Composite

Part:BBa_K4368007

Designed by: Carmen Jimenez Amores, Alonso Molina Calvo, Paola Morales Gonzalez   Group: iGEM22_UMA_MALAGA   (2022-09-26)
Revision as of 18:28, 7 October 2022 by Carmeenjmnezz (Talk | contribs) (→‎Characterization)


pcI + rbs + RFP + terminator

Description

RFP (BBa_E1010) is the gene coding red fluorescent protein which is a protein synthesized by the cnidaria Discosoma sp. In addition, this part includes the composition used by the team, which includes a strong rbs (BBa_B0030), a double terminator (BBa_B0015) as well as a promoter inducible by the cI protein of the phage lambda (BBa_R1051).

Characterization

The expression cassette sequence was digested with EcoRI and PstI enzymes and subsequently ligated with a ampicillin resistant plasmid backbone (Amp). Transforming bacteria were created with this plasmid and seeded on LB-Agar+Amp plates. After growth, colonies were selected based on their color (red) 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

UMA RFP.png

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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 633
    Illegal AgeI site found at 745
  • 1000
    COMPATIBLE WITH RFC[1000]

Contribution

  • Group: [1]
  • Author: Molina Calvo, Alonso


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Parameters
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