Team:Cambridge/Signaling/Primers

Promoter Primers
Temperatures were calculated using https://www.finnzymes.fi/tm_determination.html. The uppercase letters match the plasmid sequence, while the lowercase letters are 'bumper bases' for restriction cuts and the biobrick prefix and suffixes. Bumper bases, biobrick prefixes, and plasmid DNA are separated by spaces.

Bacillus RBS Primers
We made primer sets for 2 ribosomal binding sites in Bacillus of differing binding strengths. We expect B._sub_RBSs to bind very strongly because it is the consensus sequence for RBS in B. subtilis. Because of its small size, we also made detection primers for use with VF & VR (or temperature compatible alternates) to detect the RBS within the insert,

B. subtilis consensus RBS - http://www.ncbi.nlm.nih.gov/pubmed/10446248

B._sub_RBSs_F
 * 5' GAATTCGCGGCCGCTTCTAGAG AAAGGAGG TGTTA 3'

B._sub_RBSs_R
 * 5' CTGCAGCGGCCGCTACTAGTAACA CCTCCTTT CTCT 3'

RBS modified from ECE112 SpoVG RBS - http://www.bgsc.org/Catalogs/Catpart4.pdf

B._sub_RBSw_F
 * 5' GAATTCGCGGCCGCTTCTAGAG AGAGGTGG TGTTA 3'

B._sub_RBSw_R
 * 5' CTGCAGCGGCCGCTACTAGTAACA CCACCTCT CTCT 3'

Agr Primers

 * These primers are to extract each part of the Agr operon so that a Bacillus RBS can replace the E. coli RBS currently attached to the genes.

agr senders
Agr_B_F Agr_B_R
 * 5' GTTTCTT C GAATTC GCGGCCGC T  TCTAG ATGaactattttgacaacaa 3'
 * 5' TT C CTGCAG CGGCCGC T  ACTAGT A TTATTA  tttcagatcctctttgatg 3'

Agr_D_F
 * 5' CTT C GAATTC GCGGCCGC T  TCTAG atgaatactctgttcaatctgtttt 3'

Agr_D_R
 * 5' TT C CTGCAG CGGCCGC T  ACTAGT A TTATTA ttcatgcagctgggtcagct 3'

agr receivers
Agr_C_F
 * 5' GTTTCTT C GAATTC GCGGCCGC T  TCTAG atgattctgatgttcaccat 3'

Agr_C_R
 * 5' TT C CTGCAG CGGCCGC T  ACTAGT A TTATTA gttattgatgatttcgactt 3'

Agr_A_F
 * 5' GTTTCTT C GAATTC GCGGCCGC T  TCTAG  atggaaatcgcactggcta 3'

Agr_A_R
 * 5' TT C CTGCAG CGGCCGC T  ACTAGT A TTATTA  gattttcttgacattgcgta 3'