use of high-fidelity polymerases):
PCR products run glue
Agarose Gel DNA Recovery (Enhanced DNA Recovery Kit)
At this point, the target genes and skeleton are obtained
20220721
4. Plasmid construction
- Connect eGFP fragments to the pSB1C3 skeleton; Attach the SulAp_eGFP fragment to the pSB1C3 skeleton;
- The homology arm has been added when the primers are designed in the front to facilitate subsequent Gibson connections:
- DNA fragments and Backbone are mixed in a 3:1 ratio, using Gibson connections.
Reaction system:
Note: Linearized vectors with generally 10-50 ng/10ul reaction system, and the molar ratio of the insert fragment to the vector is 2-3:1 is the best.
Use a PCR instrument at 50°C, incubate for 1-2h or at 16°C overnight
5. Chemical Conversion:
- After being connected by Gibson:
• Take 50 μL of E. coli competent cells and place on ice to slowly thaw.
• Add the assembly product, mix well with a flick and leave in an ice bath for 20 min.
• After 42 °C thermal excitation for 60 s, quickly ice bath for 2 min, the process does not shake the EP tube.
• Add 300 μL of LB liquid medium without antibiotics and resuscitate the culture in a 37 °C shaker for 60 min.
• For plasmid transformation (the amount of plasmid DNA added to the transformation >5 ng is sufficient), take 100 μL of the recovered bacterial solution, coat on a solid plate containing Canas, and invert at 37 °C for about 16 h.
(BL21 transformation colonies are relatively dense, so do not centrifuge before coating, but blow and aspirate the bacterial solution at the bottom)
20220722
6. Verification of plasmid construction
Colony PCR
Agarose gel electrophoresis validation
• Send for sequencing
• Compare sequencing results with target sequences
The construction of the pSB1C3-j23119_eGFP and pSB1C3-SulAp_eGFP plasmids has been completed
- PUC57-SulAp-relE plasmid is converted to DH5a
• Flat coating
• PCR verification
• Pick-up
• plasmids exaction
20220723
7. SulAp_eGFP Characterization
Experimental operation:
1. Seed solution preparation: pSB1C3（43 BL21）、pSB1C3-j23119_eGFP（BL21）、pSB1C3-SulAp_eGFP（JM109）、pSB1C3-SulAp_eGFP（BL21）
2. Pick the transformed single colonies from the plate and transfer to 4 ml of liquid LB (C+), 37 °C, 220 rpm overnight incubation, use tin foil to block out the light;
3. Expand culture and protein expression: Add 10 ml lb (C+) to a 250 ml Erlenmeyer flask, take 100 ul of overnight culture liquid and add LB culture medium to OD595 = 0.4-0.5 (about 3-4 h) at 37 °C, 220 rpm, use tin foil to block out the light;
4. UV induction at different times, 37 °C, 200 rmp culture overnight or for 6-8 h, use tin foil to block out the light;
5. Collect bacterial bodies: microplate reader measures OD600, fluorescence value (excitation light 485 nm, absorbed light 535 nm, gain value 50)
- The results of the fluorescence value were positively correlated with the UV induction time
- Repeat the experiment 3 times
20220724
8. SulAp-hrpR-eGFP plasmid construction, unsuccessful
- Attach the Sulp-relE fragment to the pSB1C3 skeleton:
- Amplify SulAp-relE fragments from pUC57-SulAp-relE
- Amplifyied pSB1C3 fragments as skeletons
20220725
9. SulAp-hrpR-eGFP plasmid construction, unsuccessful
2. pSB-SulAp-relE plasmid construction
- - Gibson procedure connects SulpAp-relE fragments and pSB1C3 backbones, competently transforms and cultures
20220726
10. pSB-SulAp-relE plasmid construction:
Colony PCR validation of SulAp-relE fragments
Bacterial P verification: relE1, 3 colonies successful, sent for sequencing (1 ,3)
pSB1C3-SulAp_eGFP fragment length is incorrect (5,6)
hrpR has no stripes and construct unsuccessfully (7, 8, 9)
20220727
1.RelE1,3-extracting plasmid, PCR, gel electrophoresis detection
2.Test pSB1C3-SulAp_eGFP fragments converted on July 22
relE, strip correct, etc. sequencing results;
pSB1C3-SulAp_eGFP et al. (7.21 and 7.27) conversion results;
20220728
1.pSB1C3-relE sequencing was successfully construct
Sequencing Results: Success
pSB1C3-relE, PadR-DC_let4X (extract plasmid tomorrow)，pick up colony, ready to construct SulAp-relE-3WJ-Bro
2.Test pSB1C3-SulAp_eGFP fragments converted on July 27
No. 21 ligation product, colony 3 with the correct band, sent for sequencing
No. 27 ligation product, colony 4 with the correct band, sent for sequencing
20220729
1.pss1 PCR products sequencing
pSB1C3-SulAp_eGFP,Colony 27 convertes to colony 2
Construct plasmid Sulp-relE-3WJ-Bro
Gibosn ligation, chemical conversion
20220730
pSB1C3-SulAp_eGFP sequencing was successful
Sequencing Results: Success
