Difference between revisions of "Part:BBa K3440018"

Revision as of 19:49, 25 October 2020

PCB detection

Pconst(BBa_J23100)-RBS(BBa_B0034)-bphR2(BBa_K1413021)-Myc(BBa_K823036)-DT(BBa_B0015)-Pbphr1(BBa_K1155001)-RBS(BBa_B0034)-LuxI(BBa_C0061)

Usage and Biology

This is the final construct to detect a PCB and consequently produce a quorum sensing molecule. bphR2 is produced constitutively and can bind to a PCB. The complex PCB:bphR2 can activate bphR1 promoter, under which LuxI is placed. LuxI produces the 3OC6-HSL quorum sensing molecule.

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 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 1). Ten colonies were picked from the plate and we obtained five bands for which the band height corresponded to the expected length of the construct (2493bp).

Figure 1: Colony PCR gel for BBa_K3440018

The colonies for which the size seemed correct were sent for sequencing at Microsynth AG. The sequence obtained for colony 4 corresponded to the expected part, but it was not fully sequenced due to the limitations of the Sanger sequencing method.

This construct was further analysed by Western Blot to check whether we obtained protein expression of bphR2 thanks to the added myc-tag (Figure 2). Additionally, 10^-4 M 1,1-biphenyl was added to BBa_K3440018 to compare the differences in protein expression. No bands of the correct size (35.1 kDa) were obtained for BBa_K3440018 in uninduced nor induced conditions, therefore we could not prove that this construct can express bphR2 constitutively.

Figure 2: Western blot with p3.2 and p3.2+biphenyl as BBa_K3440018

Sequence and Features