Team:Fudan/Results
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Results
Stress resistance: hns knockout Nissle 1917
H-NS is a transcriptional regulator which controls 5% gene expression in E. coli, including the glutamate decarboxylases GadA and GadB and the glutamate/GABA antiporter gadC. To knock out hns, we decided to use Red recombinase knock-out system (Fig. 1) and designed part BBa_K3245003. Considering that, from our experience, Nissle 1917 is hard to conduct gene operations, we introduced approximately 500 bp length of homologous sequence flanking to provide higher knock-out affinity.
Fig. 1 structure of part BBa_K3245003.
Figure. 2 bacteria PCR testing gene hns knockout status. Primers are designed on up and down homologous sequences. Successful knockout would yield a 1.3 kb DNA fragment and failure to knockout would yield a 2kb DNA fragment. M: λ-DNA (EcoT14 digest). +: Plasmid containing BBa_K3245003 as template. 1-9: Different testing single colony as template.
After successfully knocking out hns in Nissle 1917, we further tested how this will affect its acid resistance ability. We conducted out experiment according to protocol listed on
Figure. 3 Acidic shock test showing the acid resistance ability of hns-knockout Nissle 1917 strains against wild type Nissle 1917. Top-left: WT Nissle 1917 after acidic shock. Top-right: ∆hns after acidic shock. Bottom-left: WT Nissle 1917 before acidic shock. Bottom-right: ∆hns after acidic shock.
Competitive capability improvement: microcin MccB17
To enable Nissle 1917 stay in patient’s gut for longer period of time, we must improve its competitiveness against symbiotics as well as harmful bacteria. MccB17 displayed antibacterial activity on some strains of E. coli, Enterobacter, Pseudomonas and Shigella, so we chose this part to enhance the competitiveness of Nissle 1917. We ordered DNA synthesis of MccB17 gene cluster in two parts and linked it to pST-BSD plasmid, a kind of Bacterial artificial chromosome with single copy. Notably, plasmid pMccB17 is also single-copied at its nature state.
Figure.4 construction of microcin MccB17 plasmid. oriV is a single-copy replicatior. MccB17 (BBa_K3245010) is divided into 2 segments and was synthesized by Generay. Co. ltd. We linked mcbA-D and McbD-G together and cloned the whole mcbA-G gene cluster into Pst-BSD plasmid. It is noteworthy that fragment pro-A consists of a natural promoter for MccB17.
Figure.6 One of the representative disk diffusion test plates. N, Nissle 1917 with MccB17. T1, DH10B with MccB17. K20, 20 mg/ml Kanamycin. mcH, sample bacteria that expresses MccB17 from author’s lab in which this part was first characterized.
However, all test groups show common symbiosis and faint effect of inhibiting E. coli DH10B. Due to the toxicity of MccB17, its concentration in cell is very low and it’s hard to gain precise data without purification. Further measurement is needed.
Quorum sensing module construction
As we have mentioned in our project description, we wanted our quorum sensing luxpR-tetR-ptetR expressing system fulfill the function of activating protection protein expression when the flora scale is small and shut off the expression of these proteins when flora scale goes high. Unfortunately, our model proved that if we stick on using wild type luxpR, our system will fail to activate ptetR as there would be too much tetR in cells even when luxpR is at its leakage state. In order to reduce the leakage intensity of luxpR so that it can initiate transcription after luxI-R dimer reaches the threshold we need, we remolded luxpR to form luxpR(HS100) and luxpR(fus100) promoter.
Figure.7 Quorum sensing test of 4 different promoters. Testing methods can be found in notebook-protocols. HS100: BBa_K3245009. 10023: BBa_K3245001. HS: BBa_K2558001. luxpR: BBa_R0062.
With the help of improved quorum sensing promoter, we successfully solved the problem of starting our system.
Though we have lowered the leakage of luxpR, we didn’t stop on what we had achieved. In order to further diminish the tetR level in whe whole system when luxpR is closed, we constructed single-copy plasmid, coupled with B0033, an RBS with only 1% percent the strength of B0034, to achieve minimum leakage.
Figure.8 Construction of plasmid pST-kanR-luxpR(HS100)-tetR to show an example of tetR expression system we use in our project.
The next question that we cared about is to ensure that our system can close once luxpR is triggered. Since we were using a super-weak expression system, we would have to check whether our system could close ptetR or not. Satisfyingly, due to the ultra-strict property of ptetR and relatively high expression efficiency of luxpR(HS100), we could discover conspicuous expression strength reduction on ptetR.