pSB1C3-SulAp_eGFP,pick up colony, extract plasmid and prepare to construct pSB1C3-SulAp-hrpR-eGFP
2.SulAp-relE-3WJ-Bro verification
1. PCR results of SulAp-relE-3WJ-Bro colony:
The first No. 4 + the second No. 3, No. 5 band approach, sent for sequencing (note that the 3F+R and 5F1+R1 primers are different)
3.Activity of Sulp after UV induction
Experimental operation:
1. Seed solution preparation: take non-recombinant BL21、pSB1C3-SulAp_eGFP (BL21);
2. Pick a single colony from the plate and transfer to 4ml liquid LB (C+), 37 °C, 220 rpm culture,use tin foil to block out the light;
3. Induction of expression: Add 10 ml lb (C+) to a 50 ml centrifuge tube, take 100 ul of overnight culture solution and add LB culture medium to OD595 = 0.4-0.5 (~3-4 h) at 37 °C, 220 rpm, use tin foil to block out the light;
4. UV induction at different times, 37 °C, 200 rmp culture, 0h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, use microplate reader to measure of OD600 and fluorescence values respectively (excitation light 485 nm, absorbed light 535 nm, gain value 50)
Experimental data is available and the results are as follows:
0730 SulAp Startup Effect (Notes)
20220731
1.Construct pSB1C3-SulAp-hrpR_eGFP (unsuccessful)
1. Extract the successfully constructed pSB1C3-SulAp_eGFP plasmid
2. Amplify hrpR fragments and pSB1C3-SulAp_eGFP skeleton
2.construct pSB1C3-T4 lysis-lacZ
1. Amplify fragments and skeletons
2. Gel recovery, Gibson ligation + transformation
20220801
1.pSB1C3-SulAp-eGFP validation
1. The sequencing is correct, the fragment size is correct, and the unseen of skeleton P is not because of plasmid
2. Build the pSB1C3-SulAp-hrpR_eGFP, the skeleton is still not amplified, and it needs to be redesigned
2.pSB1C3-T4 lysis-lacZ authentication
1. The lacZ clip is not connected
20220802
1.SulAp-relE-3WJ-Bro sequencing was successful
Plasmid profile completion:
2.Plasmid construction
1. pSB1C3-SulAp-hrpR_eGFP
2. pSB1C3-T4 lysis-lacZ
Strip correctly, cut glue recycled, Gibson connected, transformed
20220803
1.Plasmid ligation test
pSB1C3-SulAp-hrpR_eGFP has suspected bands:
pSB1C3-T4 lysis-lacZ，Colony 4 may be correct:
Colonies are too dense, dilute inoculation after transformation ，try again
2.Test the effect of relE-3WJ-Bro
Experimental Materials:
SulAp-relE-3WJ-Bro
Experimental operation:
1. Seed solution preparation: Take SulAp-relE (DH5a), SulAp-relE-3WJ-Bro (DH5a)；
2. Pick a single colony from the plate and transfer to 4ml liquid LB (C+), 37 °C, 220 rpm culture, use tin foil to block out the light；
3. Induction of expression: Add 10 ml lb (C+) to a 50 ml centrifuge tube, take 100ul of overnight culture broth and add LB culture medium to OD595 = 0.4-0.5 (approximately 3-4 h) at 220 rpm, use tin foil to block out the light;
4. UV induction at different times, 37 °C, 200 rmp culture, 0h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, use microplate reader to measure of OD600 and fluorescence values respectively
Invalid experimental data:
20220804
1.Plasmid ligation test
pSB1C3-SulAp-hrpR_eGFP：
Send for sequencing: Colonies 1, 2, 3, 6
pSB1C3-T4 lysis-lacZ：
20220806
1.The pSB1C3-SulAp-hrpR_eGFP was successfully construct
1. Sequencing result: Success
2.Colony inoculation culture
1. pSB1C3-SulAp-hrpR_eGFP (colony 3, Extract plasmid, construct Amp30E)
2. pSB1C3-T4 lysis-lacZ (colony 8, cultured and then extracted plasmid PCR test, banded and then sequenced)
3. pSB-SulAp-relE-3WJ-Bro (Extract plasmid, PCR verified)
4. pSS5（DH10B）
5. pSB1C3（DH10B）
20220807
1/Experimental preparation
1. Extract plasmid
2. Prepare different concentrations of arabinose
20220808
1.pSB1C3-SulAp-Amp30E_eGFP construct
1. Expansion of skeletons and fragments
No strips
2.pSB1C3-T4 lysis-lacZ construction
Recycle fragments, connect and convert Gibson
20220809
1.pSB1C3-SulAp-Amp30E_eGFP construction
Recycle fragments, connect and convert Gibson
2.pSB1C3-T4 lysis-lacZ authentication
send Colonies 3 and 8 for sequencing
3.pSS5new_pSB1C3_pBAD_T4 lysis device test
- transfer pSS5 plasmid to the DH10B competent state
- Transfer pSB1C3 plasmid to the DH10B competent state
- Different concentrations of arabinose 0, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250mM (10μL)
1. seed solution preparation: take pSS5, pSB1C3, pick up a single colony from the plate, transfer to 4 ml of liquid LB(C+), 37 °C, 220 rpm overnight culture;
2. Expand culture and protein expression: add 20 ml of LB (C+) to a 50 ml centrifuge tube, take 100 ul of overnight culture broth and add LB culture medium to OD595 = 0.4-0.5 (~3-4 h) at 37 °C, 220 rpm;
