IGEM:MIT/2008/Notebook/Yogurt/Notes
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JUN 10
JUN 16
Summary of individual tasks to research:
- S. Mutans metabolism pathway-Asad
- L. bulgaricus metabolism pathway-Allin
- secretion (full or partial)-John
- other secretion systems (introduction of)-Sara
- T7 as a promoter (L. bulgaricus)-Prarthna
- L. bulgaricus transformation/protocols-Derek
- binding assay protocols-Andrew
- 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)
- 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.
Parts/Construction
- Construction
- GFP
- Synthetic peptide
- L. bulgaricus vector
- Assay
- L. bulgaricus
- 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