Difference between revisions of "Part:BBa K3519010"

The araC protein is constitutively produced by the medium strength Anderson promoter (J23118) and the strong RBS (B0034) and production of any downstream gene (gene of interest) is controlled by the P-araBAD.

The AraC protein forms a dimer, each molecule binds to the araI1 and araO2 regulatory regions of the DNA and prevents the binding of RNA polymerase to the P-araBAD promoter region and hence represses the transcription of the downstream gene.

In the presence of Arabinose (and low amount of glucose sugar), the Arabinose binds to the AraC protein dimer, brings about a conformational change and binds to the regions araI1 and araI2 regions of the DNA releasing the P-araBAD promoter. Now, RNA polymerase binds to the P-araBAD promoter and proceeds with transcription. The incorporation of RNA polymerase at the P-araBAD promoter is enhanced by the CAP-cAMP complex bound to the CAP binding site. As the concentration of cAMP is inversely proportional to the concentration of glucose, for a constant amount of CAP protein, lesser the concentration of glucose, more will be the concentration of cAMP molecules, hence enhanced transcription of any downstream gene.



Figure 1: The designed arabinose inducible promoter system. The araC is constitutively transcribed in the opposite direction. This promoter system was designed as a tightly regulated, user modulated, inducible system and is suitable for use in a ‘kill switch’.

References:

1. Guzman, Luz-Maria, et al. "Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter." Journal of bacteriology 177.14 (1995): 4121-4130
2. https://parts.igem.org/Part:BBa_K2442101

Team: BNDS_China 2022 Contribution

Characterization design

The arabinose-induced GFP expression plasmid—pGR-ECK120015170—which contains araC operon, araBAD promoter, RBS, GFP, ECK120015170 terminator, and RFP (Fig. 1) from Ying-Ja Chen’s work was used to characterize terminator ECK120015170. The GFP gene was set upstream and RFP was placed downstream of the ECK120015170 terminator.

Fig 1. The design of the main part of the genetic circuit

We titrated the induction of arabinose by varying the concentration of the inducer. The ratio of GFP relative intensity to ABS600 value was the amount of protein expression despite the effect of strain concentration.

Quantitative characterization

Previous studies have shown the feasibility of using green fluorescent protein (GFP) as a quantifiable reporter gene, so it was available to use the intensity of GFP fluorescence to reflect the amount of protein expression upstream. The GFP intensity over OD value can show the amount of protein expression. The ratio due to the different concentrations of arabinose is visualized in Fig. 2.

Fig 2. The arabinose titration curve

Limitation of experiment

When the inducer arabinose was added, the way in which arabinose enters the cytosol was not taken into account. Unlike other inducers, arabinose requires a transporter protein called araE to assist its entry into the cytosol. Therefore, probably because this was not taken into consideration, our gradient induction results were not rigorous enough.

Sequence and Features