Team:Cambridge/Oligos

Biobrick forward primer

>BBa_G00100 Part-only sequence (20 bp) tgccacctgacgtctaagaa

Biobrick backwards primer

>BBa_G00101 Part-only sequence (20 bp) attaccgcctttgagtgagc

The Vibrio Lux operon

[http://www.ncbi.nlm.nih.gov/nuccore/5726577 AF170104-1]

The Phosphoreum Lux operon

[http://www.ncbi.nlm.nih.gov/nuccore/166797857 DQ988873]

[http://partsregistry.org/cgi/partsdb/dna.cgi?part_name=BBa%20I0500 Pbad Promoter Part]

[http://www.fasebj.org/cgi/reprint/7/11/1016 Phosphoreum LuxL gene upstream of LuxC]

The phosphoreum oligos we ordered

Notes on alterations to the SLOCK pHK724 clone

• The sequence which SLOCK sent us is just the sequence which is found on [http://www.ncbi.nlm.nih.gov/nuccore/AF170104.1 NCBI] (found by sequence alignment of the two sequences)
• We do not know exactly what is in the plasmid, maybe some information can be found in the references here
• From the annotated start of the lux ICDABEG operon from NCBI there are no restriction sites in conflict with the BioBrick standards.
• Editing ideas:
  • Remove I and G regions from the operon (are these useful?)
  • Attach suffices and prefixes
  • Is this sufficient to then put into a BioBrick? what Chassis should we use?
• A good [http://www.bio.cmu.edu/courses/03441/TermPapers/97TermPapers/lux/operon.html summary] of the lux operon with quality references
• How to design a [http://partsregistry.org/Ribosome_Binding_Sites/Design RBS]

Oligo Order for Vibrio Fischeri on 13.08.10

Oligo Order for Vibrio Fischeri on 17.08.12

Firefly Oligos to be ordered

• Mutagenesis Oligos to be ordered before DNA 2.0 comes through
• Oligos to biobrick our DNA 2.0 order

Our first attempts at oligo design (a little excessive)

Oligos to BioBrick the whole lux operon (never ordered as too expensive)

luxICDABE operon sequence from NCBI

LuxR sequence from NCBI

Thioesterase

ygcB gene from E. Coli K12 to over-express thioesterase: GTGAATACAACGCTGTTTCGATGGCCGGTTCGCGTCTACTATGAAGATACCGATGCCGGTGGTGTGGTGTACCACGCCAGTTACGTCGCTTTTTATGAAA GAGCACGCACAGAGATGCTGCGTCATCATCACTTCAGTCAGCAGGCGCTGATGGCTGAACGCGTTGCCTTTGTGGTACGTAAAATGACGGTGGAATATTA CGCACCTGCGCGGCTCGACGATATGCTCGAAATACAGACTGAAATAACATCAATGCGTGGCACCTCTTTGGTTTTCACGCAACGTATTGTCAACGCCGAG AATACTTTGCTGAATGAAGCAGAGGTTCTGGTTGTTTGCGTTGACCCACTCAAAATGAAGCCTCGTGCGCTTCCCAAGTCTATTGTCGCGGAGTTTAAGC AGTGA

Forward Primer:

gaattcgcggccgcttctagaGTGAATACAACGCTGTTTC

Complement (for Gibson):

GAAACAGCGTTGTATTCACtctagaagcggccgcgaattc

Bacward primer:

ctgcagcggccgctactagtaTCACTGCTTAAACTCCG

Complement:

CGGAGTTTAAGCAGTGAtactagtagcggccgctgcag