Part:BBa_K3440006

RhlI-Myc under constitutive promoter

Pconst(BBa_J23100) - RBS(BBa_B0034) - RhlI(BBa_C0070) - Myc(BBa_K823036)

Usage and Biology

In our project, we used this part to test production of LuxI under a constitutive promoter.

LuxI can produce C4-HSL, a lactone that can be used as a signalling molecule for which the sender is RhlI and the receiver is RhlR.

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 corresponding to this part (see figure 1 for the plate) and PCR-amplified them using the VR and VF2 primers contained in the plasmid backbone pSB1C3.We then ran electrophoretic gels at 180V for 30 mins (Figure 2). In particular, we obtained G1 and G7 for which the band height corresponded to the expected length of the construct (1045bp).

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  Figure 1: Transformation plate for BBa_K3440006(G)
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  Figure 2: Colony PCR gel for BBa_K3440006(G) and BBa_K3440008(J)

G1 and G7 were therefore sent for sequencing to Microsynth AB. The sequence obtained for colony G7 corresponded to the expected part and was further analysed by Western Blot (Figure 3) to check whether we obtained protein expression of RhlI thanks to the added myc-tag. We obtained a band of the correct size (23,8kDa), which proved that this construct could express LuxI constitutively.

Figure 3: Western blot with G7 as BBa_K3440006

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We further analysed G7 by co-cultivating it with Chromobacterium violaceum (Figure 4). Chromobacterium violaceum turns purple in the presence of lactones (AHLs), which was proved by a control test where we added 2µL of a solution of 10mM synthetic 3C4-HSL in acetonitrile onto a plate with Chromobacterium violaceum colonies (Figure 5). Thus, because this construct expresses LuxI and produces lactone C4-HSL, we expected a co-culture of Chrombacterium violaceum and G7 to turn purple. However, the plate did not show any color. Given that expression occurs as shown by the Western Blot, we can conclude that the concentration was probably too low for a color to appear on the plate.

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  Figure 4: Co-culture of chromobacterium + BBa_K3440006(G)
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  Figure 5: Chromobacterium under AHL

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 722
  Illegal BglII site found at 760
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]