IGEM:MIT/2008/Notebook/Yogurt/Notes: Difference between revisions
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**includes TEV cut site, half of linker, and 20 bp of GFP | **includes TEV cut site, half of linker, and 20 bp of GFP | ||
**length = 76 bp | **length = 76 bp | ||
**melting temp = | **melting temp = 71.8 C | ||
**GC content % = 51.3 | **GC content % = 51.3 | ||
Revision as of 13:32, 16 June 2008
JUN 10
JUN 16
sequence of signal - epitope - p1025 - epitope
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 ATG CGT AAA GGA GAA GAA C
- includes TEV cut site, half of linker, and 20 bp of GFP
- length = 76 bp
- melting temp = 71.8 C
- GC content % = 51.3
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