3. Different concentrations of arabinose induction, 37 °C, 200 rmp culture.
4. Bacterial measurement: induction 0h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, respectively, microplate reader measurement OD600, record data.
20220810
1.pSB1C3-SulAp-Amp30E_eGFP authentication
Colonies 1 and 2, send for sequencing
20220811
1.pSB1C3-T4 lysis-lacZ sequencing comparison
1. Sterilization verification
20220812
1.pSB1C3-T4 lysis-lacZ construction
2.pSB1C3-SulAp-Amp30E_eGFP construction
20220813
1.pSS5new_pSB1C3_pBAD_T4 lysis device repeat the experiment
20220814
1.pSB1C3-T4 lysis-lacZ authentication
Colony No. 8 (posterior), Band No. 1 is weak Send sequencing, experimental verification
2.pSB1C3-SulAp-Amp30E_eGFP verify
20220815
1.pSB1C3-SulAp-Amp30E_eGFP test
1. PCR, strip correct, send for sequencing tomorrow
2.pSB1C3_pBAD_T4 lysis-lacZ test
1. PCR, strip correct, send for sequencing tomorrow
2. Colors appear
20220817
1.pSB1C3-SulAp-Amp30E_eGFP the test was successful
2.pBAD_T4 lysis-lacZ the test was successful
3.Amp30E amplification system effect verification
Experimental operation:
1. Seed solution preparation: take non-recombinant BL21, pSB1C3-SulAp_eGFP (BL21); pSB1C3-Amp30E_eGFP (BL21), pick up a single colony from a plate and transfer to 4 ml of liquid LB(C+), 37 °C, 220 rpm culture, use tin foil to block out the light;
2. Induction of expression: Add 10 ml lb (C+) to a 50 ml centrifuge tube, take 100 ul of overnight culture liquid and add LB culture medium to OD595 = 0.4-0.5 (about 3-4 h) at 37 °C, 220 rpm, use tin foil to block out the light;
3. UV induction at different times, 37 °C, 200 rmp culture, 0h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, respectively use microplate reader measurement of OD600 and fluorescence values (excitation light 485 nm, absorbed light 535 nm, gain value 50)
Experimental data need to be duplicated
20220819
1.relE-3WJ-Bro construct
2.Amp30E-relE-3WJ-Bro construct
Recycle the connection
20220820
1.Test the scale-up system
1. The steps are the same, the experiment is successful, and the data is available (just visualize):
0821-Amp30E Effect Test - Fluorescence ÷OD
2.relE-3WJ-Bro inspection
Colonies 5/6/10/17 are available and will be amplified tomorrow for testing
3.Amp30E-relE-3WJ-Bro inspection
Colonies 2/4/17 are available and will be amplified tomorrow for testing
20220821
DH10B、T4 lysis（DH10B）
Arabinose induction lysis device (data available)
DH10B、T4lysis-lacZ（DH10B)
Arabinose induces lacZ color reaction
pSB1C3（DH10B）
pSB1C3-relE（DH10B）
Existing (colonies 1 and 9 are ok)
pSB1C3-Amp30E-relE-3WJ-Bro（DH10B）
20220823
1.Amp30E-relE-3WJ-Bro（DH5a）Conversion results
Select colonies 10 and 11 (expect 4200 bp)
2.Amp30E-relE-3WJ-Bro（DH10B）Conversion results
Select colonies 1 and 3 (expected 4200 bp)
