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). It gives a green color (emission at 530nm) when excited at 483nm. LuxR, originally from Vibrio Fischeri, 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). We obtained the expected size for the bands (bp) for O14 and O19.

Figure 2: Colony PCR Gel for BBa_K3440014(Q)

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 Q 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_K34440014 (N1,N3,N6)

Figure 4: Fluorescent measurements for BBa_K3440014(Q)

We also performed simpler tests by putting synthetic C6-HSL into eppendorfs containing colonies Q (Figure 5). Those tests proved that this circuit works and that Plux can be activated by the subpart containing LuxR.

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

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