Team:Fudan/Demonstrate
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Demonstrate
Our work mainly focused on three modules: The Acid Resistance Module, the Antimicrobial Peptide Module and the Secretion module. In addition, we have made great effort in optimizing our quorum sensing control system in both experimental and theoretical way.
Acid Resistance Module
We managed to knockout original hns with Red recombinase toolkit as shown in Results. H-NS is an important transcriptional regulator in E. coli which has been proved to be correlated with acid tolerance.
Fig 1. Plate count test of Δhns. A, ∆hns before acidic shock. B, ∆hns after acidic shock. C, WT Nissle 1917 before acidic shock. D, WT Nissle 1917 after acidic shock. All groups are diluted to the same concentration.
Obviously, common ∆hns group does not show remarkable deficiency compared with WT group. But after acidic shock ∆hns group remains good vitality while the count of WT group decreases sharply. The result indicates that hns knockout leads to improvement of Nissle's acid resistance.
Antimicrobial Peptide Module
We ordered DNA synthesis of the gene cluster in two parts and linked it to pST-BSD plasmid, a kind of Bacterial artificial chromosome with single copy. Plasmid pMccB17 is single-copied in its nature state. We also took advantage of this unique requirement to solve regulation incompatible problem in the next module. After transferring into E. coli DH10B we carried out disk diffusion tests on soft agar double-layer and solid culture plates.
Fig.2 One of the representative disk diffusion test plates. N, Nissle 1917. T1, DH10B with MccB17. K20, 20 mg/ml Kanamycin. mcH, sample bacteria from author’s[1] lab.
However, all test groups shows common symbiosis and faint effect of inhibiting E. coli DH10B. Due to the toxicity of MccB17, its concentration in cells is very low and it’s hard to gain precise data without purification. Further measurement is needed.
Secretion module and the QS control system
We constructed two new hybrid promoters and their properties are significant.
luxpR-fus100 ( BBa_K3245001 ) shows higher expression level and it’s still under QS signal control in similar condition to the wild type. This will bring remarkable increase in lacZ expression and lead to better competence of digesting lactose.
luxpR-HS100 ( BBa_K3245009 ) shows lower leakage at the cost of a lower expression level. The latter is acceptable while the former is important in demanding circuits. In our case we applied this improved promoter to link upstream QS regulation with tetR suppression effect downstream. It’s a successful trial since tetR-ptetR system is rather strict and luxpR ( WT ) is unsuitable due to its high leakage which will deactivate ptetR at low bacteria density.
Fig. 3 shows characterization of the promoters.
Fig. 4&5 luxpR-tetR-ptetR regulation test. Left side is the photo of experimental groups. Test tubes from left to right are ptetR-EsfGFP, pST-HS100, p15A-WT and DH10B groups respectively. The plus mark represents for DH10B with ptetR-EsfGFP.
Obviously, leakage of the wild type luxpR caused tetR expression and lead to inhibition of the downstream reporter. Application of luxpR-HS100 with low leakage shows great improvement in function. Finally we adopted a BAC plasmid with single copy to carry luxpR-HS100-tetR controller and the leakage was perfectly solved.
[1] Frederic Collin, Anthony Maxwell. The Microbial Toxin Microcin B17: Prospects for the Development of New Antibacterial Agents. Journal of Microbiology and Biotechnology, YJMBI-66164; No. of pages: 26; 4C.