Team:WHU-China/Notebook
Notebook for Quenching Module
Notebook for Sensing Module
Notebook for Chemotaxis
Notebook for TA systems
Notebook for Cell-free quorum sensing
31/8/2020
Picked 3 single colonies of recovered DH5α strains and transferred them into 3 test tubes with LB liquid medium.
2/9/2020
Added 50μL of DH5α into a new test tube with 5mL LB liquid medium and then cultured in a shaker for 3h(OD600=0.941).
Prepared competent bacteria according to the protocol of One Step Competent cell Preparation Kit, Beyotime (D0301) and transferred GFP, rhlR, AiiA and sensor parts from previous iGEM kits respectively into the competent bacteria (100μL/each), then cultured under 37℃ overnight. Failed to grow single colonies.
5/9/2020
3μL sensor part from previous iGEM kit was transferred into 100μL newly purchased DH5α strains according to the instruction of the kit, then spread them on the plates with chloramphenicol under 37℃ overnight.
The absorption peak of E0040 (GFP) and sensor parts solution were both 270nm after measuring.
25/9/2020
Dissolved the AiiC gene dry powder from IDT to 10ng/μL and the primers to 100μM. Used PCR to get the sequence containing AiiC and Gibson overlap sequence, Ta was set to 48℃. Failed to get the target band.
26/9/2020
Used EcoRI and PstI to digest AiiC and PSB1A3 (from previous iGEM kit).
Incubated under 37℃ overnight, then inactivated the enzymes under 80℃ for 5 min.
27/9/2020
Purified AiiC and PSB1C3 according to the protocol of TIANquick Midi Purification Kit, Tiangen (DP204), then used agarose gel electrophoresis to observe the result of purification. Only can see PSB1A3 band.
Used agarose gel electrophoresis to observe the original AiiC gene solution. Got the target band.
Used the Rapid DNA Ligation Kit to ligate AiiC and PSB1A3.
After ligation, transferred them into competent bacteria according to the kit and spread it on a plate with ampicillin.
29/9/2020
Used colony PCR to check the AiiC in the 3 strains inoculated on 28/9.
2/10/2020
Extracted the plasmids containing AiiA from TOP10 (ordered from Genscript) according to the instruction of TIANquick Midi Purification Kit, Tiangen (DP204).
Used agarose gel electrophoresis to check the plasmids containing AiiA. Observe one band which is much larger than the target band.
Used colony PCR to get pvdq from PAO1.
Transferred plasmids containing AiiA into prepared competent BL21, then cultured on LB plates with kanamycin under 37℃ overnight.
3/10/2020
Used agarose gel electrophoresis to check the result of pvdq colony PCR. Failed to get target band.
Haoyu Zheng helped us pick single colonies containing AiiA into liquid LB medium.
4/10/2020
Used colony PCR to get pvdq from PAO1. Prepared 8 PCR tubes of reaction systems in total for different Ta from 50-56℃.
Used agarose gel electrophoresis to check the results of pvdq colony PCR. Failed to get target band.
5/10/2020
Used colony PCR to get pvdq from PAO1. Prepared 3 PCR tubes of reaction systems in total for different Ta from 51.5-56.4℃.
Used agarose gel electrophoresis to check the result of pvdq colony PCR. Failed to get target band.
Took 1mL of AiiA-pET28a strains from each of the 3 tubes, then amplified and cultured in LB liquid medium (about 120 mL with 0.6 mL kanamycin) overnight under 37℃ and 200 rpm.
6/10/2020
Added 5mL bacteria solution from each conical flask prepared on 5/10 into 120 mL LB liquid medium, then cultured under 200 rpm for 1h (OD600=0.456). Added 60μl IPTG (1M) each bottle and incubate under 28℃ (negative control: added 10ml bacteria solution without adding IPTG into 50 ml EP tube).
Used colony PCR to check the AiiA in the three flasks of the bacteria solutions. Used agarose gel electrophoresis to check the result of AiiA colony PCR. Only got one band over 12kb. Then stopped induced expression and picked 3 original TOP10 single colonies and transferred them into LB liquid medium (5mL LB with 25μL kanamycin) respectively, cultured overnight under 37℃,200 rpm.
Used EcoRI and PstI to digest 17 μL AiiC (about 10 ng/μL) and 8 μL PSB1C3 (about 25 ng/μL). Prepared 2 reaction systems of AiiC and 1 of PSB1C3.
