Part:BBa_K4472991

Split ribozyme detecting hcat and expressing GFP

This composite part is build out of the split ribozyme halfs, guide RNAs complementary to the stable endogenous E.coli mRNA hcat and a GFP reporter. We assembled this part using golden gate assembly and transformed it into E.coli DH5alpha.

In our colony PCRs we were able to confirm that we have assembled the construct correctly as all our PCRs had bands at the expected height of around 1600-1700 bp.

Figure 1: Gel of Colony PCR of assembled split ribozyme - reporter constructs. Bands expected at around 1600-1700bp. Ladder: 1kb benchtop from Promega. Marked is the PCR of 2 clones with BBa_K4472990.

The promoters were too weak to get any significant signal in our plate reader measurements afterwards (figure 2). Our model showed that stronger promoters would have had a much stronger effect, see here: https://2022.igem.wiki/uni-hamburg/model

Figure 2: Fluorescence measurement of GFP at emission 485 nm and excitation 510 nm normalized against the negative control (bacteria without GFP construct). Dilution indicated in legend.

The fluorescence of GFP was measured at 485nm emission and 515nm excitation. The platereader read a significant amount of the emission laser at 510 nm for the excitation detection, rendering the data useless because all samples—including the negative control—had high values. At 515 nm we could detect just a very small difference between the negative control containing bacteria lacking GFP and the bacteria carrying our split ribozyme GFP construct. The positive control of just the one promoter BBa_J23115 in front of the GFP is very low as well.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal NheI site found at 331
  Illegal NheI site found at 354
  Illegal NheI site found at 686
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 588
  Illegal XhoI site found at 135
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1495