Revision as of 10:41, 9 October 2023

P_Tet* promoter + RBS

Introduction

The 2023 UT Austin iGEM Team’s Parts Collection includes a multitude of parts necessary for engineering bacteria to secrete microcins, a type of small antimicrobial peptide. Specifically, our team has designed parts that allow us to engineer a modular two-plasmid microcin secretion system that secretes putative novel microcins predicted by bioinformatics analysis. The first plasmid—the ‘microcin’ plasmid—contains the microcin and a signal peptide, while the second plasmid—the ‘secretion system’ plasmid—contains genes for two proteins of the E. coli microcin V type I secretion system (T1SS) machinery collectively referred to as cvaAB. Our parts can be easily assembled into transcriptional units to express any of our current novel microcins either constitutively or under inducible control.

Usage and Biology

This is an inducible promoter part compatible with Golden Gate Assembly using the Bee Toolkit (BTK) syntax (Leonard et al., 2018). This part undergoes BsaI digestion to produce a linear part with overhangs characteristic of a Type 2 part in the BTK syntax (see Figure ?????????). This part consists of the P_tet promoter upstream of a hammerhead ribozyme (HHRz), the latter of which helps insulate the transcript from sequence-specific interference effects. The P_tet promoter can be bound by the repressor protein TetR, leading to a decrease in transcription of genes downstream of this part. TetR may be removed from the promoter when bound by the inducer molecule anhydrotetracycline (aTc), allowing for the selective induction of transcription on plasmids containing both P_tet and the tetR gene.

Figure 1. BTK/YTK part types

Sequence and Features