3.The T4-lacZ build is complete and the blue effect is displayed
1. Transfer pBAD_T4 lysis-lacZ plasmid into DH10B competent state;
2. Prepare 8 Erlenmeyer flasks (250 ml) containing M9 liquid medium (50 ml) and sterilize;
3. Add 100ul lacZ bacterial solution to the medium (+25 μl chloramphenicol), overnight culture at 37 °C, 220 rpm;
4. Different conditions induce expression, and samples in Erlenmeyer flasks are used to observe the blue phenomenon.
5. Put into a shaker for culture and observe the phenomenon
20220824
1.Amp30E-relE-3WJ-Bro fragment amplification for testing
Fail
2.Amp30E-relE (DH5a) colony PCR
Take colonies 2 and 9 for culture, tomorrow PCR, and if successful, extract the plasmid
3.pSB1C3（DH10B）、pSB1C3-relE（DH10B）、pSB1C3-Amp30E-relE（DH10B）Induce expression
(Using pSB1C3-relE9 colony, Amp30E-relE-3WJ-Bro1 colony)
The data needs to be duplicated
20220825
1.pSB1C3-Amp30E-relE Clips are sent for testing
2.pSB1C3（DH5a）、pSB1C3-relE（DH5a）、pSB1C3-Amp30E-relE（DH5a）Induce expression
Repeat yesterday's experiment to continue testing, experimental results are not satisfactory, continue to test with DH10B
20220826
1. pSB1C3-Amp30E-relE Sequencing results
The relE fragment is lost, design the primer , and test whether there is a relE fragment on the ligation product tomorrow
Transfer Sulp-relE-3WJ-Bro to DH10B ， use PCR to screen colonies tomorrow
20220827
1.RelE toxin protein and tag system testing
pSB1C3-SulAp-Amp30E-relE has a band that conforms to the length of relE (with Colony 2)
2.pSB1C3-SulAp-relE-3WJ-Bro（DH10B）colony PCR
Obtain pSB1C3-SulAp-relE (DH10B) available (colonies 1 and 9 are ok)
pSB1C3（DH10B）
pSB1C3-SulAp-relE-3WJ-Bro（DH10B）
pSB1C3-SulAp-Amp30E-relE-3WJ-Bro（DH10B）
20220828
1.pSB1C3（DH10B）、pSB1C3-relE-3WJ-Bro（DH10B）、pSB1C3-Amp30E-relE-3WJ-Bro（DH10B）Test
Repeat the experiment, the results need to be debugged:
pSB1C3-SulAp-relE-3WJ-Bro（DH10B）Previously constructed Colony 9 is not available
pSB1C3-Amp30E-relE-3WJ-Bro（DH10B）Colony No. 1 is available and data are available for testing
20220829
1.pSB1C3-SulAp-relE-3WJ-Bro (DH10B) Second Build Test (Successful)
1. Colonies all have target bands
2. Take the PCR reaction product above and detect whether there are relE and 3WJ-Bro fragments respectively
The relE fragment is correct:
3WJ-Bro fragment correct:
2.pSB1C3-Amp30E-relE-3WJ-Bro（DH10B）【succeed】
1. Detect the presence of releE, 3WJ-Bro, and Amp30E fragments
Colony 1 (data available): left reelE, right 3WJ-Bro
Colony 3: Left reelE, right 3WJ-Bro
Amp30E fragment: Both 1 and 3 colonies have the correct bands
Colony 1 was tested, and the experimental data matched the trend, Preserve bacteria
3.pSB1C3（BL21）、pSB1C3-relE-3WJ-Bro（BL21）、pSB1C3-Amp30E-relE-3WJ-Bro（BL21）Test
pSB1C3（BL21）possess
pSB1C3-relE-3WJ-Bro（BL21）Both a and b have bands, start with colony bpSB1C3-Amp30E-relE-3WJ-Bro（BL21）In transformations of 1 and 3 , colony 1 is used first
20220830
1.pSB1C3-Amp30E-relE-3WJ-Bro (BL21) Colony PCR Verification (Incorrect)
2.pSB1C3-Amp30E-relE (DH10B) colony PCR Verificationvalidation (no relE fragments)
1. With Amp30E strip (2366bp)
2. No relE stripe (383bp)
20220831
1.RelE experimental data
0830-Amp30E-relE-Absorbance
2.pSB1C3-SulAp-relE-3WJ-Bro (DH10B) build and test colonies 9 and 11 for the second time
Both relE and 3WJ-Bro bands were correct, perform follow-up experiments were performed with colony 11, which was successfully tested yesterday
20220901
Test the mSarlet-I-3WJ-Bro label
20220902
1.pSB2C3-Amp30E-relE-3WJ-Bro（BL21）
1. With Amp30E strip (2366bp)
2. With 3WJ-Bro strip (aroundabout 900bp)
3. RelE band (aroundabout 300bp)
20220906
1. Arabinose-induced T4 lysis device
Get replicated experiment data
20220909
1.Arabinose-induced T4 lysis device
Get replicated experiment data