Part:BBa_K3440008

RhlR with myc tag under constitutive promoter

Pconst(BBa_J23100) - RBS(BBa_B0034) - RhlR(BBa_C0071) - Myc(BBa_K823036) - DT(BBa_B0015)

Usage and Biology

In our project, we used this part to test production of RhlR under a constitutive promoter. RhlR gene is originally from Pseudomonas aeruginosa (UniProtKB - P54292) and produces RhlR, which together with C4-HSL (lactone signalling molecule) can activate Rhl promoter Prhl. In our project, this system is used in our oscillating module.

We added a myc-tag to the part in order to be able to characterize it by Western Blotting.

Characterization

Due to the pandemics, we haven’t been able to use biobricks to create the iGEM Stockholm 2020 parts. Those parts were ordered as gene blocks from Integrated DNA Technologies Inc.. As a result, the sequences of the biobricks used are the same, but the scars between biobricks might differ, as well as the final size of the part.

After insertion into a pSB1C3 plasmid and transformation of E. coli TOP10 cells using heat shock, we picked several colonies (see Figure 1 for the plate) corresponding to this part and PCR-amplified them using the VR and VF2 primers contained in the plasmid backbone pSB1C3.

Figure 1: Transformation plate for J

We then ran electrophoretic gels at 180V for 30 mins(Figure 2). We obtained two bands, J2 and J3, corresponding to the length of the construct (1299bp).

Figure 2: Colony PCR gel for BBa_K3440007(G) and BBa_K3440008(J)

J2 and J3 were sent for sequencing to Microsynth AB. The sequence obtained for colony J3 corresponded to the expected part except for a single mismatch, and it was further analysed by Western Blot (Figure 3) to check whether we obtained protein expression of RhlR thanks to the added myc-tag. We did not obtain a band of the correct size (28,8 kDa), therefore we could not prove that this construct can express RhlR constitutively.

Figure 3: Western blot with J3 as BBa_K3440008

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
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 880
  Illegal BamHI site found at 301
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 776