Team:BIT-China/Results
Killer device
Since the lethal efficiency of killer genes will decide the capacity of general circuit, so we have to:
1. Prove that the toxin protein we selected can successfully express and the
lethal effect is obvious
2. Adjust the translation efficiency of toxin proteins through replace ribosome binding site (RBS), thus to adjust the threshold
3. Construct gene circuits connecting killer device and inhibitor device
4. Find another possible way to produce the lethal effect
2. Adjust the translation efficiency of toxin proteins through replace ribosome binding site (RBS), thus to adjust the threshold
3. Construct gene circuits connecting killer device and inhibitor device
4. Find another possible way to produce the lethal effect
Results:
1. Successfully constructed the testing circuits of toxin proteins [link to parts]- AraC+PBAD+B0032+mazF, AraC+PBAD+B0032+hokD
2. After transformation, we measured the OD600 to draw the growth curve and observed the function of toxin protein.
3. In addition to B0032, we selected a stronger RBS B0034 and a weaker one B0031. Through measuring OD600, we demonstrated that we can adjust the lethal efficiency through replacing RBS.
4. Successfully constructed the testing circuit of toxin proteins according to two kinds of inhibitors. [link to parts]
5. Tested the lethal effect of producing DSB through coupling sgRNA and Cas9
2. After transformation, we measured the OD600 to draw the growth curve and observed the function of toxin protein.
3. In addition to B0032, we selected a stronger RBS B0034 and a weaker one B0031. Through measuring OD600, we demonstrated that we can adjust the lethal efficiency through replacing RBS.
4. Successfully constructed the testing circuit of toxin proteins according to two kinds of inhibitors. [link to parts]
5. Tested the lethal effect of producing DSB through coupling sgRNA and Cas9
1. Constructing the testing circuits
The DNA Agarose Gel electrophoresis showed that the whole length of the first circuit is about 1500bp, the second circuit is about 1400bp.
Fig.1 DNA Agarose Gel electrophoresis results of mazF
Fig.2 DNA Agarose Gel electrophoresis results of hokD
2. Testing the lethal effect of toxin proteins MazF and HokD by measuring the growth curve.
The testing groups are the two circuits above (mazF and hokD), while the control is the empty vector. Add arabinose or not, there is another comparison. We used 0.01% arabinose to induce the PBAD promoter when the OD600 is 0.6. We measured the OD600 every hour until the bacteria reached the flat stage.
Fig.3 Compared with the negative control (the empty pSB1C3 vector), OD600 of the two circuits containing toxin proteins are obviously lower, and the difference is evident as time going on. No obvious difference observed among the three groups with no induction. It showed that toxin proteins didn’t leak out.
Through the growth curve, we concluded that toxin protein HokD and MazF have different lethal efficiency. Depending on different situations, we can choose either of them.
3. Replacement of the RBS to adjust the lethal effect as well as the threshold
Through one-step mutation, we have separately replaced the B0032 (33.96%) with B0031 (12.64%) and B0034 (100%). Here are the sequencing results.
Fig.4 The successful sequencing results of 4 mutations: B0031+mazF; B0034+mazF; B0031+hokD; B0034+hokD
We used 0.01% arabinose to induce the PBAD promoter when the OD600 is 0.6. We measured the OD600 every hour until the bacteria reached the flat stage.
Fig.5 For the toxin protein MazF, we concluded that the influence of strong RBS is more evident.
Fig.6 For the toxin protein HokD, the replacement of RBS will influence the expression of hokD. The weaker RBS B0031 leads to lower expression of toxin protein HokD.
We tested the lethal efficiency of HokD and MazF under different RBS. Compared with the positive control (pSB1C3 empty vector), the three experimental groups showed obvious difference. For MazF, the strong RBS B0034 was proved to have highest lethal efficiency. For HokD, the weak RBS B0031 was proved to have the lowest lethal efficiency.
