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

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==JUN 16==
==JUN 16==


sequence of signal - epitope - p1025 - epitope


ATGTACGTCTTCTTTTTTAGGCGTGCGGTAAACTTGTTTCACCGACTTAATCGGCGTCGTGACGAGGATAGTCGCTCAGGTGACCGCCCTTGAAAGACTACAAAGACGACGACGACAAAGGTGGTGGTCAGCTGAAAACCGCTGACCTGCCGGCTGGTCGTGACGAAACCACCTCTTTCGTTCTGGTTGGTGGTGGTTACCCGTACGACGTTCCGGACTACGCTGGTGGTGGTTCTGGTGGTGGTTCTGGTGGTGGTTCTnnn


===Summary of individual tasks to research:===
===Summary of individual tasks to research:===
Line 16: Line 14:
# L. bulgaricus transformation/protocols-Derek
# L. bulgaricus transformation/protocols-Derek
# binding assay protocols-Andrew
# binding assay protocols-Andrew
# epitope sequence


===Linear Sequence/Design===
===Linear Sequence/Design===
Line 57: Line 54:
*reagents
*reagents


===Reverse Primer for GFP===
===TEV Protease cut site===
[[Image:reversegfp.jpg]]
 
*reverse sequence: CTGCAGCGGCCGCTACTAGTATTATTAAGAGATGTGAGAGATGTGAGAGATGTGAGAGATGTGAGAGATGTGAGAGATGTGTTTGTATAGTTCATC
* Amino Acid Sequence : Glu-Asn-Leu-Tyr-Phe-Gln-Gly (Cuts between Gln-Gly)
* Base Pair Sequence: GAAAACCTGTACTTCCAGGGT


===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


===TEV Protease cut site===
==JUN 17==
===Links to sites from individual research topics===
*T7 as a promoter in L. bulgaricus/Other viable promoter options
**LacS promoter
***http://www.patentstorm.us/patents/5639644/description.html
***http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=161534
**http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=123959
**http://jb.asm.org/cgi/content/full/184/4/928
*Secretion signal peptide
**[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=178052&blobtype=pdf cleavage site]
 
===P4 Revised===
[[image:P4-4.jpg]]
**reverse sequence: CTG CAG CGG CCG CTA CTA GTA AGA GAA TAT AAA AAG CCA GAT TAT TAA TCC GGC TTT TTT ATT ATT TTT ATT AGT GGT GAT GGT GAT GAT GTT TGT ATA GTT CAT CCA TGC
**length = 111 bp
**melting temp = 69.6 C
**GC content % = 36.0
 
==JUN 18==
* [[../lbulgaricus |''Lactobacillus delbrueckii'' subsp. ''bulgaricus'' Information & Protocols]]
 
* [[../toothbindingassay | Tooth binding assay protocol]]
 
*http://www.freewebs.com/naguiar/ (S. mutans Information)


* Amino Acid Sequence : Glu-Asn-Leu-Tyr-Phe-Gln-Gly (Cuts between Gln-Gly)
*http://pubs.acs.org/cgi-bin/abstract.cgi/jafcau/2002/50/i05/abs/jf010958t.html (S. mutans adsorption to HA Beads)
* Base Pair Sequence: GAAAACCTGTACTTCCAGGGT


===Forward Primer for Promoter===
*http://www.blackwell-synergy.com/doi/full/10.1111/j.1399-302X.2007.00412.x (Influence of Starch and Sucrose on S. mutans)
[[Image:forward_prom.jpg]]
**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===
===Friday Meeting breakdown===
[[Image:reverse_pep.jpg]]
#Andrew: primers
**reverse sequence: 5'- AAA AGT TCT TCT CCT TTA CGC ATA GAA CCA CCA CCA CCC TGG A -3'
#Derek: L. bulgaricus procedures
**length = 43 bp
#Allin: metabolism
**melting temp = 67.3 C
#John: binding assay
**GC content % = 46.5
#Sara: secretion
#Asad: S. Mutans/Binding Assay
#Prarthna: promoters

Latest revision as of 05:26, 20 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

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

TEV Protease cut site

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


JUN 17

Links to sites from individual research topics

P4 Revised

    • reverse sequence: CTG CAG CGG CCG CTA CTA GTA AGA GAA TAT AAA AAG CCA GAT TAT TAA TCC GGC TTT TTT ATT ATT TTT ATT AGT GGT GAT GGT GAT GAT GTT TGT ATA GTT CAT CCA TGC
    • length = 111 bp
    • melting temp = 69.6 C
    • GC content % = 36.0

JUN 18

  • [[../lbulgaricus |Lactobacillus delbrueckii subsp. bulgaricus Information & Protocols]]
  • [[../toothbindingassay | Tooth binding assay protocol]]

Friday Meeting breakdown

  1. Andrew: primers
  2. Derek: L. bulgaricus procedures
  3. Allin: metabolism
  4. John: binding assay
  5. Sara: secretion
  6. Asad: S. Mutans/Binding Assay
  7. Prarthna: promoters
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