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Completion-Killing

Purpose

The killing part in our fourth C, "completion", is to eliminate the Streptococcus Mutans that phages are unable to infect. When the amount of the S.mutans is so large that the phages can't afford the loading, the signal-detecting competence simulating peptide(CSP) would pass the threshold value and turn on the communication signal, thereby simulating the promoter of the killing part. The signal sequence and endolysin downstream would be secreted and this will eliminate the excess S.mutans.

Background

To start with, N-acyl homoserine(AHL) is a general quorum sensing material in gram negetive bacteria. The AHL we found is exists particularly in V. fischeri, and has been successfully utilized in many gram negative bacteria, including E.coli, in many papers and previous iGEM teams. It can move through E.coli membrane without any extra help. Also, as a 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, and the most common ones would be typeI and typeII. However, common experimental strain doesn't apply to the typeI pathway. Furthermore, single signal sequence in type II secretion only leads the recombinant protein to the periplasm region. Therefore, it is difficult to choose a short sequence efficiently to bring our product through the E.coli membrane. Endolysin is an enzyme expressed by phage-infected bacteria. The C-terminus bind to the host cell wall, while the N-terminus is enzymatic domain. Working with holin, which lyses cell membrane, endolysin could break the cell wall of bacteria. While it usually serves as peptidase, endolysin sometimes tends to work on gram negative species, which have layers of peptidoglycan cell wall. Though originally intended to be utilized inside the cell wall, endolysin can also be applied outside the cell as many papers have shown. Furthermore, since the lipid layer of the bacteria composes the cell wall, gram negative bacteria is not as harmful as it was previously.

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 has found that recombinant protein linked to the C-terminus of this signal sequence can be effectively secreted to extra-cellular region, with various size and hydrophobicity, though not cleaved. Though the mechanism is not well understood, we assume that it would lead to periplasm with the signal domain in the front region of the protein, and later secreted 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 proteins, along with other diverse passenger characters.

M102-ORF19
There is a set of open reading frames in S. mutans phage M102 that is 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 19th and 20th opening reading frame are able to contact and lyse cell wall. While the phage usually encodes one endolysin and comprises all ability to lyse cell wall, M102 has two open reading frames, the 19th and 20th, allowing it to encode two different endolysin to break the cell wall of S. mutans. The endolysin translated by the 19th opening reading frame is 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 is usually involved with endolysin. In our experiment, we would first test if the 19th open reading frame is enough to do harm to the Strepptoccocus Mutans. If it is not fatal, we would consider the 20th open reading frame for further experimentation.

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

Functional Measurement

Result

Reference

1. (2006)"Extracellular accumulation of recombinant proteins fused to the carrier protein YebF in Escherichia coli"
2. (2007)"ACES™ Signal Sequence and YebF Expression Systems Technical Brief"
3. (2012)"A Protein Export Pathway Involving Escherichia coli Porins"
4. (2006)patent" PROTEIN PRODUCTION METHOD UTILIZING YEBF "
5. (2007)"Genome sequence of Streptococcus mutans bacteriophage M102"
6. (2012)"Biology and Genome Sequence of Streptococcus mutans Phage M102AD"

Completion-Antibiofilm

Purpose

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

Background

Extracellular polymeric substance (EPS) is important in the formation of biofilm. The EPS matrix consists of polysaccharides, proteins and nucleic acids. The dense extracellular matrix of the biofilms and the outer layer of cells protect the interior of the community, and in some cases, even increase antibiotic resistance. Even though the biofilm seem to be robust, we found that enzymatic degradation is able to weaken the biofilm structure.

As mentioned before, we are going to use antibiofilm enzyme as our tool. 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

Promoter (Asr promoter)
The Asr promoter comes from Escherichia coli strain MG1655, and has the ability to be induced by external acidification (pH 4.0 ~ 5.0). The sequence of Asr promoter includes an open reading frame coding for a polypeptide consists of 111 amino acids. Besides, according to computer-assisted analysis, the predicted polypeptide also contains a typical signal sequence of 30 amino acids, and it might represent either a periplasmic protein or an outer membrane protein.

Signal protein (yebF with RBS)
As described in the killing part, yebF is a transporting protein naturally secreted by Escherichia coli, and it can also be used in experimental strains which are rarely involved in protein secretion.