Incubated under 37℃ overnight, then inactivated the enzymes under 65℃ for 25min.
7/10/2020
Extracted the plasmids containing AiiA from TOP10 according to the instruction of TIANquick Midi Purification Kit, Tiangen (DP204).
Used agarose gel electrophoresis to check the products of digestion of PSB1C3, the result of AiiC digestion, plasmids containing AiiA. Got one band over 12kb from plasmids extracted from TOP10.
Used colony PCR to check AiiC (3 reaction systems) from previous bacteria solutions which may contain AiiC ligated with pSB1A3(do the same thing as before, but change annealing temperature this time).
Used agarose gel electrophoresis to check the result of AiiC colony PCR. Failed to get target band.
Used EcoRI and PstI to digest 17 μL 12kb plasmids from TOP10.
Incubated under 37℃ for 2h, then used agarose gel electrophoresis to check the result of digestion. Got 2 bands of 6-8kb.
Purified AiiC and PSB1C3 according to the instruction of the DNA Purification Kit.
8/10/2020
Transferred 100μL BL21 into 5mL LB liquid medium and then cultured in a shaking table for until OD600 reached 0.510.
Used PCR to get the sequence containing AiiC and Gibson overlap sequence (3 reaction systems for 56-65.6℃).
Prepared competent BL21 (OD600=0.510).
Used the Rapid DNA Ligation Kit to ligate AiiC and PSB1C3, then transferred the product into competent BL21, then cultured on LB plates overnight under 37℃.
Used agarose gel electrophoresis to check the result of PCR. Failed to get the target band.
9/10/2020
Used EcoRI and PstI to digest plasmids from company to check AiiA. 37℃ water bath for 2h.
Used agarose gel electrophoresis to check the result of digestion. The negative control had a 12kb band while the enzyme-digested product had a 1kb band and a 7kb band.
Picked 6 single colonies from the 3 plates cultured on 8/10 (the colonies had grown into lawn), then transferred into 6 tubes of LB liquid medium (6mL LB with chloramphenicol).
Prepared competent BL21 (OD600=0.34).
Used the Rapid DNA Ligation Kit to ligate AiiC and PSB1K3, PSB1C3. Transferred the production into competent BL21, then cultured on LB plates overnight under 37℃.
10/10/2020
Picked 3 single colonies (1 containing AiiC-PSB1C3 and 2 containing AiiC-PSB1K3) and transferred them respectively into 3 test tubes of 6mL LB liquid medium (containing 50ng/μL antibiotics(Cm and Kan separately)), cultured overnight under 37℃, 200rpm.
11/10/2020
Built a cell-free system according to the protocol. After incubation in 37℃ for 1h, added 100μL cell-free system into 400μL nickel column.
Used SDS PAGE to check the expression of AiiA in the cell-free system.
Took each 1.5 mL bacteria solution from LB liquid medium cultured on 10/10 (2 tubes of PSB1K3-AiiC and 1 tube of PSB1C3-AiiC), extracted the plasmids (PSB1K3-AiiC and PSB1C3-AiiC) according to the instruction of TIANprep Mini Plasmid Kit (final volume: 100μL each).
Used EcoRI and PstI to digest PSB1K3-AiiC and PSB1C3-AiiC.
Used agarose gel electrophoresis (1%) to check PSB1C3-AiiC plasmids, PSB1K3-AiiC plasmids and the result of digestion. Got target band of PSB1C3-AiiC plasmids and only one band of the enzyme-digested products of PSB1C3-AiiC (about 2000bp).
12/10/2020
Used agarose gel electrophoresis (0.6%) to check the result of digestion again. Got only one band of the enzyme-digested products of PSB1C3-AiiC.
Transferred 60μL BL21 solution into 5mL LB liquid medium, cultured under 37℃, 200rpm until OD600 reached 0.305, then prepared competent BL21. Transferred AiiA plasmids in to the competence bacteria, then plated them on the plates with corresponding antibiotics overnight under 37℃.
Transferred bacteria containing PSB1C3-AiiC into 120 mL LB liquid medium (containing 200μL chloramphenicol) .
Checked the results of SDS-PAGE done on 11/10. Only find blurry or no target band. Conducted SDS-PAGE again. But still no target bands.
13/10/2020
Prepared competent BL21.
