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

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===Peptide Sequence Manipulations===
===Peptide Sequence Manipulations===
*http://slam.bs.jhmi.edu/gd/
*http://slam.bs.jhmi.edu/gd/
===Primer Analyzer Tool (check temperature & GC content)===
*http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/Default.aspx
#  primers should be 17-28 bases in length; 
#  base composition should be 50-60% (G+C); 
#  primers should end (3') in a G or C, or CG or GC: this prevents "breathing" of ends and increases efficiency of priming; 
#  Tms between 55-80oC are preferred; 
#  3'-ends of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesised preferentially to any other product; 
#  primer self-complementarity (ability to form 2o structures such as hairpins) should be avoided; 
#  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===
===Parts/Construction===
#Construction
#Construction

Revision as of 11:37, 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
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