Team:NKU China/Notebook
Laboratory Notes
☞☟ Week1 (May 16–May 22)
In order to make sure the efficiency of our "consumer", we should first knock out the luxS gene in our engineering bacteria GR286(a simplified strain of Bacillus amyloliquefaciens LL3). We used a markerless gene replacement method to knock out the luxS gene.
Construction of targeting vector : the upstream and downstream of luxS gene were combined by over-lapping PCR and ligated into plasmid pKSU.
pKSU-ΔluxS was transformed into GR286, and positive clones were selected.
| 2× Taq Master Mix | 10μL |
| pKSU-F | 1μL |
| pKSU-R | 1μL |
| Bacterium solution | 1μL |
| ddH2O | 7μL |
| Total | 20μL |
| 94oC | 10 min | |
| 94oC | 30 sec | |
| 58oC | 30 sec | 30 cycles |
| 72oC | 1 min 30 sec | |
| 72oC | 10 min | |
| 16oC | ∞ |
Selection of positive clones by PCR
The transformants were cultured at 42oC with chloramphenicol to select single-crossover clones.
| 2× Taq Master Mix | 10μL |
| luxS-up-F | 1μL |
| luxS-dn-R | 1μL |
| Bacterium solution | 1μL |
| ddH2O | 7μL |
| Total | 20μL |
| 94oC | 10 min | |
| 94oC | 30 sec | |
| 56oC | 30 sec | 30 cycles |
| 72oC | 2 min | |
| 72oC | 10 min | |
| 16oC | ∞ |
Selection of single-crossover clones by PCR
(No.1-4 are single-crossover strains, No.5 is positive control.)
☞☟ Week2 (May 23–May 29)
The single-crossover strains were then cultured in LB medium and passaged every 12 hours for 4 generations.
The last generation was cultured in medium with 5-fluorouracil to select double-crossover clones. Regretfully, we didn't get the double-crossover clones.
| 2× Taq Master Mix | 10μL |
| luxS-up-F | 1μL |
| luxS-dn-R | 1μL |
| Bacterium solution | 1μL |
| ddH2O | 7μL |
| Total | 20μL |
| 94oC | 10 min | |
| 94oC | 30 sec | |
| 56oC | 30 sec | 30 cycles |
| 72oC | 2 min | |
| 72oC | 10 min | |
| 16oC | ∞ |
Selection of double-crossover clones by PCR
(No.1-5 are experimental groups, No.6 is wild GR286. The result showed that we failed to get the double-crossover clones.)
☞☟ Week3 (May 30–Jun 05)
Transformants were cultured at 42oC with chloramphenicol again and the single-crossover clones were selected successfully.
| 2× Taq Master Mix | 10μL |
| luxS-up-F | 1μL |
| luxS-dn-R | 1μL |
| Bacterium solution | 1μL |
| ddH2O | 7μL |
| Total | 20μL |
| 94oC | 10 min | |
| 94oC | 30 sec | |
| 56oC | 30 sec | 30 cycles |
| 72oC | 2 min | |
| 72oC | 10 min | |
| 16oC | ∞ |
Selection of single-crossover clones by PCR
(No.1&2&4 are single-crossover strains,No.5 is positive control. )
The single-crossover strains were then cultured in LB medium and passaged every 12 hours for 4 generations.
The last generation was cultured in medium with 5-fluorouracil to select double-crossover clones. We finally obtained our aimed strain—GR286ΔluxS.
| 2× Taq Master Mix | 10μL |
| luxS-up-F | 1μL |
| luxS-dn-R | 1μL |
| Bacterium solution | 1μL |
| ddH2O | 7μL |
| Total | 20μL |
| 94oC | 10 min | |
| 94oC | 30 sec | |
| 56oC | 30 sec | 30 cycles |
| 72oC | 2 min | |
| 72oC | 10 min | |
| 16oC | ∞ |
Selection of ΔluxS clones by PCR
(The No.4 is the aimed strain―GR286ΔluxS)
☞☟ Week4 (Jun 06–Jun 12)
The GR286ΔluxS strain was cultured and made competent for future use.