Lysostaphin producer (iGEM part BBa_K748002)
Part BBa_K748002 comes from 2012 HIT-Harbin iGEM team, and it encodes lysostaphin. Lysostaphin is naturally secreted by Staphylococcus simulans. It is a zinc-containing metalloenzyme of 27 kDa, and has activities of three different enzymes, which are glycylglycine endopeptidase, endo-β-N-acetyl glucosamidase and N-acetyl-muramyl-L-alanine amidase. Glycylglycine endopeptidase hydrolyzes glycylglycine bonds in the polyglycine bridges that form cross links between glycopeptide chains in the cell wall peptidoglycan. As for endo-β-N-acetyl glucosamidase and N-acetyl-muramyl-L-alanine amidase, they can cleave the bonds between N-acetyl glucosamine, N-acetyl muramic acid and alanine respectively. Moreover, lysostaphin is able to lyse actively growing and non-dividing cells in biofilms rapidly, whereas most antibiotics works effectively only on actively dividing cells. Last but not least, lysostaphin has low toxic and cause nearly no side effect on human body. Therefore, we select it as our lytic enzyme against the biofilms of Streptococcus mutans.

Terminator (iGEM part BBa_B0015)
A double terminator composed of B0010 and B0012. It has used by many previous teams, and have strong terminating force.

Functional Measurement

For testing our circuit’s function, we designed a set of experiments.

Stage one— Test the Asr promoter
Asr promoter(with RBS)+RFP+Terminator

Transform the circuit to Escherichia coli, and grow the modified E. coli on agar plates overnight.

Steps:

1. Pick both red and non-red colonies, and grow the E.coli in separate liquid cultures.
2. Prepare 2 sets of 2 plates, one pH 4 and one pH 7. Spread the red strain liquid culture on one set of the ph4 and 7 plates, and repeat on the other set with the non-red strain.
3. Select the strain that expresses growth differences from the previous step. Prepare two of each liquid culture tubes for pH 3.5, 4, 4.5, 5, 5.5, 6, 6.5, and 7, and grow the selected strain in all tubes. Use the growth data from each tube to plot the precise effect of pH on asr promoter.
4. Measure the OD value (RFP/bacterium number).

Stage two— Test the signal protein yebF
(1) J23100+yebF(with RBS)+RFP+Terminator
(2) J23100+RFP+Terminator

Transform both circuits to Escherichia coli, and grow the modified E.coli liquid culture overnight.

Steps:

1. Adjust the two cultures to the approximate OD value.
2. Centrifuge the cultures and extract the supernatants.
3. If the YebF signal sequence does work, the E.coli supernatant containing first circuit should be red while the second circuit having normal culture color.

Stage three—Test the lysostaphin producer iGEM part BBa_K748002 with yebF
(1) J23100-(RBS)yebF-lysostaphin-terminator
(2) J23100-(RBS)yebF-terminator

Steps:

1. Transform both circuits to Escherichia coli, and grow the modified E.coli liquid culture overnight. At the meantime, culture Streptococcus mutans biofilms, and add crystal violet to label the location of the biofilms.
2. Adjust the two cultures to the approximate OD value.
3. Centrifuge the cultures and extract the supernatants.
4. Finally, add the solution into the liquid culture of Streptococcus mutans. Use confocal microscope to check the crystal violet stain. The amount of crystal violet stain can show if the biofilms are degraded or not.

Result

Reference

Completion-Indicator

Purpose

Background

Chassis

Design

Functional Measurement

Result

Reference

1. 2006 MIT iGEM team and iGEM part registry BBa_J45008 / BBa_J45009 / BBa_J45014
2. NCBI Blast: TPA_inf: Saccharomyces cerevisiae S288c chromosome X, complete sequence
3. NCBI Blast: TPA_inf: Saccharomyces cerevisiae S288c chromosome IV, complete sequence
4. NCBI Blast: TPA_inf: Saccharomyces cerevisiae S288c chromosome XV, complete sequence
5. (2012)"Beyond Streptococcus mutans: Dental Caries Onset Linked to Multiple Species by 16S rRNA Community Analysis"