Transferred plasmids containing AiiA into the competent BL21.
Added 5 mL bacteria (containing PSB1C3-AiiC) solution into 120 LB liquid medium (containing 200 μL chloramphenicol), cultured under 37℃, 200rpm for 2h. Then added 60 μL IPTG into the culture, cultured overnight under 28 ℃.
14/10/2020
Picked 4 single colonies (pET28a-AiiA) cultured on 12/10, then added them respectively into 4 tubes of 5mL LB liquid medium (containing 25μL kanamycin).
Extracted pET28a-AiiA plasmids according to the instruction of TIANprep Mini Plasmid Kit, Tiangen (DP103), then used EcoRI and PstI to digest the product.
Used agarose gel electrophoresis (1%) to check the result of digestion. Both the original solutions of plasmids and solutions of plasmids with enzyme digestion had no bands.
Checked the result of PSB1C3-AiiC induced expression.
SIAT shared their stock of nissle1917 strain. Stored at 4°C after receipt.
23/8/2020
Inoculated three single colonies of nissle1917 into 5ml LB liquid medium and cultured overnight in a 30°C shaker.
24/8/2020
Took one of nissle1917 culture to streak on the LB plate, cultured overnight at 30°C. Other stored at 4°C.
25/8/2020
The cultivation plate of nissle1917 was sent to Tongji.
30/8/2020
Took 50 μl glycerol stock of DH5α and spread on the LB plate, cultured overnight at 30°C
Picked three single colonies of BL21 from the LB plate stored in the laboratory, inoculated them into 5ml LB liquid medium and cultured overnight in a 30°C shaker.
31/8/2020
Prepared the solution of ampicillin (50mg/ml), kanamycin (10mg/ml) and chloramphenicol (34mg/ml, ethanol), filtered and sterilized through 0.22 micron syringe filters.
1/9/2020
Prepared competent cells of DH5α according to the protocol of One Step Competent cell Preparation Kit, Beyotime (D0301).
Used 10 μl ddH2O to dissolve the DNA of BBa_I746361, BBa_B0010, pSB4A5 and BBa_J23100 in the dry powder plate.
Transformed the target gene and spread on the LB plate containing the corresponding antibiotic, cultured overnight at 37°C.
2/9/2020
No transformed colonies on the plate, ordered from GenScript instead.
8/9/2020
Picked three single colonies of BL21 from the LB plate stored in the laboratory, inoculated them into 5ml LB liquid medium and cultured overnight in a 37°C shaker.
15/9/2020
Colony PCR to obtain CDS of pqsR gene according to the protocol of PCR Kit with Taq, Beyotime (D7232).
16/9/2020
Detected PCR products by TAE agarose gel electrophoresis (1%, 120V, 30min) and found no target band.
18/9/2020
Repeated the colony PCR but reduced Ta to 52°C.
19/9/2020
Detected PCR products by 1% agarose gel electrophoresis (TAE, 120V, 30min) and found no target band.
24/9/2020
Repeated the colony PCR but used primers with fewer overhangs and extended pre-denaturation time to 5min.
Detected PCR products by 1% agarose gel electrophoresis (TAE, 120V, 30min) and found target band.
Gel extracted according to the protocol of TIANgel Midi Purification Kit, Tiangen (DP209).
Used 10 μl ddH2O to dissolve the DNA of TEVprotease and TEVprotease+histag.
Took 4 μl of TEVprotease, TEVprotease+histag and pSB1A3 linearized vector, digested them with EcoRI and PstI overnight at 37°C according to the protocol of EcoRI, Beyotime (D6329) and PstI, Beyotime (D6565).
25/9/2020
Detected gel extracted products by 1% agarose gel electrophoresis (TAE, 120V, 30min) and found target band.
Detected digested products of TEVprotease, TEVprotease+histag and pSB1A3 by 1% agarose gel electrophoresis (TAE, 120V, 30min) and found target band.
Extracted digested products of TEVprotease, TEVprotease+histag and pSB1A3.
Repeated the PCR but used extracted products of pqsR CDS as template and used the first pair of primers to add biobricks prefix and suffix.
26/9/2020
Detected PCR products by 1% agarose gel electrophoresis (TAE, 120V, 30min) and found no target band.
Streaked on the LB plate containing kanamycin to resuscitate glycerol stock of TOP10 with plasmids which inserted BBa_B0010 and expression devices of phiR73 delta and GFP, cultured overnight at 30°C.
