Part:BBa_K2941001

GFP-TAG-RFP (improved from BBa_K567018)

Intro

This part is improved from the biobrick BBa_K567018 by removing the T7 promoter sequence. Since this part lacks the T7 promoter sequence, the biobrick can be directly used to test incorporation of noncanonical amino acids (ncaa) in amber stop codons in Escherichia coli hosts lacking T7 like the amberless C321.deltaA.exp strain. The original iGEM team, STJU-BioX Shanghai have explained, that the idea of this part was to be able to report the functionality of their tRNA synthetases. We used it to optimize our production.

What is it and how should it work?

The part consists of green fluorescent protein (GFP) and red fluorescent protein (RFP) connected by 11 glycines with an amber stop codon in between. Only GFP would be present, when the fluorescent reporter is expressed. Ideally RFP would be present only, when a tRNA synthetase with ncAA recognizing TAG codons is expressed. Theoretically the strain we used would not have been able to express even GFP without the tRNA synthetase due to the absence of the release factor 1 (RF1).

How did it work in our experiments?

We cloned this in pUC19 vector, which we found to have a bit leaky expression under the lac promoter. GFP signal was pretty high already when we started the inducements and surprisingly even RFP was expressed before the inducement, which could potentially be also caused by our strain, vector and the environment. However, while the GFP was visibly detectable, RFP was detectable only with a fluorometry. We excited GFP at 395 nm and RFP at 570 nm wavelength, after which the emission was measured at 509 nm and 615 nm respectively. Therefore we highly recommend to not panic when you cannot see any redness in the cultures, and to measure the signals with a fluorometric device. In addition to this, there is only 11 amino acids separating GFP and RFP, so a human eye would not see the fusion protein as clearly red and green colors. Due to the leaky inhibition, we compared the change in the signals over time. We substracted the signals at the moment of inducement from the signals measured after the inducement. Then we divided the signals at the end of the incubation by the ones near the inducement.

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 1302
  Illegal AgeI site found at 1414
  
• 1000
  COMPATIBLE WITH RFC[1000]