Part:BBa_K1897014

GFP with pLuxR

This part was made up of promoter pLuxR BBa_R0062, ribosome binding site (RBS) BBa_B0030, green fluorescent protein (GFP) coding sequence BBa_E0040 and double terminator BBa_B0015.

The GFP protein originated from jellyfish Aequeora victoria, and has a dual-peak absorption-excitation spectrum with a major peak at 396 nm and a smaller peak at 475 nm. Excitation at both peaks results in flurorescence at 503 or 509 nm (Mishin et al., 2010).

Usage and Biology

This part can be used to visually verify that the whole quorum sensing system with LuxR (BBa_K1897008) and LuxI (BBa_K1897008) is working, and that the genes under the control of pLuxR are being expressed.

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 84
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 740
  

Construction of composite GFP

The full composite GFP was made from overlap PCR of 3 fragments - namely the "front" fragment, comprising of the biobrick prefix, promoter, RBS, as well as some bases from the GFP coding sequence; the "middle" fragment, comprising of a little of the RBS, the coding sequence of GFP, the HA tag as well as a few bases of the terminator sequence; and the "terminator" fragment, comprising of a small part of the GFP coding sequence, the HA tag, the double terminator and the biobrick suffix.

Verification of composite GFP

Figure 1: DNA gel electrophoresis photo of overlap PCR to stitch the GFP fragment with front and terminator fragments. The reaction was successful as seen from the observed band size (approximately 1000 bp) for the complete composite LuxR, which is close to the expected band size of 993 bp (Box A). Each lane in Box A consists of duplicate overlap PCR reaction for GFP.

In order to verify that the complete GFP composite has been made via overlap PCR, DNA gel electrophoresis was performed, and the expected band size was 993 bp. As seen from Figure 1, the band size of product termed 'Full GFP' has approximately the correct band size of around 1000 bp.