27/9/2020
Ligated digested products of TEVprotease, TEVprotease+histag and pSB1A3 separately according to the protocol of Rapid DNA Ligation Kit, Beyotime (D7002).
Transformed the ligated products and spread on the LB plate containing ampicillin, cultured overnight at 30°C.
28/9/2020
Normal recombinants appeared on the plates, picked three single colonies, inoculated them into 5ml LB liquid medium containing ampicillin and cultured overnight in a 30°C shaker.
29/9/2020
Used recommended standard vector sequencing primers to build colony PCR system to check the recombinant plasmid.
Detected PCR products by 1% agarose gel electrophoresis (TAE, 120V, 30min) and found target band.
Repeated the PCR of pqsR CDS using longer primers but extended pre-denaturation time to 5min.
30/9/2020
Detected PCR products by 1% agarose gel electrophoresis (TAE, 120V, 30min) and found no target band.
2/10/2020
Took 12 μl of pSB1A3 and pSB1C3 linearized vector, plasmid inserted BBa_B0010 and GFP expression device, digested them with EcoRI and PstI overnight at 37°C according to the protocol of EcoRI, Beyotime (D6329) and PstI, Beyotime (D6565).
Took 12 μl of plasmid inserted TEVprotease, plasmid inserted BBa_B0010 and GFP expression device, digested them with XbaI and PstI overnight at 37°C according to the protocol of XbaI, Beyotime (D6713) and PstI, Beyotime (D6565).
Repeated the PCR of pqsR CDS but change primers.
3/10/2020
Detected digested products by 1% agarose gel electrophoresis (TAE, 120V, 30min) and found target band.
Gel extracted according to the protocol of TIANgel Midi Purification Kit Tiangen (DP209).
Detected PCR products by 1% agarose gel electrophoresis (TAE, 120V, 30min) and found target band.
Repeated the PCR of pqsR CDS but used the first pair of primers.
4/10/2020
Detected PCR products by 1% agarose gel electrophoresis (TAE, 120V, 30min) and found target band.
Gel extracted according to the protocol of TIANgel Midi Purification Kit Tiangen (DP209).
5/10/2020
Ligated digested products of GFP expression device and pSB1A3 according to the protocol of Rapid DNA Ligation Kit, Beyotime (D7002).
Transformed the ligated products and spread on the LB plate containing ampicillin, cultured overnight at 30°C.
7/10/2020
No transformed colonies on the plate.
Repeated but failed again.
2020/9/4
Subculture of HL-60
1. The HL-60 cell line (culture bottle) was obtained from the Preservation Center（CCTCC）
2. Transfer 2 / 3 cell suspension to another two culture bottles, three culture bottles together (37 ℃, 5% carbon dioxide), the total amount of cell suspension in each bottle is 6ml.
Medium: RPMI-1640 (10% FBS)
2020/9/5
Subculture of HL-60
1. The cell density was more than 90% in three HL-60 culture bottles under inverted microscope.
2. In three culture bottles, two parts of 3 ml cell suspension were taken from each bottle and injected into 10 cm culture dish respectively, and 7 ml RPMI-1640 (10% FBS) was added to each dish. No. 1-6.
3. Under the inverted microscope, the cell density decreased significantly, about 20%.
4. 37 ℃, 5% carbon dioxide incubator.
5. Order 500 ml RPMI-1640.
2020/9/6
HL-60 culture
1. The culture dish was observed in the morning, and the cell density was not high, so it was not passaged. Put them back into the incubator for cultivation.
2020/9/7
Medium preparation & HL-60 subculture
A. Medium preparation
RPMI-1640: 45 ml, FBS: 5 ml, Total volume: 50 ml
Multiple tubes were prepared and packed in 50 ml centrifuge tubes.
B. Subculture of HL-60
1. The density of No. 1-6 culture dishes was more than 90%.
2. After blowing each dish, take 5 ml cell suspension and add it into a new culture dish numbered X-1 (x is the original number), and add 5 ml medium to each dish.
3. The cell density of the old and new culture dishes decreased compared with before, but the cell density of the old one was significantly higher than that of the new one. It is speculated that the reason is that the air mixing is not even enough. Put it into the incubator for cultivation.
