Part:BBa_K3245006

Usage and biology:

This part is used to express tetR under the control of our engineered quorum sensing promoter luxpR(HS100). This part features low leakage when luxpR is not induced, approximately one fifth of that of BBa_K234005. To use it, we recommend you to couple it with BBa_K3245013 to achieve 2-step regulatory expression (luxpR-tetR-ptetR). You should clone BBa_K3245006 into a low-copy plasmid and BBa_K3245013 into a high-copy plasmid to achieve maximum regulatory effect.

Design:

This year, our team aims to construct a luxpR-tetR-ptetR secondary regulative system. The main problem we encountered was that ptetR is a strict regulative promoter while luxpR has unnegligible leakage. If we try to express tetR with wild-type luxpR and couple them with downstream ptetR, the leakage of WT luxpR is already enough for ptetR to stop working. Under this circumstance, we must design a low-leakage tetR expression system. To achieve this, we put BBa_K3245009 (luxpR-HS100) upstream of C0040 instead of R0062 (wild type luxpR).

Characterization:

Protocol:

1. measurement of C0040 leakage:

Required earlier steps: clone part BBa_K3245006 and BBa_K3245013 into plasmids and transform them into E. coli DH10B. We recommend you to clone BBa_K3245006 into a low-copy plasmid (single-copy plasmid is the best choice), and BBa_K3245013 into a high-copy plasmid. Use bacteria PCR to confirm that both plasmids are successfully transformed, then incubate in liquid LB medium overnight. Cryopreserve this strand.

1) Take 10µl culture out of preservation tube, streak the culture on an LB plate and incubate at 37˚C for 18 hours.

2) Pick up 3 colonies and incubate each of them in liquid 2*YT medium at 37˚C overnight.

3) Take 500 µl culture out of each culture, centrifuge at 12000 rpm for 1 minute.

4) Dispose supernatant fluid and re-suspend the sediment with 1000 µl PBS to prepare suspension.

5) Add 100 µl suspension into a 96-well plate. Use 100 µl PBS as blank control. Read fluoresce at 420nm activation wavelength and 528nm absorbance wavelength. Also measure OD600.

6) Use iGEM 2019 Data Analysis Template: Fluorescence Standard Curve Protocol to calibrate data according to your standard curve and calculate MEFL/particle value.

Result:

Fig.1 Comparison of tetR (C0040) leakage of BBa_K3245006 in different vectors. +: DH10B transformed with only ptetR-B0034-GFP (BBa_K3245013) in a pBR322-ori plasmid, serve as positive control. PST-HS100: DH10B transformed with both luxpR(HS100)-B0033-tetR (BBa_K3245006) in a single-copy plasmid PST-BSD and ptetR-B0034-EsfGFP (BBa_K3245013) in a pBR322-ori plasmid. p15A-HS100: DH10B transformed with both luxpR(HS100)-B0033-tetR (BBa_K3245006) in a medium-copy plasmid p15A and ptetR-B0034-GFP (BBa_K3245013) in a pBR322-ori plasmid. p15A-WT: DH10B transformed with both luxpR(WT)-B0033-tetR (BBa_K3245006) in a medium-copy plasmid p15A and ptetR-B0034-GFP (BBa_K3245013) in a pBR322-ori plasmid. DH10B: wild type E. coli DH10B, serves as negative control.

We expected that our luxpR(HS100)-tetR expression system would show less inhibiting effect on ptetR than the luxpR(WT) system. To confirm this, we measured the leakage of luxpR(HS100)-tetR expression system by coupling it with ptetR-EsfGFP and measuring how it inhibits the strength of ptetR. From figure one we can conclude that luxpR (HS100)-tetR system has been significantly improved compared to luxpR (WT)-tetR system in that it has over two times weaker influence on ptetR under its leaking stage compared to the WT system. Further improvement of tetR expression system is conducted by changing low copy replicons to reach nearly no inhibiting effect on ptetR.