Team:Fudan/Design
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Design
Overview
We used a substrain of E. coli called Nissle 1917 as our chassis and added several modules to it so that it could fulfill our goal to solve lactose intolerance stably for a long time.
The first is the Acid Resistance Module. In order to make our bacteria survival as they pass through the highly acidic environment of the stomach, we focused on a gene called H-NS. We improved the acid resistance system by knocking out the key gene H-NS in the acid resistance system 2 (AR 2) of Nissle 19171 so that the acid resistance ability of Nissle 1917 was significantly improved and it wouldn’t undergo huge loss when passing through gastric acid.
The second is the Antimicrobial Peptide Module. We adopted microcin B17 (Mcc B17) to inhibit the reproduction of other flora moderately, which improved the competitiveness of Nissle 1917 by secretion of the antibacterial peptide. And it could multiply to a certain number in despite of the intense competition with gut flora.
The third is the Secretion module. In order to make the lactose break down more efficiently, we chose the latter between increasing its activity and expression. The fusion of luxpR and high-intensity promoter J23100 significantly increased lacZ gene’s expression.
We use Quorum Sensing System (QS system) to control our separate modules. When our engineering bacteria reach the stomach, they can survive because of the Acid Resistance Module. When they are in the intestine, the Antimicrobial Peptide Module works so that our bacteria have advantage and multiply rapidly. When the population reaches a certain level to trigger the QS system, the Antimicrobial Peptide Module is turned off and the secretion of lactase is started. In this way, we reduce the burden on the bacteria, allowing them to multiply before secreting lactase. And when the quantity is not enough, they can start reproduction again, repeating the previous cycle to make their population remain stable.
Acid Resistance Module
H-NS is one of the seven global regulators and it is able to influence the expression of 5% of the genes in E.coli. It has been proved that we can improve the acid resistance of bacteria significantly by knocking out H-NS. H-NS acts almost exclusively as a repressor and it binds to about 250 loci. The lack of H-NS might lead to the decrease in the inhibition level of the expression of key proteins in acid resistant system 2 so that the acid resistance system 2 is enhanced. And the strain lacking H-NS bears a high survival rate after the acid shock compared with the wild type.2
Antimicrobial Peptide Module
In our system, we adopt microcin B17 ( Mcc B17 ), which targets DNA gyrase in bacteria. It contains an operon encoding seven proteins: McbA, McbB, McbC, McbD, McbE, McbF and McbG. The McbA gene is the structural gene, McbBCD encodes the components of synthase that post-translationally modifies McbA to introduce the thiazole and oxazole moieties in the peptide backbone. And it is the moieties with thiazole and oxazole that target bacterial gyrase to block DNA replication and transcription. The genes McbE, McbF and McbG are responsible for export and immunity.3 The expression of immunity gene McbG can prevent MccB17 from binding to gyrase and can be combined with the efflux pump McbE, McbF that control concentration of MccB17 in the cell, conferring immunity to the MccB17. In our system, we only shift its excretion gene and immunity gene to the downstream of the QS related negative promoter so that the bacteria will become sensitive to the microcin and control the population.
The gene cluster of microcin B17.4
Quorum Sensing System
Quorum sensing is basically a way that bacteria control their behavior by detecting the density of their colonies around them. In our system, we use luxI/luxR to accomplish the quorum-sensing module.5 LuxI and luxR must form a heterodimer to perform its bioactivity.6 The tetR Gene expression products can inhibit the ptetR promoter.7 In order to reduce the leakage intensity of the luxpR upstream of tetR gene so that it can initiate transcription after the dimer reaches the threshold we need, we fuse luxpR with the HS100 promoter8. The expression of immunity gene McbG can prevent MccB17 from binding to gyrase and can be combined with the efflux pump McbE, McbF that control concentration of MccB17 in the cell, conferring immunity to the MccB17. And the LacZ is a Lactase gene that can be regulated by luxpR promoter.
When the population of our bacteria is low, there are not enough luxI and luxR to form a heterodimer, thus the luxpR promoter cannot promote downstream genes. The tetR cannot be expressed and it cannot inhibit the expression of McbEFG, so our bacteria have immunity to their toxic action of Antimicrobial peptide. And they use toxic action to give themselves an edge in the competition so that they have enough resources to reproduce.
When the population of bacteria reaches a certain level, the heterodimer triggers the downstream controllable promoter. And the immunity gene is inhibited so that the advantage in the competition of our engineering bacteria is disappeared and their quantity cannot continue to grow as before. At the same time, the LacZ gene begins to express lactase.
Reference
[1], [2] Xianxing Gao, Xiaofeng Yang, Jiahui Li, Yan Zhang, Ping Chen and Zhanglin Lin Engineered global regulator H-NS improves the acid tolerance of E. coli. Microbial Cell Factories (2018) 17:118
[3], [4] 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.
[5] Fuqua W C , Winans S C , Greenberg E P . Quorum Sensing in Bacteria: The LuxR-LuxI Family of Cell Density-Responsive Transcriptional Regulators. Journal of Bacteriology, 1994, 176(2):269-275.
[6] Harshad L , Diby P , Hyang K J . N \r, -Acyl Homoserine Lactone-Mediated Quorum Sensing with Special Reference to Use of Quorum Quenching Bacteria in Membrane Biofouling Control. BioMed Research International, 2014, 2014:1-25
[7] N-Acyl Homoserine Lactone Production by Klebsiella pneumoniae Isolated from Human Tongue Surface. Sensors, 2012, 12(12):3472-3483.
[8] https://2018.igem.org/Team:Tsinghua