2020/9/8
Culture of HL-60 by changing medium
1. Take out 6-7 ml cell suspension from each plate of 12 plates, add 6-7 ml medium, and put them back into the incubator for culture
2020/9/9
HL-60 was cultured by changing medium and the acquisition of THP1
1. HL-60 liquid change.
2. The THP1 cell line was obtained from the Preservation Center and cultured in the incubator.
2020/9/10
HL-60 changing medium and THP1 subculture
1. When HL-60 was cultured in different medium, it was found that there were small particles around the cells, which may be the fragments left by cell death, but the risk of contamination was not ruled out.
25/9/2020
2. THP1 subculture: the cell suspension (about 9 ml) in the culture bottle was divided into 3 times, 3 ml suspension was taken each time to a new 10 cm cell culture dish, and 7 ml RPMI-1640 (10% FBS) was added to each culture flask, and then cultured in the incubator, labeled as plates 1, 2 and 3.
2020/9/11
HL-60 changing medium and subculture of THP1
1. After changing the solution of HL-60, it was found that the particles still existed and the cell morphology was slightly irregular.
2. THP1 subculture: take 2.5 ml cell suspension from each dish into a new dish, and add 7.5 ml medium to each dish, labeled as 1-1, 1-2, 1-3, 1-4, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-3, 3-4. Put it into the incubator for cultivation.
2020/9/12
Differentiation induction of HL-60
A. Preparation of cell suspension
1. Select the culture dish 1, 2, 3, 2-1, 4-1, 6-1 of HL-60 cell line, and draw 50 ml cell suspension (packed in two 50 ml centrifuge tubes, 25 ml in each tube).
2. 1 ml cell suspension was taken out from each centrifuge tube and counted by cell counting plate and trypan blue exclusion method to estimate cell density.
3. Take 10 ml suspension from each centrifuge tube to two 10 ml centrifuge tubes, and take 1 ml cell suspension from each tube to two new 10 ml centrifuge tubes, and add 9 ml culture medium.
4. Repeat operation 3 to make the final cell concentration reach about 5.5e6/ml.
B. Preparation of differentiation inducible medium
RPMI-16401: 45 ml, FBS: 15 ml, DMSO: 1.9 ml
Total volume: 150 ml
C. Establishment of differentiation inducible culture system
1. Take out 1 ml diluted cell suspension and inject it into a new 10 cm culture dish, and add 6.5 ml medium with differentiation induction.
2. Repeat operation 1 until all cell suspensions are used up. 20 culture plates, labeled 1-20, are obtained and cultured in the incubator.
2020/9/13-2020/9/16
Replacement of HL-60 and THP1
2020/9/17
Chemotaxis of CXCL1 to THP1, a preliminary experiment
A. Preparation of concentration gradient of CXCL1 solution
B. Cell suspension preparation
C. Transwell Migration Assay
Sampling sequence: the first row: Control1, control2, control3, 10nM-1, 10nM-2, 10nM-3
The second row: 1nM-1, 1nM-2, 1nM-3, 0.1nM-1, 0.1nM-2, 0.1nM-3
2. Put into the incubator (37 ℃, 5% carbon dioxide) for 15 h (overnight)
D. Result count
Results: it was found that the number of cells in each counting room was almost less than 10, and the data was not statistically valid. It was considered to select a higher concentration of chemokines, a longer culture time and more cells in the formal experiment, and use flow cytometry to count cells.
2020/9/21-2020/9/24
Changing medium of HL-60 and THP1
1. It was found that all THP1 cell suspensions except one dish were turbid and pinkish white. The growth of THP1 cells was poor by microscopic examination, which was determined as fungal contamination.
2. Cryopreserved cells: the only one dish of THP1 in good condition
2020/9/27
HL-60 cells were cultured and passaged
1. Fungal contamination was also found in HL-60 cells
2. Cryopreserved cells: HL-60 in good condition
3. Order a new batch of HL-60 and THP1 cells from the Preservation Centre
2020/10/1
Passage culture of THP1 cells
1. THP1 cell line, about 7 ml cell suspension, was obtained from the Preservation Center
2. Subculture: 3 ml and 4 ml cell suspensions were respectively injected into two 10 cm culture dishes, and each dish was supplemented with medium to 15 ml
3. Prepare 40 ml medium with antibiotics
RPMI-1640: 36 ml, FBS: 4 ml, Double antibody: 0.4 ml
2020/10/2
Passage culture of THP1 cells
1. Observation found that one dish of cells in poor condition, suspected of contamination, so abandoned
2. Another dish of cells was introduced into 5 new culture dishes (2 ml cell suspension was left in each dish, and the medium was added to 17 ml)
2020/10/3
Observation of THP1 cell culture
1. No contamination was found, but the cell growth was slow, so the solution was not changed.
2020/10/4
Observation of THP1 cell culture
1. No pollution was found, but a large number of cells agglomerated in the shape of mulberry, and the agglomerations decreased significantly after blowing and mixing.
