Part:BBa_K4368005

pcstA + rbs + cI + terminator

Description

It is a composition based on the cI repressor coding region of bacteriophage lambda, which features an LVA tail for rapid degradation of the protein (BBa_C0051). In the case of our team, it has been placed under the control of the pcstA promoter (BBa_K118011), together with a strong rbs (BBa_B0030) and double terminator (BBa_B0015). This part of our group's collection, iGEM22_UMA_MALAGA, is used as a mechanism of gene regulation, as the transcription and subsequent translation of the repressor protein will be influenced by the concentration of glucose generated in the medium by the transformed bacteria.

Characterization

The expression cassette sequence was digested with EcoRI and PstI enzymes and subsequently ligated with a chloramphenicol resistant plasmid backbone (Cm). Transforming bacteria were created with this plasmid and seeded on LB-Agar+Cm plates. After growth, colonies were selected based on their color (white) and DNA extraction was performed using the Promega PureYield Plasmid Miniprep System kit. The resulting DNA is used for further digestion with EcoRI and PstI. The digests are then run on a 0.75% agarose gel at 90 mV voltage and constant amperage. BioRad brand RedSafe is used as an intercalating developing agent.

Enzyme digestion

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
  COMPATIBLE WITH RFC[1000]

Contribution

• Group: [1]
• Author: Jiménez Amores, Carmen