Part:BBa_K3024009:Experience

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Applications of BBa_K3024009

This part contains the C protein gene downstream of an inducible promoter pBad. This gives us the ability to increase its expression by administrating the monosaccharide arabinose in the growth medium. Due to very limited literature on the dynamics of C inhibition on Pe, the promoter of cox factor, we designed this part to test the concentration-dependent effect of C on the native regulatory region of the P2 switch.

User Reviews

UNIQ5f90b58befb52b98-partinfo-00000000-QINU UNIQ5f90b58befb52b98-partinfo-00000001-QINU

••••• iGEM Stockholm 2019

We created an inducible construct for the C-protein enabling the control of of this protein rather than having a constitutive expression, as is naturally found in the P2 phage switch. To do so, we used a pBad promoter, which can be controlled with a arabinose-based gradient. Our growth curve results showed little to no arabinose-induced toxicity, although higher concentrations of arabinose lead to lower cell proliferation in general. Western blot results showed a distinct increase in C-protein expression, which was in line with our expectations. The RFP reporter was hard to distinguish in the Western blot, and did not reach threshold expression levels in spectrophotometric measurements. Figure 1. Growth curve in different Arabinose concentrations (0%, 0.05%, 0.1%, 0.2%, 0.4%) over the course of 10 hours. AraC3, empty vector was used as negative control. Absorbance OD600 was measured in 5 min cycles in a FluOstar plate reader, set to orbital shaking before each cycle. Figure 2. ClariOstar measurement of mRFP(ex:550-20/em:605-40). Cells were grown in a plate-reader for 10 hours, 120 cycles with 5 min intervals, 20 flashes for each cycle. AraC3 empty vector was used as negative control. As a positive control we used TOP10 cells transformed with the RFP coding device, BBa_J04450 in the pB1C3 backbone. Figure 14. Western blot of BBa_K3024009. Transfected TOP10 cells were grown in LB-media with different concentrations of arabinose from starting OD 0.05 for 5 hours at 37C. Samples were collected at stationary phase after 300 min. L= ladder -C= negative control. +C= positive control for RFP only. Anti-GAPDH was used as a loading control. See detailed protocol in the iGEM Stockholm 2019 wiki.