The lsrACDB gene from Bacillus thuringiensis was cloned and ligated to T-vector.
| 2× Taq Master Mix | 25μL |
| lsrACDB-F | 2μL |
| lsrACDB-R | 2μL |
| Bacterium solution | 2μL |
| ddH2O | 19μL |
| Total | 50μL |
| 94oC | 10 min | |
| 94oC | 30 sec | |
| 57oC | 30 sec | 30 cycles |
| 72oC | 4 min 30 sec | |
| 72oC | 10 min | |
| 16oC | ∞ |
| 10× DNA Ligase Buffer | 2μL |
| T4 DNA Ligase | 1μL |
| pMD19 T-Simple Vector | 1μL |
| lsrACDB | 3μL |
| ddH2O | 13μL |
| Total | 20μL |
| Reaction condition: 16oC overnight | |
The T-lsrACDB was transformed into DH5α and plate was coated, and then positive clones were selected.
| 2× Taq Master Mix | 10μL |
| M13F | 1μL |
| M13R | 1μL |
| Bacterium solution | 1μL |
| ddH2O | 7μL |
| Total | 20μL |
| 94oC | 10 min | |
| 94oC | 30 sec | |
| 59oC | 30 sec | 30 cycles |
| 72oC | 4 min 30 sec | |
| 72oC | 10 min | |
| 16oC | ∞ |
Selection of positive clones by PCR
(No. 3&4 are positive results)
After restriction enzyme digestion verification, the positive clones were then sequenced. Unfortunately, the sequencing result showed some mutations inside the target gene.
| 10× FastDigest Buffer | 2μL |
| BamH Ⅰ | 1μL |
| T-lsrACDB | 1μL |
| ddH2O | 7μL |
| Total | 20μL |
| Reaction condition: 37oC for 40 min | |
Restriction enzyme digestion verification
(No.1 are lsrACDB fragement, No.2 are linearized T-vector.)
The gene cloning process was repeated but there were still some mutations.
We finally decided to request the gene company to synthesize the lsrACDB gene.
☞☟ Week5 (Jun 13–Jun 19)
This week, we started to construct another controller―supplier.
A strong promoter C2 was cloned from former kit and luxS gene was cloned from GR286.
| 2× Taq Master Mix | 25μL |
| C2-F | 2μL |
| C2-R | 2μL |
| p-C2 | 2μL |
| ddH2O | 19μL |
| Total | 50μL |
| 94oC | 10 min | |
| 94oC | 30 sec | |
| 58oC | 15 sec | 30 cycles |
| 72oC | 30 sec | |
| 72oC | 10 min | |
| 16oC | ∞ |
| 2× Taq Master Mix | 25μL |
| luxS-F | 2μL |
| luxS-R | 2μL |
| Bacterium solution | 1μL |
| GR286 | 2μL |
| ddH2O | 19μL |
| Total | 50μL |
| 94oC | 10 min | |
| 94oC | 30 sec | |
| 59oC | 30 sec | 30 cycles |
| 72oC | 30 sec | |
| 72oC | 10 min | |
| 16oC | ∞ |
PCR cloning product of promoter C2 and gene luxS
(NO.1&2 are C2, No.3&4 are luxS)
Two segments were fused together by fusion PCR and ligated into T-vector. After that, the vector was transformed into DH5α.
| 2× Taq Master Mix | 25μL |
| C2-F | 2μL |
| luxS-R | 2μL |
| C2 | 2μL |
| luxS | 2μL |
| ddH2O | 17μL |
| Total | 50μL |
| 94oC | 10 min | |
| 94oC | 30 sec | |
| 59oC | 30 sec | 30 cycles |
| 72oC | 40 sec | |
| 72oC | 10 min | |
| 16oC | ∞ |
| 10× DNA Ligase Buffer | 2μL |
| T4 DNA Ligase | 1μL |
| pMD19 T-Simple Vector | 1μL |
| C2-luxS | 4μL |
| ddH2O | 12μL |
| Total | 20μL |
| Reaction condition: 16oC overnight | |
Positive clones were selected by colony PCR.
| 2× Taq Master Mix | 10μL |
| M13-F | 1μL |
| M13-R | 1μL |
| Bacterium solution | 1μL |
| ddH2O | 7μL |
| Total | 20μL |
| 94oC | 10 min | |
| 94oC | 30 sec | |
| 59oC | 30 sec | 30 cycles |
| 72oC | 40 sec | |
| 72oC | 10 min | |
| 16oC | ∞ |
PCR cloning product of promoter C2 and gene luxS
4 positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.
| 10× FastDigest Buffer | 2μL |
| BamH Ⅰ | 1μL |
| T-lsrACDB | 1μL |
| ddH2O | 7μL |
| Total | 20μL |
| Reaction condition: 37oC for 40 min | |
Restriction enzyme digestion verification
(No.1 are linearized T-vector, No.2 are C2-luxS fragment.)