4. sgRNA targeting genome of strains with no NHEJ repair system
To test the effects of Cas9 and targeting sgRNA, we separately constructed two plasmids containing sgRNA and Cas9 protein. We co-transformed the two plasmids into E.coli TOP10, and added 10mM arabinose to induce the expression of Cas9. In order to validate the effect of Cas9 and sgRNA, we did quantitative analysis of colony formation after induction to confirm CRISPR/Cas9-Mediated DSB in E.coli TOP10.
Fig.7 experimental procedure
The cells were diluted 102–104 -fold and plated onto agar plates containing AmpR and CmR. The results showed that the control group with no induction was growing better than the testing group when both of them were diluted 103 -fold, which means the DSB did have the lethal effect and can be used as a candidate for killer device.
Fig.8 Confirmation of CRISPR/Cas9-Mediated DSB in E.coli TOP10
Recombination
Aim
Considering the factor of the potential deficiency of executor killer due to the completely lost of plasmids and the number of "in-promoters" will affect our system, we decide to integrate the killer device into the genome. This group mainly provided a tool for genome integration. The insertion fragment is our testing device, including the expression cassette of rfp and killer gene. With this recombination system, we can get the upgraded type of our P-SLACKiller.
Results
1. Coupled CRISPR/Cas9 system with λ-Red recombineering to integrate the donor fragments, but we haven’t finished the second plasmid construction
2. Tried the traditional lambda Red recombination and successfully inserted four testing devices into the genome, locus of LacI.
2. Tried the traditional lambda Red recombination and successfully inserted four testing devices into the genome, locus of LacI.
Coupling CRISPR/Cas9 system with λ-Red recombineering
1. Plasmid construction
We designed a two-plasmid system applied for it. There was a low-copy plasmid pSTV29 for Cas9 and λ-Red expression from the arabinose-inducible promoter PBAD respectively and a high-copy plasmid pUC19 for sgRNA expression from a constitutive promoter J23119.
Fig.1 Double plasmids to meet different requirements for each element. Equip the high copy number plasmid pUC19 with sgRNA targeting site on genome and donor fragment with homologous arms. The low copy number plasmid are equipped with expression cassette of Cas9 protein and λ-Red recombinase
We mainly used
Gibson assembly
to construct these two plasmids. The pUC19 plasmid containing sgRNA and donor fragment was successfully constructed. Separately, we successfully constructed the lambda Red protein and Cas9 protein on pSTV29.
However, we didn’t finish the second plasmid construction due to the limit of time. Instead, we decided to employ the traditional lambda Red recombination system to carry out the integration process since we received the plasmid pKD46 from Dr. Bo Lv.
Lambda red recombination
For facile blue and white colony screening, the target site we chose is the 5 ’end of LacZ gene encoding the active site of β-galactosidase. At first, we used strain TOP10 as our host. To test the experiment conditions which is optimal for transformation, we did the pre-experiment and used the resistance Kana gene as the donor fragment to target the gene locus of LacZ. We have tried several times, but failed.
Considering that the complementary sequence between donor fragment and target site is one of the most important factors deciding the successful rate of recombination, we re-designed our experiment. The whole sequence of TOP10 is not open access and the specific sequence for LacZ has been modified and we couldn’t confirm the modified one. So we decided to replace the host to DH5α.
1. Transformed the plasmid pKD46 into host and made it competent cell after induced by L-arabinose
We transformed the pKD46 into competent cell DH5α, and added arabinose to induce the expression of recombinase. When the OD600 is about 0.6 which means the bacteria is at the log phase, we made it competent for electroporation transformation.
2. Preparation of donor fragment
We designed homologous arms with the length of 50bp and 500 bp. The short one was designed on the primer, while the long arm was constructed through over-lap extension PCR
(OE-PCR).
After several times of try-error, we confirmed the comparatively optimal condition for lambda Red recombination. The target site is LacI and the host is DH5α. The homologous arm of 50 bp is enough for recombination.
Using the pKD3 as template, we added the cat expression cassette behind the testing parts, the whole donor fragment was sandwiched by 50bp left and right homologous arm.