2020/10/6
Observation of THP1 cell culture
1. Individual clusters of THP1 became larger, and the cell density was still not high after blowing and dispersing
2020/10/7
Observation of THP1 cell culture & acquisition of Escherichia coli K-12 MG1655
1. The density of THP1 increased by visual inspection, but it was not enough for Transwell migration experiment
2. One strain of E. coli K-12 MG1655 was obtained from Zhixiong Xie's laboratory and cultured overnight in 37 ℃ incubator
2020/10/8
Exchange of THP1 cells & cryopreservation of Escherichia coli K-12 MG1655
1. It was found that the density of THP1 in one dish was enough for Transwell migration experiment
2. All THP1 culture dishes were changed
3. The Escherichia coli K-12 MG1655 plate was stored in 4 ℃ refrigerator
2020/10/9
Chemotaxis of THP1 by chemokines (formal experiment) 1
A. Preparation of chemokine mother liquor
Volume of sterile water: 6.33 μl (CXCL1, cxcl2, cxcl3), 5.95 μl (CXCL8), 5.75 μl (CCL2), 6.41 μl (CCL3), 5.62 μl (CCL8).
The mother liquor of 1E5 nmol / L of seven chemokines was obtained
B. Cell suspension was prepared
C. Transwell Migration Assay
Sampling order:
First board: first row: 1Control 1, 1Control 2, 1Control 3, cxcl1-1, cxcl1-2, cxcl1-3
The second row: 1Control 3, 1Control 4, 1Control 5, cxcl2-1, cxcl2-2, cxcl2-3
Starting time: 10:12
The second board: the first row: 2control 1, 2control 2, 2control 3, cxcl3-1, cxcl3-2, cxcl3-3
The second row: 2control 4, 2control 5, 2control 6, cxcl8-1, cxcl8-2, cxcl8-3
Starting time: 10:42
The third board: the first row: 3control 1, 3control 2, 3control 3, ccl2-1, ccl2-2, ccl2-3
The second row: ccl3-1, ccl3-2, ccl3-3, ccl8-1, ccl8-2, ccl8-3
Starting time: 10:24
2. Put it into the incubator for overnight cultivation
dHL-60 and HL-60 RNA were extracted
Failure, reason: did not shake well after adding isopropanol
2020/10/10-2020/10/11
Chemotaxis of THP1 by chemokines (formal experiment) 2
Count the number of cells in the lower chambers by flow cytometry to calculate the chemotactic index
The number of cells was recorded with SSc as the horizontal axis and FSC as the longitudinal axis.
