Part:BBa_K3440012

pRhl promoter with GFP activated by RhlR expression and AHL

Pconst(BBa_J23100) - RBS(BBa_B0034) - RHlR(BBa_C0071) - Myc(BBa_K823036) - DT(BBa_B0015) - Prhl(BBa_R0071) - RBS(BBa_B0034) - GFP(BBa_K1949060)

Usage and Biology

This part expresses GFP in the presence of C4-HSL. GFP is a fluorescent reporter gene originally found in Aequorea victoria (Jellyfish)(UniProtKB - P42212). It gives a green color (emission at 530nm) when excited at 483nm. RhlR is constitutively expressed uner J23100 promoter. In the presence of C4-HSL (a lactone produce by RhlI from Pseudomonas aeruginosa -UniProtKB - P54291), RhlR:C4-HSL complex can activate Prhl, which controls production of GFP. A myc tag was also added to monitor the expression of RhlR. In our project, this part was used to show that Prhl could be activated in the presence of C4-HSL thanks to the presence of RHlR receptor in the first subpart.

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 heat shock transformation of the pSB1C3 plasmid containing the BBa_K3440011, we picked colonies from plates (Figure 1) and PCR amplified them with primers VF and VR2.

Figure 1: plate of O

We ran gels of the product at 180V and for 30 mins (Figure 2). We obtained the expected size for the bands (2107bp) for O14 and O19.

Figure 2: Colony PCR Gel for BBa_K3440012(O)

We therefore prepared plasmid preparations and glycerol stocks of those and sent them for sequencing to Microsynth AG. The sequence obtained corresponded to the expected part for both O14 and O19.

We then proceeded to test the activity of the constitutive promoter thanks to the GFP reporter added in the part. We measured fluorescence intensity (excitation 483nm, emission 530nm) of GFP for O14,O19 and calibrated the values with OD600 measurements. Figure 3 shows the fluorescence intensity without a lactone for inducer, it is therefore used as a blank. As expected, the fluorescence intensity is very low (<1000AU). Figure 4 also shows the fluorescence intensity in the presence of C4-HSL, and a small increase in fluorescence intensity can be observed after induction, although not as high as expected. Results were a bit higher for synthetic AHL than AHL by RhlI from BBa_K3440006.

Figure 3: Fluorescence measurements for BBa_K34440011 (N1,N3,N6)

Figure 4: Fluorescent measurements for BBa_K3440012(O)

We also performed simpler tests by putting synthetic C4-HSL and RhlI into eppendorfs containing colonies O14 and O19 (Figure 5 and figure 6). Those tests proved that this circuit works and that Prhl can be activated by the subpart containing RhlR, and it also proved that RhlI could produce the lactone.

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  Figure 6: Fluorescence of BBa_K3440012 (O14) in the presence of C4-HSL under UV light
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  Figure 7: Fluorescence of BBa_K3440012 (O19) in the presence of C4-HSL under UV light

Finally, we performed Western blots to check whether we could observe protein expression of RhlR (Figure 8). However, no band was shown at 28,8kDa. Given the fluorescence results, we can hypothesize that RhlR expression is very low and cannot be seen on a Western Blot.

Figure 8: Western blot with O14 and O19 as BBa_K3440012

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
  Illegal BsaI.rc site found at 1754