☞☟ Week6 (Jun 20–Jun 26)
The sequencing result showed that there was a correct strain and thus it could be used for the following experiments. We obtained the correct plasmid T-C2-luxS from DH5α. Then the fragment C2-luxS was obtained by digestion and gel extraction.
| 10× FastDigest Buffer | 4μL |
| BamH Ⅰ | 2μL |
| T-C2-luxS | 25μL |
| ddH2O | 9μL |
| Total | 20μL |
| Reaction condition: 37oC for 40 min | |
The C2-luxS fragment was ligated to linearized plasmid pWH1520, and then the ligation product was transformed into DH5α.
| 10× DNA Ligase Buffer | 2μL |
| T4 DNA Ligase | 1μL |
| pMD19 T-Simple Vector | 1μL |
| C2-luxS | 5μL |
| ddH2O | 11μL |
| Total | 20μL |
| Reaction condition: 16oC overnight | |
The plasmid pWH-C2-luxS was extracted from DH5α. To prevent the plasmid from DAM&DCM methylation, we transformed it into E.coli JM110.
The plasmid pWH-C2-luxS was extracted from JM110,and then it was treated with BamH Ⅰ methylase.
| 10× BamH Ⅰ methyltransferase Buffer | 10μL |
| BamH Ⅰ methyltransferase | 1μL |
| S-adenosylmethionine | 0.5μL |
| pWH-C2-luxS | 80μL |
| ddH2O | 8.5μL |
| Total | 100μL |
| Reaction condition: 37oC for 1 hour | |
The plasmid was transformed into GR286 by electroporation.[Failed]
Selection of positive clones by colony PCR
(No.1 is positive control, No.2-6 are experimental groups. The result showed that we failed to transformed the plasmid pWH-C2-luxS into GR286)
☞☟ Week7 (Jun 27–Jul 03)
This week, we tried to use different voltages to transform the plasmid. Sadly, all of these attempts rendered negative results.
We considered whether the luxS gene is toxic for GR286, and the bacteria tends to reject the gene when a strong promoter is inserted upstream of it. So, we planned to use inducible promoter to reconstruct our expression vector.
The plasmid pWH1520 contains a strong xylA promoter originating from Bacillus megaterium, and transcription initiated by this promoter is xylose-inducible. Also, the gene of interest carries its own ribosome binding sequence (RBS) and translation initiation codon. Based on these points, we redesigned primers.
☞☟ Week8 (Jul 04–Jul 10)
luxS gene was cloned from GR286 using our new primers.
| 2× Taq Master Mix | 25μL |
| YD-luxS-F | 2μL |
| YD-luxS-R | 2μL |
| Bacterium solution | 2μL |
| ddH2O | 19μL |
| Total | 50μL |
| 94oC | 10 min | |
| 94oC | 30 sec | |
| 58oC | 30 sec | 30 cycles |
| 72oC | 40 sec | |
| 72oC | 10 min | |
| 16oC | ∞ |
PCR cloning product of gene luxS
The luxS fragment was purified by gel extraction, and ligated into linearized pWH1520. Then the vector was transformed into DH5α.
| 10× FastDigest Buffer | 4μL |
| BamH Ⅰ | 2μL |
| pWH1520 | 25μL |
| ddH2O | 9μL |
| Total | 40μL |
| Reaction condition: 37oC for 40 min | |
| 10× DNA Ligase Buffer | 2μL |
| T4 DNA Ligase | 1μL |
| linearized pWH1520 | 1μL |
| luxS | 3μL |
| ddH2O | 13μL |
| Total | 20μL |
| Reaction condition: 16oC overnight | |
Positive clones were selected by colony PCR.
| 2× Taq Master Mix | 10μL |
| pWH-F | 1μL |
| pWH-R | 1μL |
| Bacterium solution | 1μL |
| ddH2O | 7μL |
| Total | 20μL |
| 94oC | 10 min | |
| 94oC | 30 sec | |
| 58oC | 30 sec | 30 cycles |
| 72oC | 40 sec | |
| 72oC | 10 min | |
| 16oC | ∞ |
Selection of positive clones by colony PCR
4 positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.
| 10× FastDigest Buffer | 2μL |
| BamH Ⅰ | 1μL |
| pWH-luxs | 10μL |
| ddH2O | 7μL |
| Total | 20μL |
| Reaction condition: 37oC for 40 min | |
Restriction enzyme digestion verification
(No.1 are linearized pWH1520, No.2 are luxS fragments.)