2020/10/13
Liquid culture of Escherichia coli K-12 MG1655
1. Select a single colony of E. coli K-12 MG1655 into the test tube containing 5 ml LB medium. 37 ℃, 200 rpm overnight
2020/10/14
MazE gene colony PCR, pET-28a plasmid extraction
A. Colony PCR of MazE gene
1. The experimental group and negative control group were set up. The formula and operation were shown in colony PCR protocol
2.1% agarose gel electrophoresis, see agarose gel electrophoresis protocol
The order of sampling: marker, MazE experimental group, MazE negative control group
Results: in the experimental group, there were obvious target bands between 200 bp and 300 bp, and there were obvious primer dimer bands in both groups
B. Extraction of pET-28a plasmid
A total of 100 μl plasmids were extracted from two tubes of bacterial liquid (1.5 ml each tube) with TIANprep Mini Plasmid Kit, Tiangen (DP103)and then refrigerated in 4 ℃
2020/10/15
Colony PCR of PemI and HipB genes
1. The bacterial solution used on October 14 continued to be cultured overnight, and colony PCR was carried out with the bacterial solution. The two genes were respectively set up in the experimental group and the control group, and the PCR conditions were the same as those in 2020/10/14
Sample order: Marker, P2, P1, P-negative, H, H-negative
Results: there was no target band and there was a very obvious primer dimer band
Analysis: the primer design is not ideal, the annealing temperature is not suitable, and the concentration of bacterial solution is too high
2. The bacterial liquid was stored at 4 ℃
2020/10/16
Colony PCR of PemI and HipB genes
1. 10.15 refrigerated bacterial solution was used for colony PCR. PemI was divided into P1 (annealing temperature 61 ℃), P2 (annealing temperature 57 ℃) and negative control, and HipB was divided into H (annealing temperature 57 ℃) and negative control. The other PCR conditions were the same as 10.14
Results: there was no target band, and there were obvious primer dimer bands in all groups, but it was weaker than that of 10.15
2. Another E. coli K-12 MG1655 colonies were cultured overnight at 37 ℃ at 200 rpm in a tube containing 5 ml LB medium
2020/10/17
PemI and HipB colony PCR & pET-28a identification by electrophoresis
1. 10.16 inoculated bacterial solution was used for colony PCR. The annealing temperature of PemI group was 61 ℃ and that of HipB group was 57 ℃. The other PCR conditions were the same as those of 10.14
2.10.14 the two tubes of pET-28a were identified by electrophoresis, and a band between 3KB and 4KB was obtained, which was supposed to be the super helix pET-28a
2020/10/18
PemI and HipB electrophoresis, MazF colony PCR and pET-28a single restriction digest
1. PemI and HipB electrophoresis is conducted, and no target bands was identified
2. MazF colony PCR, annealing temperature was 46℃, other conditions are the same as 10.15
3. pET-28a single restriction digest by NotI enzyme for 12 hours
2020/10/19
MazE PCR and MazF, PemK, HipA colony PCR
1. MazE PCR was conducted again with another pair of primers to add the homologous arm at the 3’ end. The annealing temperature was 58.0℃ and the extension time was 1 min 20 s. Other PCR conditions are the same as 10.15
2. MazF, PemK and HipA colony PCR were conducted. The annealing temperature for MazF was 60.9℃, for PemK was 53℃ and for HipA was 53.9℃
Result: No target bands were detected.
9.11 preparation of culture media (LB, LA, 2x YTPG)
9.15 3A Assembly of plasmids (transcription factors overexpression-T, las activator-L, rhl activator-R) and transformation
9.16 single colonies observed in the Ampr plates
9.17 colony PCR, failed to see any right band
9.18 ligation of remaining digested fragments, colony PCR (failed), and another round of digestion
9.19 single colonies observed in the Ampr plates
9.20 colony PCR, failed to see any right band; ligation of digested fragments, transformation
9.22 preparation of culture media (LB, LA); failed to detect any colony
9.24 PCR amplification of fragments using primer pairs synthesized by Sangon Biotech, failed
9.26 massive digestion of plasmids and fragments, failed to detect any band of fragments in the agarose gel
9.29 PCR amplification of fragments using redesigned primer pairs, successful; ligation using Hieff Clone® Plus Multi One Step Cloning Kit and transformation
9.30 colony PCR, positive for colonies T1, T2, T5, C1-C6, negative for colonies L and R
10.1 plasmid extraction of C; PCR amplification of fragments, ligation and transformation of plasmids L and R
10.2 plasmid extraction of T1 and T5; colony PCR, positive for colonies L6, L7 and R4
10.3 plasmid extraction of T5, L6, L7 and R4
10.10 plasmid F (fluorescence reporter) construction using previously described seamless assembly method
10.11 colony PCR, positive for F2 and F7; preparation of mother solution for CFPS premix
10.12 plasmid extraction of F2, F7, L6, R4; cell-free assays using commercial kit E. coli S30 Extract System for Circular DNA (Promega)
10.13 plasmid extraction of C, R4, F2
10.15 transformation of plasmid T to E. coli BL21 (DE3); preparation of S30 buffer
10.16 recombinant BL21 (DE3) single colonies picked for inoculation; preparation of CFPS premix
10.17 E. coli lysates preparation using sonication
10.18 cell-free assays using self-made E. coli lysates pre-enriched with transcription factors (LasR, RhlR); SDS-PAGE
10.19 plasmid samples (T, L, R, F, C) and pSB1A3 sequencing primers sent to Sangon Biotech; later verification of inserted genes by sequencing
