Team:MIT/Organizational Daily Notebook

PROTOCOLS

 * DNA Ligations
 * Top10 chem competent cell transformation
 * DNA sequencing
 * PCR
 * Running DNA Gels
 * Restriction Digests
 * Preparing Antibiotic Stocks

Summary of individual tasks to research:

 * 1) S. Mutans metabolism pathway-Asad
 * 2) L. bulgaricus metabolism pathway-Allin
 * 3) secretion (full or partial)-John
 * 4) other secretion systems (introduction of)-Sara
 * 5) T7 as a promoter (L. bulgaricus)-Prarthna
 * 6) L. bulgaricus transformation/protocols-Derek
 * 7) binding assay protocols-Andrew

Linear Sequence/Design
Ex-promoter-RBS-ATG-signal-epitope-p1025-linker (TEV cleavage site)-GFP-His-TAATAA-termination-SP (see top of page)
 * Base pairs: 35-10-3-90-30-60-45-700-15-6-8
 * potential eptiopes
 * FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys)
 * C-myc (E Q K L I S E E D L)
 * HA (Y P Y D U P D Y A: #2)

DNA/Plasmid Editor

 * http://www.biology.utah.edu/jorgensen/wayned/ape/

Peptide Sequence Manipulations

 * http://slam.bs.jhmi.edu/gd/

Primer Analyzer Tool (check temperature & GC content)

 * http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/Default.aspx


 * 1)  primers should be 17-28 bases in length;
 * 2)  base composition should be 50-60% (G+C);
 * 3)  primers should end (3') in a G or C, or CG or GC: this prevents "breathing" of ends and increases efficiency of priming;
 * 4)  Tms between 55-80oC are preferred;
 * 5)  3'-ends of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesised preferentially to any other product;
 * 6)  primer self-complementarity (ability to form 2o structures such as hairpins) should be avoided;
 * 7)  runs of three or more Cs or Gs at the 3'-ends of primers may promote mispriming at G or C-rich sequences (because of stability of annealing), and should be avoided.
 * http://bioweb.uwlax.edu/GenWeb/Molecular/Seq_Anal/Primer_Design/primer_design.htm

Parts/Construction

 * 1) Construction
 * 2) GFP
 * 3) Synthetic peptide
 * 4) L. bulgaricus vector
 * 5) Assay
 * 6) L. bulgaricus
 * 7) S. Mutans

To Do/To Buy:

 * finalize DNA sequence
 * primer
 * synthesis
 * reagents

TEV Protease cut site

 * Amino Acid Sequence : Glu-Asn-Leu-Tyr-Phe-Gln-Gly (Cuts between Gln-Gly)
 * Base Pair Sequence: GAAAACCTGTACTTCCAGGGT

Links to sites from individual research topics

 * T7 as a promoter in L. bulgaricus/Other viable promoter options
 * LacS promoter
 * http://www.patentstorm.us/patents/5639644/description.html
 * http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=161534
 * http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=123959
 * http://jb.asm.org/cgi/content/full/184/4/928
 * Secretion signal peptide
 * cleavage site

P4 Revised

 * reverse sequence: CTG CAG CGG CCG CTA CTA GTA AGA GAA TAT AAA AAG CCA GAT TAT TAA TCC GGC TTT TTT ATT ATT TTT ATT AGT GGT GAT GGT GAT GAT GTT TGT ATA GTT CAT CCA TGC
 * length = 111 bp
 * melting temp = 69.6 C
 * GC content % = 36.0

JUN 18

 * Lactobacillus delbrueckii subsp. bulgaricus Information & Protocols


 * Tooth binding assay protocol


 * http://www.freewebs.com/naguiar/ (S. mutans Information)


 * http://pubs.acs.org/cgi-bin/abstract.cgi/jafcau/2002/50/i05/abs/jf010958t.html (S. mutans adsorption to HA Beads)


 * http://www.blackwell-synergy.com/doi/full/10.1111/j.1399-302X.2007.00412.x (Influence of Starch and Sucrose on S. mutans)

Friday Meeting breakdown

 * 1) Andrew: primers
 * 2) Derek: L. bulgaricus procedures
 * 3) Allin: metabolism
 * 4) John: binding assay
 * 5) Sara: secretion
 * 6) Asad: S. Mutans/Binding Assay
 * 7) Prarthna: promoters

JUN 20
Powerpoint [linked here]

JUN 24

 * Ordered MRS broth and agar for L.bulgaricus from Difco

AUG 1

 * Initial outline for powerpoint:

1. Overview
 * Large synthetic biology -> food overview
 * Our goal
 * Why important
 * Teeth
 * Globally
 * Plan of Attack
 * forming DNA
 * lactobacillus bulgaricus
 * binding assay

2. Binding Assay
 * New method
 * (Controls to prove eficacy of method)

3. DNA work
 * Gene construct
 * Brief reason for parts in teh construct
 * signal seq for l.bulgaricus
 * Promoter
 * characterizing bad T7
 * BioBricks

4. L.bulgaricus
 * Transforming
 * Restriction Mechanism
 * Emphasize how hard it is

5. Final results

6. Our contribution (biobricks, l.bulgaricus, binding assay for cells)

7. Final overview
 * This is specific, but applies to a lot strains / peptides