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Purpose

The killing part in completion is to eliminate the Streptococcus Mutans that phages are unable to infect. When the amount of the signal detecting competence simulating peptide (CSP) pass the threshold value, it would also turn on the communication signal. If the phage couldn’t lower down the amount of S. mutans, the communication signal would accumulate and there by stimulate the killing part in the helper Probiotics. The signal sequence and endolysin downstream would be secreted and eliminate the excess S. mutans.

Background

To start with, N-acyl homoserine(AHL) is a generally quorum sensing material in gram negative bacteria. The AHL we found is particularly existed in V. fischeri, and had been successfully complete utilized in many gram negative bacteria including E.coli in many papers and previous iGem teams. It can pass through the membrane of E. coli without any extra help. As gram negative bacteria, E. coli doesn't naturally secrete substance into extracellular environment, except for some toxins. Therefore, secreting proteins out of membrane in non-toxin experimental strain would be much harder. There are few types of secretion pathway in bacteria, the most common ones would be type I and type II. However, common experimental strain doesn't apply the pathway. Furthermore, single signal sequence in type II secretion only leads the recombinant protein to the periplasm area. Therefore, choosing a short sequence efficiently to bring our product out of the E.coli membrane is a hard process.

Endolysin is an enzyme expressed by phage-infected bacteria. The N-terminus binds to the host cell wall, while the C-terminus is enzymatic domain. Working with holin, which lyses cell membrane, endolysin could break the cell wall of bacteria. Usually serves as peptoglycose, endolysin tends to work on gram negative species, which have layers of peptoglycan cell wall. Though originally intended to be utilized inside the cell wall, endolysin can also be applied outside cell according to previous research. Furthermore, it is even less harmful to gram negative bacteria this way because of the lipid layer of those bacteria composes cell wall.

Design

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LuxpR promoter
The promoter of the circuit is in charge of the communication between phage and E. coli communication. Induced by AHL-luxR complex, the promoter could turn on the expression of downstream coding sequence once the SOS signal(AHL) is spread by phage.

(RBS)yebF
yebF is a protein that is naturally secreted by E.coli. Research had found that recombinant protein linked to the C-terminus of this signal sequence could be effectively secreted to extracellular region, with various size and hydrophobicity, though not cleaved. Though the mechanism not well understood, it is assumed to be lead to periplasm by the signal domain in the front region of the protein, and later on secreted extra cellular through porin on the cell wall. The advantage of the signal sequence is that it can be secreted in experimental strains which usually do not secrete protein, and the diverse passenger characters.

M102-ORF19
There are a set of open reading frames in S. mutans phage M102 responsible for endolysin production and activation, which is the region containing opening reading frame 18-20. With the eighteenth open reading frame producing holin that destroys the cell membrane, the endolysins expressed by the later on 19th and 20th opening reading frame are able to contact and lyse cell wall. Different from normal scenario, which phage usually encode one endolysin comprises all ability to lyse cell wall, M102 has two open reading frame, the 19th and 20th, encoding two different endolysin to break the cell wall of S. mutans. The endolysin translated by the nineteenth opening reading frame is a glucosidase, which can break the polysaccharide capsule of Strepptoccocus cell wall. The N-terminus, in charge of cell wall binding, has different amino acid coding with other endolysins, indicating its specificity. The 20th opening reading frame encodes pepdoglycandase, and has CHAP domain which endolysin is normally involved. In our experiment, we would first test if the nineteenth open reading frame is enough to do harm to the Strepptoccocus Mutans, if it's not fatal, we would consider the twentieth open reading frame for further experiment.

B0015
A double terminator composed of B0010 and B0012. It has used by many iGEM teams, and have strong terminating force.

Result

Reference

Purpose

Streptococcus Mutans will form biofilms, which may cause plaque, tooth decay and gum infection. Also, biofilms make it hard for our engineered probiotics to capture and kill the streptococcus mutans inside. Nowadays, the most basic way of removing biofilm is by physical methods such as brushing one's tooth regularly. However, there are many people who are not able to do so by themselves, so we want to induce a biological method to get rid of biofilms with our engineered probiotics. Biofilm is a polymeric conglomeration generally consists of extracellular DNA, proteins, and polysaccharides. Therefore, we are going to design a circuit that produce several enzymes to destroy biofilm structure.

Background

Formation of a biofilm Some species are not able to attach to a surface on their own but are sometimes able to anchor themselves to the matrix or directly to earlier colonists. Once colonization has begun, the biofilm grows through a combination of cell division and recruitment. Polysaccharide matrices typically enclose bacterial biofilms. In addition to the polysaccharides, these matrices may also contain material from the surrounding environment, including but not limited to minerals, soil particles, and blood components, such as erythrocytes and fibrin. The biofilm is held together and protected by a matrix of secreted polymeric compounds called EPS. EPS is an abbreviation for either extracellular polymeric substance or exopolysaccharide, although the latter one only refers to the polysaccharide moiety of EPS. In fact, the EPS matrix consists of polysaccharides, proteins and nucleic acids. A large proportion of the EPS is more or less strongly hydrated, however, hydrophobic EPS also occur; one example is cellulose which is produced by a range of microorganisms. This matrix encases the cells within it and facilitates communication among them through biochemical signals as well as gene exchange. The EPS matrix is an important key to the evolutionary success of biofilms. One reason is that it traps extracellular enzymes and keeps them in close proximity to the cells. Thus, the matrix represents an external digestion system and allows for stable synergistic microconsortia of different species. The dense extracellular matrix of the biofilms and the outer layer of cells protect the interior of the community. In some cases antibiotic resistance can be increased a thousandfold. Lateral gene transfer is greatly facilitated in biofilms and leads to a more stable biofilm structure. Enzymatic degradation is able to weaken the biofilm structure and release microbial cells from the surface. As mentioned before, we are going to use antibiofilm enzyme as our tool in this part. According to 2012 INSA-Lyon and HIT-Harbin iGEM teams, we decided to use lysostaphin as our ideal enzyme. Lysostaphin has activities of 3 enzymes, including glycylglycine endopeptidase, endo-β-N-acetyl glucosamidase and N-acteyl muramyl-L-alanine amidase. Those enzymes can cleave the glycine–glycine bonds which form cross links between glycopeptide chains in the cell wall peptidoglycan, and cleave the peptidoglycan of a backbone made up of alternating β-1,4 linked N-acetylglucosamine and N-acetylmuramic acid residues as well.

Design

Result

Reference