Part:BBa_K3215010

CmR Cassette + P(LacI)

This part is composed of a selectable marker cassete (Chloramphenicol Resistance) and a LacI regulated promoter, together with a flanking region that enables the user to change the phn Operon promoter, utilizing a recombination technique.

The major advantage of this part is that the user can remove the selection marker later, using a recombination system, since the CmR Cassette used in this part (BBa_K3215006) contains a 48bp sequence that can be used as a homology source for recombinases.

Part construction

To construct this part, parts BBa_K3215006 and BBa_R0010 were synthesized already fused with parts BBa_K3215017 and BBa_K3215018, respectively, and are referenced in this "Part construction" section as "CmR Cassette" and "promoter", although they are fused with those before mentioned parts.

To fuse the promoter with the CmR resistance, we first PCR amplified it with a chimeric primer in order to add a flanking sequence of 30 bp homologous to the CmR Cassette. Size was confirmed by gel electrophoresis. DNA was purified from gel.

Fig. 1. Size confirmation of the promoter + flanking sequences (FS) with 30 bp. Results from parts "CmR Cassette + Strong Promoter" (BBa_K3215007) and "CmR Cassette + P(Kat)" (BBa_K3215009) are also represented.

The CmR Cassette was also PCR amplified with a chimeric primer in order to add a 20 bp flanking sequence homologous to the promoter part. Size was confirmed by gel electrophoresis. DNA was purified from gel.

Fig. 2. Size confirmation of the CmR (1322 bp) + flanking sequences (FS) with 20 bp. Results from parts "CmR Cassette + Strong Promoter" (BBa_K3215007) and "CmR Cassette + P(Kat)" (BBa_K3215009) are also represented.

We fused the promoter fragment derived from the homologous flanking sequence PCR with the CmR Cassete + flanking sequences, by Single-Joint PCR (primerless PCR followed by a second-round PCR with external primers) generating the composite part BBa_K3215010. Fragments were confirmed by gel electrophoresis and purified from gel.

Fig. 3. Second-round PCR using external primers. Size confirmation of composite parts. Results from parts "CmR Cassette + Strong Promoter" (BBa_K3215007) and "CmR Cassette + P(Kat)" (BBa_K3215009) are also represented.

We then used the purified DNA to transform E. coli K12 harboring a PKD46 plasmid, by arabinose-induced Lambda-Red Recombinase method. Transformation was plated on LB + chloranfenicol plates. Colonies were screened by Colony PCR (cPCR). Colonies that showed promising amplification had its DNA extracted by alkaline lysis and PCR was done to confirm them.

Fig. 4. PCR of positive colonies from cPCR. Expected size: Kat (879 bp); Lac (1034 bp); J23100 (869 bp). Results from parts "CmR Cassette + Strong Promoter" (BBa_K3215007) and "CmR Cassette + P(Kat)" (BBa_K3215009) are also represented.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 33
  Illegal XbaI site found at 852
  Illegal XbaI site found at 922
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 33
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 33
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 33
  Illegal XbaI site found at 852
  Illegal XbaI site found at 922
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 33
  Illegal XbaI site found at 852
  Illegal XbaI site found at 922
  
• 1000
  COMPATIBLE WITH RFC[1000]