3. Transformed the donor fragment through electroporation into the competent cell we prepared before
After transformation, we observed clones on the chloramphenicol plates. We selected the positives clones through colony PCR.
According to our design, the donor fragment is about 2000 bp, while the substituted fragment on genome is 1083bp, which means the successful integration will result in over 1000 bp increasing of length on the locus of LacI. We used the verifying primers to test whether we have successfully integrated these donor fragments.
Fig.5 The positive clones are 1~7, separately indicating the testing circuits above, and 8 is the negative control whose template is the original genome without any integration.
Compared with the negative control with no integration occurred, the colony PCR showed that we have successfully integrated the four circuits into the LacI locus of genome.
Site-directed promoter mutation
We have two combinations of inhibitor and "in-promoter": TetR-PTet, CI-PR. The strength of "in-promoter" will decide the expression level of killer gene. Directly applied these two combinations, we got the initial threshold. In order to adjust the threshold, we planned to mutate the "in-promoter" and got mutants with various strengths. We chose PTet as our target promoter. Through literature research, we chose the -35 region as our mutation region. We mainly used RFP to indicate the promoter strength in our wet experiment.
Results
1. Tried to use one-step mutation, but because of the secondary structure of promoters, we didn’t get positive results
2. Built a library of mutated PTet promoters and characterized the promoter activity through measuring the red fluorescence intensity
3. Selected 4 mutants and got the successful sequencing results
2. Built a library of mutated PTet promoters and characterized the promoter activity through measuring the red fluorescence intensity
3. Selected 4 mutants and got the successful sequencing results
1. One step mutation--plan A
At first, we used “Quickchange”, using the methylated plasmid as a template, which was extracted from E.coli, and we designed a pair of 25pb primers containing the desired mutation to amplify the DNA through PCR, and we got the demethylated plasmids. Finally, we treated the products with Dpn1, which was specific for methylated DNA and was used to digest the parental DNA template and selected for mutation-containing synthesized DNA. And we used the method of thermal stimulation to transform it into E.coli.
But we didn’t we get the positive clone of the plasmid. And we thought as a result of the characteristics of the promoter structure, viewing it from the secondary structure, there appears a cervical-loop structure. Also a mutation site just appears in the loop, which results in primers having a comparatively long homologous region. So the complementary pairing occurs between upstream and downstream primer, forming primer dimer, which lead PCR amplification to failure.
2. Digestion and ligation—plan B
To avoid forming the cervical-loop structure, we gave up the one-step mutation approach. Instead, we designed the mutation on the primers and planned to construct the testing circuit through the traditional digestion and ligation.
We designed random primers containing NNNNNN in -35 region. Through PCR amplification, we purified the PCR product. We used DpnI to digest the template. Constructed the fragment on the vector pSB1C3, we transformed the ligation product into E.coli TOP10. After incubation at 37℃for 16h, we observed the formation of single clone.
Through colony PCR, we selected the clone on the plate and had successfully got the positive clones. To facilitate the characterization of promoter strength, we constructed circuits containing rfp. The whole length is 923bp, counting the region between VF and VR, the positive length should be over 1000. Results shown in Fig.1.
Then we selected the positive clones, incubated it at the tube, and measured the RFP intensity. Under the same condition, the fluorescence intensity of original promoter was 5000, and the PTet-1, PTet-2 and PTet-3 were about 200, while the selected No.25 was about 500.
Fig.2 RFP intensity measurement results. The original PTet has highest activity, while the strength of mutant PTet-25 is about 1/10 of the original one. Other mutants has considerably low activity.
Compared with the original promoter, the promoter activity after mutation was sharply decreased while the difference was obvious between the testing group (data over 200) and the negative control (data below 100). Since we employed PTet to control the expression of toxin proteins, it’s better that we can reduce the background expression as much as possible. The sequencing results confirmed the successful mutation of four mutants.
Fig.3 The sequencing results. Mutation occurred only in -35 region.
Through measuring the RFP intensity, we selected four mutants. They can be used to control the expression of toxin proteins.
