IGEM:Cambridge/2008/Turing Pattern Formation/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.

Primer name	Parent vector	init. length	init. Tm	init. GC%	final length	final Tm	final GC
pXyl promoter forward	pSG1154	26	58.7+3	15.3%	50	80.0+3	34.0%
at gaattcgcggccgcttctagag TTCATGAAAAACTAAAAAAAATATTG
pXyl promoter reverse	pSG1154	26 bp	63.0+3	26.6 %	50	79.5+3	44.0%
gat ctgcagcggccgctactagta TATGTCATATTGTAAGTAAGTTGCAC
pSpac promoter forward	pMUTIN-YFP	22	58.5+3	36.3%	46	82.6+3	45.6%
at gaattcgcggccgcttctagag AGAACAACCTCTGCTAAAATTC
pSpac promoter reverse	pMUTIN-YFP	21	59.3+3	38.0%	45	80.4+3	46.6%
tat ctgcagcggccgctactagta AAGCTTAATTGTTATCCGCTC
pPac promoter forward	pMUTIN-YFP	21	61.7+3	38.0%	45	84.5+3	46.6%
at gaattcgcggccgcttctagag AAACGAGGTCATCATTTCCTT
pPac promoter reverse	pMUTIN-YFP	26	57.7+3	26.6%	50	82.1+3	46.0%
cgc ctgcagcggccgctactagta CAAATGTAGTCTTTGAAAGTATTACA
pUpp promoter forward	pAD45-25	22	58.8+3	27.2%	46	82.6+3	41.3%
at gaattcgcggccgcttctagag GATGAATAAATTTTGGCGATAT
pUpp promoter reverse	pAD45-25	25	60.9+3	36.0%	49	80.3+3	44.8%
tat ctgcagcggccgctactagta GAGGATCAAATACATACAGTTTTCC

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.

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
*5' GTTTCTT C GAATTC GCGGCCGC  T  TCTAG ATGaactattttgacaacaa 3'
 
Agr_B_R 
*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'