IGEM:MIT/2008/Notebook/Yogurt/Notes: Difference between revisions

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**FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys)
**FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys)
**C-myc (E Q K L I S E E D L)
**C-myc (E Q K L I S E E D L)
***              (- _ + _ _ _ - - - +)
**HA (Y P Y D U P D Y A: #2)
**HA (Y P Y D U P D Y A: #2)
***      (_ _ _ - _ _ - _ _)


===DNA/Plasmid Editor===
===DNA/Plasmid Editor===

Revision as of 12:05, 16 June 2008

JUN 10

JUN 16

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
  8. epitope sequence

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

Peptide Sequence Manipulations

Primer Analyzer Tool (check temperature & GC content)

  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.

Parts/Construction

  1. Construction
    1. GFP
    2. Synthetic peptide
    3. L. bulgaricus vector
  2. Assay
  3. L. bulgaricus
  4. S. Mutans

To Do/To Buy:

  • finalize DNA sequence
  • primer
  • synthesis
  • reagents

Reverse Primer for GFP

  • reverse sequence: CTGCAGCGGCCGCTACTAGTATTATTAAGAGATGTGAGAGATGTGAGAGATGTGAGAGATGTGAGAGATGTGAGAGATGTGTTTGTATAGTTCATC

Forward Primer for GFP

forward sequence: GAA AAC CTG TAC TTC CAG GGT GGT GGT GGT TCT GGT GGT GGT TCT GGT GGT GGT TCT TAC GCA TTT CCT CTT CTT

    • includes TEV cut site, half of linker, and 20 bp of GFP
    • length = 76 bp
    • melting temp = 72 C
    • GC content % = 51.3
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