Difference between revisions of "Part:BBa K3867009"

Latest revision as of 15:28, 20 October 2021

amilCP device

AmilCP coding device driven by Plac. LacI can bind with its promoter to negatively regulate gene expression. It expresses amilCP with IPTG induction.
we constructed a new amilCP device (BBa_K3867009) containing LacI gene, which is driven by Plac promoter. Both LacI and amilCP are connected to the downstream of Plac, which is regulated by both LacI repressor and IPTG inducer. This new amilCP device could be self-regulated because LacI protein can inhibit its self-expression. When amilCP needs expression, IPTG is required to be added into the culture. This induction expression could be considered as an economic expression way. Using the absorbance at 588nm (the maximum wave length of amilCP), we detected the response of this device to different concentration of IPTG, indicating that it could be inhibited by LacI, and induced well by IPTG.
In order to construct BBa_K3867009, overlapping PCR method was used to amplify the genes of Plac, LacI and BBa_K592015. Then the PCR product was inserted into plasmid pSB1C3 to create the new part BBa_K3867009. The identification result is showed in Fig.1.

Fig.1. The identification result of BBa_K3867009 construction. M: Marker; 1: PCR result of amilCP device from BBa_K3867009; 2: Plasmid of BBa_K3867009.

We detected the effect of IPTG induction on the two amilCP devices, using different concentration of IPTG. We set 4 groups: the original amilCP device (BBa_K592015), the new amilCP device (BBa_K3867009) with or without IPTG, and one negative control without amilCP expression. When all groups’ OD600 approximately reached to 0.6, a certain concentration of IPTG was added to the culture medium, incubated cells at 37℃ for 16h. Measure the absorbance values at 588 nm and 600 nm for each group every 2h, using an automatic microplate reader. The results are showed as follows (Fig.2-Fig.3).

Fig.2. The amilCP expression of BBa_K592015 and BBa_K3867009 with and without IPTG induction. The relative expression of amilCP is standardized with the ratio of OD588 value to the OD600 value for each group every 2h.

The figure showed that both BBa_K592015 and BBa_K3867009 expressed amilCP, and different concentration of IPTG had the same inducing trend for K3867009. The results indicated that BBa_K592015 expressed a mountain of amilCP without IPTG. However, BBa_K3867009 expressed a high level of amilCP only with IPTG presence. The leakage expression of amilCP is very low, and it is sensitive to the IPTG induction.

Fig.3. The comparison of amilCP expressed by BBa_K592015 and BBa_K3867009 with and without IPTG induction. The relative expression of amilCP is standardized with the ration of OD588 value to OD600 value for each group every 2h. “*”, significant difference compared to k38672009 without IPTG induction.

This figure indicated that the original amilCP device (BBa_K592015) can express amilCP with a high constant level. However, the new part amilCP device BBa_K3867009 express a high level of amilCP only with the IPTG induction, which is considered as an economic way.

Sequence and Features