Part:BBa_K3440014

GFP under Plux in presence of LuxR and AHL

Pconst(BBa_J23100) - RBS(BBa_B0034) - LuxR(BBa_C0062) - Myc(BBa_K823036) - DT(BBa_B0015) - Plux(BBa_B0015) - RBS(BBa_B0034)-GFP(BBa_E0040)

Usage and Biology

This part was designed to produce GFP in the presence of 3OC6-HSL. 3OC6-HSL can from a complex with LuxR, which is expressed constitutively. the complex can activate Plux promoter, under which GFP is placed. 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. LuxR, originally from Vibrio Fischeri (UniProtKB - P12746), is constitutively expressed under J23100 promoter. In the presence of 3OC6-HSL (a lactone produced by LuxI from Vibrio Fischeri), the LuxR:3OC6-HSL complex can activate Plux, which controls production of GFP. In our project, this part was used to show that Plux could be activated in the presence of 3OC6-HSL thanks to the presence of LuxR 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_K3440014, we picked colonies from plates (Figure 1) and PCR amplified them with primers VF and VR2.

Figure 1: plate of Q

We ran gels of the product at 180V and for 30 mins (Figure 2 and Figure 3). We obtained the expected size for the bands (2135bp) for Q2 (Figure 3) and Q3 (Figure 2).

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  Figure 2: Colony PCR Gel for BBa_K3440014(Q), showing successful Q3
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  Figure 3: Colony PCR Gel for BBa_K3440014(Q), showing successful Q2

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 Q3.

We then proceeded to test the activity of the part. We measured fluorescence intensity (excitation 483nm, emission 530nm) of GFP for Q3 and calibrated the values with OD600 measurements. Figure 4 shows the fluorescence intensity in the presence of 3OC6-HSL (both from BBa_K3440000 and from synthetic AHL) and without it. As expected, the fluorescence intensity is very low (<1000AU) without inducer but a significant increase in fluorescence intensity can be observed after induction by 3OC6-HSL, both synthetic and produced.

Figure 4: Fluorescent measurements for BBa_K3440014(Q3)

This result was also confirmed when we performed simpler tests by putting synthetic 3OC6-HSL and LuxI into eppendorfs containing colonies of Q (Figure 5). To conclude, those tests proved that this circuit works and that Plux can be activated by the subpart containing LuxR, and it also proved that LuxI could produce the lactone.

Figure 5: Fluorescence of BBa_K3440014 in the presence of 3OC6-HSL under UV light

Finally, we performed Western blots to check whether we could observe protein expression of LuxR (Figure 6). The experiment confirmed that LuxR could be produced by the first subpart thanks to a visible band at the correct size (29,8kDa).

Figure 6: Western blot with Q3 as BBa_K3440014

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 874
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1023
  Illegal BsaI.rc site found at 1750