Part:BBa_K3440000

LuxI-Myc

Pconst(BBa_J23100) - RBS(BBa_B0034) - LuxI(BBa_C0061) - Myc(BBa_K823036) - DT(BBa_B0015).

Will generate LuxI constitutively.

Usage and Biology

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

LuxI can produce 3OC6-HSL, a lactone that can be used as a signalling molecule for which the sender is LuxI and the receiver is LuxR.

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 BBa_K3440000(A)

We then ran electrophoretic gels at 180V for 30 mins(Figure 2). In particular, seven colonies were picked from plate A5 and we obtained six bands for which the band height corresponded to the expected length of the construct (1158bp).

Figure 2: Colony PCR gel for BBa_K3440000

Other gels were also ran, and those for which the size seemed correct were sent to sequencing at Microsynth AB. The sequence obtained for colony A3 corresponded to the expected part and was further analysed by Western Blot to check whether we obtained protein expression of LuxI thanks to the added myc-tag . We obtained a band of the correct size (23,1kDa), which proved that this construct could express LuxI constitutively.

Figure 3: Western blot with A3 as BBa_K3440000

We further analysed A3 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 3OC6-HSL in acetonitrile onto a plate with Chromobacterium violaceum colonies (Figure 5). Thus, because this construct expresses LuxI and produces lactone 3OC6-HSL, we expected a coculture of Chrombacterium violaceum and A3 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: Coculture of chromobacterium + BBa_K3440000(A)
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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 698
  Illegal BglII site found at 736
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]