Here is the sequences for all of the parts we have used in our project with the biobrick prefix and suffix. When we ordered these sequences as gBlocks from IDT we include a pre-preffix and post-suffix for our original Forw. and Rev. primers to bind to.
Pre-prefix and biobrick prefix (ATG):
Pre-prefix and biobrick prefix:
Post-suffix and biobrick suffix:
TAT Csp1 HH: (BBa_K1980000)
TAT Csp1 sfGFP HH: (BBa_K1980001)
MymT HH: (BBa_K1980002)
MymT sfGFP HH: (BBa_K1980003)
pCusC promoter: (BBa_K1980004)
pCopA sfGFP: (BBa_K1980005)
pCopA sfGFP with divergent CueR: (never submitted)
pCopA with divergent CueR: (BBa_K1980006)
pCusC mKate2: (BBa_K1980007)
pCopA CueR sfGFP: (BBa_K1980008)
pCusC CusR mKate2: (BBa_K1980009)
pCopA TAT Csp1 sfGFP with divergent CueR: (BBa_K1980010)
pCopA MymT with divergent CueR: (BBa_K1980011)
pCopA MymTsfGFP with divergent CueR: (BBa_K1980012)
We amplified all out Gblock first using the primers named Forw. and Rev. If this failed then we often used the primers His Rev (new reverse) and Constituative CueR forw (new forw) which are complementary to the His tag together with some of the biobrick suffix and the end of CueR and some of the biobrick prefix respectively.
For sequencing all parts in the shipping vector we used VF2 and VR. To sequence pCusC mKate, as soon as we received it, we used pCusC sequencing forw. and pCusC sequencing rev.
As our part pCusC CusR mKate was so long the sequencing using VR and VF2 primers failed to overlap on a number of runs. We therefore designed the Feedback pCusC mKate too big sequencing primers (FCKTB forw and FCKTB rev) that allowed us to sequence the middle of the part.
Constitutive CueR forw:
pCusC sequencing forw:
pCusC sequencing rev:
To chop the MymTsfGFP out of pCopA MymT sfGFP with divergent CueR to form pCopA with divergent CueR we used primers Anthony forw and Anthony rev. (don't ask) To chop pCusC out of pCusC mKate we used pCusC chop forw and pCusC chop rev.
pCusC chop forw:
pCusC chop rev:
To move both TAT Csp1 HH and TAT Csp1 sfGFP HH into the expression vector pBAD we used the primers Ex P TAT Csp1 forw and Ex P Rev. To move MymT sfGFP into the expression vector pBAD we used the primers Ex P MymT forw and Ex P Rev. To chop MymT out of MymT sfGFP and then ligate it into shipping (refused to ampfly from gBlock) we used the primers MymT only cut rev and MymT shipping. To chop MymT out of MymT sfGFP and then ligate it into pBAD we used the primers MymT only cut rev and Ex MymT forw:
Ex P TAT Csp1 forw:
Ex P Rev:
Ex MymT forw:
MymT only cut rev:
We received pCusC mKate in a plasmid with chloramphenicol resistance and a ColE1 origin (here with the pCusC mKate included). All other parts were ligated and tested in the pSB1C3 shipping vector. For the parts MymT, MymT sfGFP, TAT Csp1 and TAT Csp1 sfGFP we amplified them from the shipping vector, digested with BspH1 and Pst1 and ligated into the pBAD HisB expression vector that had been digested with Nco1 and Pst1.
pCusC arrival plasmid with pCusC RFP: