IGEM:MIT/2008/Brainstorming

This is the new brainstorming page for our iGEM project. The previous brainstorming page is at IGEM:MIT/2008/Brainstorming Old

Technical Overseers Page

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Expression of a p1025-containing peptide in E. coli

References

PMID 9920267
A synthetic peptide adhesion epitope as a novel antimicrobial agent.

Materials

• The entire transcription unit will include

1. T7 promoter for over-expression (IPTG inducible)
2. ribosomal binding site
3. secretion signal (~30AAs) for L. bulgaricus; whether or not it works in E. coli is not critical
4. 1st epitope (~10AAs)
5. p1025 peptide (20AAs)
6. a flexible linker region (~20AAs) mostly with Gly and Ser residues. Include a 2nd epitope and a TEV protease site in the middle of the linker in case we want to remove GFP and the His-tag
7. GFP (~230AAs)
8. 6xHis tag for affinity purification and on-column digestion with TEV protease
9. double STOP codon
10. transcription terminator

Adhesion of Streptococcus mutans to hydroxyapatite (HA)

Reference

PMID 9062560
Inhibition of Streptococcus mutans adsorption to hydroxyapatite by low-molecular-weight chitosans.

The materials and methods section describes an indirect plating assay to quantify binding of S. mutans to HA. Results in Table 1 indicates that this method can be used to quantify inhibition of binding by a competitor molecule. In their study, the molecules are sugar polymers.

Materials

• HA beads (from Fluka, 80-200 micro-meter grain size)
• saliva as source of the glyco-protein (one vendor is found through Google)
• S. mutans and handling protocol (Chia is contacting Daniel Smith and Martin Taubman of the Forsyth Institute to acquire a suitable strain)
• synthetic p1025 and p1125 as positive and negative controls?

Secretion of a p1025-containing peptide from Lactobacillus bulgaricus/lactis

References

PMID 12788739 (a secretion signal sequence described; can use the promoter, RBS and secretion signal for this protein for p1025)
Determination of the domain of the Lactobacillus delbrueckii subsp. bulgaricus cell surface proteinase PrtB involved in attachment to the cell wall after heterologous expression of the prtB gene in Lactococcus lactis.

PMID 11772607
Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with various plasmids.

Materials

• Lactobacillus bulgaricus or lactis (check with Tom Knight first. Otherwise request from another lab or purchase from ATCC)
• growth medium: Ligia recommends purchasing the pre-mix powder for the MRS medium for technical simplicity (vendor?)
• Gas Pak to create an anaerobic/micro-aerobic environment. (Email bio related mailing lists to see if we can borrow the jars or incubators. We may need to purchase the disposable chemical envelopes that reduce oxygen content.)
• Expression vector: BBa_I742103 in registry is a shuttle vector for E. coli and Lactobacillus. Let's look up this vector to see whether people have used it for protein expression. Alternatively we can get some of the plasmids described in PMID 11772607.

Project road map

Stage 1

• Construct parts for E. coli for production of the p1025 fusion peptide. Obtain antibodies against epitopes and maybe p1025 itself.

• Get the hydroxyapatite(HA)-S. mutans binding assay to work in our hands.

• Practice growing and transformation of Lactobacillus. Design parts for Lactobacillus.

Stage 2

• Make E. coli produce the p1025 fusion peptide.

• (GOAL #1) See if the p1025 fusion peptide from E. coli can inhibit binding of S. mutants to HA beads. Remove GFP if necessary.

• Engineer Lactobacillus

Stage 3

• (GOAL #2) Verify production and secretion of p1025 fusion peptide by Lactobacillus by Western (and/or microscopy if we decide to have a GFP-containing part in parallel).
• (GOAL #3) Can the yogurt reduce binding of S. mutans?

Fundrai$ing!

Drew has said that he can get in touch with some MIT alumni and the MIT development office. We can send them the brochure and a letter to explain our goal, project and budget.