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

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===Forward Primer for GFP===
===Forward Primer for GFP===
forward sequence: GGT GGT TCT GGT GGT GGT TCT ATG CGT AAA GGA GAA GAA C
forward sequence: GGT GGT TCT GGT GGT GGT TCT   ATG CGT AAA GGA GAA GAA C


**includes 21 bp of 2nd half of linker, and 19 bp of GFP
**includes 21 bp of 2nd half of linker, and 19 bp of GFP

Revision as of 14:01, 16 June 2008

JUN 10

JUN 16

sequence of signal - epitope - p1025 - epitope


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: GGT GGT TCT GGT GGT GGT TCT ATG CGT AAA GGA GAA GAA C

    • includes 21 bp of 2nd half of linker, and 19 bp of GFP
    • length = 40 bp
    • melting temp = 66.2 C
    • GC content % = 50%

TEV Protease cut site

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

Forward Primer for Promoter

    • foward sequence: TAA TAC GAG TCA CTA TAG GGA ATT AAA GAG GAG AAA ATG TAC GTC TTC TTT TTT AGG CGT GCG GTA AAC TTG TTT CAC CGA CTT AAT CGG CGT CGT GAC GAG GAT AGT CGC TCA GGT GAC CGC CCT TGA AA
    • length = 131 bp
    • melting temp = 71.4 C
    • GC content % = 45.0

Reverse Primer for Peptide

    • reverse sequence: 5'- AAA AGT TCT TCT CCT TTA CGC ATA GAA CCA CCA CCA CCC TGG A -3'
    • length = 43 bp
    • melting temp = 67.3 C
    • GC content % = 46.5
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