Part:BBa_K1927003

Beta - lactamase AmpR

This part is an ampicillin resistant gene with the J04500 inducible promotor.

Applications of BBa_K1927003

The UiO team have submitted a similar part <a href="https://parts.igem.org/Part:BBa_K1927002">BBa_K1927002</a> But since the pSB1C3 vector is not an expression vector, we could not use this for calibrating our diagnostic tool. We therefore looked into the iGEM library and found BBa_J04500 which has a ribosomal binding site and IPTG inducible promotor. By combining AmpR gene downstream for the BBa_J04500 we could theoretically express the gene product which is a β-lactamase class A. This was done by restriction digestion of part BBa_J04500 with Spel and Pstl and the AmpR-insert with Xbal and PstI. Restriction digest was followed by ligation and transformation of TOP10 cells and grown on LB agar plate with chloramphenicol. Colony PCR was performed to verify successful ligation with our insert. Restriction digestion of miniprepped DNA was performed to confirm prior to sequencing.

<img src=<img> <figcaption> Figure3: Gel analysis of colony PCR with BBa_K1927003. The insert was inserted into the shipping vector pSB1C3 by using 2 different approaches; 3A assembly and a single restriction digest followed by ligation. Lane 1 and 10 are DNA ladder (GeneRuler 1 kb DNA ladder, Thermo Fisher Scientific). Lane 2 and 11 are positive control (validated part BBa_K1927000). Lane 3-7 are different colonies from 3A assembly. Lane 8-9 and 12-14 are restriction digest and ligation approach. Lane 4, 6-8, 12-13 shows the appropriate band at approx. 1000bp.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 942