☞☟ Week9 (Jul 11–Jul 17)
The sequencing result showed there's three positive strains. So one positive strain was chosen to be used for the following experiments. The pWH-luxS plasmid was extracted from the chosen strain. To prevent the plasmid from DAM&DCM methylation, we transformed it into E.coli JM110.
the plasmid pWH-luxS was extracted from JM110,and it was treated withBamH Ⅰ methylase.
| 10× BamH Ⅰ methyltransferase Buffer | 10μL |
| BamH Ⅰ methyltransferase | 1μL |
| S-adenosylmethionine | 0.5μL |
| pWH-C2-luxS | 80μL |
| ddH2O | 8.5μL |
| Total | 100μL |
| Reaction condition: 37oC for 1 hour | |
The plasmid was transformed into GR286 by electroporation, and positive clones were selected.
Selection of positive clones by colony PCR
The construction of supplier was accomplished!
☞☟ Week10 (Jul 18–Jul 24)
We have received the product of synthetic lsrACDB gene. We first used restriction-ligation method to ligate lsrACDB to plasmid pWH1520, but we failed to select positive after several tries.
| 10× DNA Ligase Buffer | 2μL |
| T4 DNA Ligase | 1μL |
| linearized pWH1520 | 1μL |
| lsrACDB | 3μL |
| ddH2O | 13μL |
| Total | 20μL |
| Reaction condition: 16oC overnight | |
Considering that the lsrACDB gene is a large fragment (4500bp), we used ClonExpress technique to clone the gene again to improve the efficiency of ligation. The lsrACDB sequence was divided into two parts and they were cloned separately. Then the two segments were ligated to the plasmid pWH1520 and the recombinant vector was transformed into DH5α. After that, verification PCR was used to select the positive clones. However, we didn't get a good result.
Selection of positive clones by colony PCR
(No.6 is positive control, No.1-5 are experimental groups)
☞☟ Week11 (Jul 25–Jul 31)
We learnt a new method called circular polymerase extension cloning (CPEC) for high-throughput cloning of complex and combinatorial DNA libraries, and we decided to use this method to try to ligate our lsrACDB gene. It's encouraging that we succeeded to ligate the lsrACDB gene to the plasmid pHT-01.
Selection of positive clones by colony PCR
(No.3-6 are positive clones)
Restriction enzyme digestion verification</div>
(No.2&3 are positive results.)
☞☟ Week12 (Aug 1–Aug 7)
Since we have already successfully constructed "supplier" and part of "consumer", we decided to measure the growth curve to explore the function of our "controller".
| GR286 | wild strain as control group |
| GR286ΔluxS | GR286 without luxS gene |
| pWH-luxS | luxS overexpression plasmid in GR286; without induced by xylose |
| pWH-luxS + xyl | luxS overexpression plasmid in GR286; induced by xylose |
| pWH1520 | empty plasmid in GR286 as control group |
| pHT-lsrACDB | lsrACDB overexpression plasmid in GR286ΔluxS |
| pHT-01 | empty plasmid in GR286ΔluxS as control group |
OD600 of different groups at specific times
Growth curve of GR286 and GR286ΔluxS
Growth curve of pWH-luxS, pWH-luxS + xyl and pWH1520
Growth curve of pHT-lsrACDB and pHT-01
Cultured media of our supplier was tested for the presence of AI-2 by inducing luminescence of Vibrio harveyi reporter strain BB170.
Fluorescence intensity of different groups at specific times
Relative fluorescence intensity of different groups
☞☟ Week13 (Aug 8–Aug 14)
For our consumer, we should also overexpress the lsrK and lsrFG gene for phosphorylating and degrading phosphorylated AI-2. We used ClonExpress technique to clone the two genes and ligate them to plasmid pHT-01 successfully.
Restriction enzyme digestion verification
(No.1&3&4 are positive results. )
We co-cultured the supplier with BB170 and tested the fluorescence intensity to explore the function of supplier. (negative result)
Fluorescence intensity of BB170 and co-culture medium
☞☟ Week14 (Aug 15–Aug 21)
