Latest revision as of 21:43, 13 October 2022

P hag

The P hag promoter controls the hag gene, which is responsible for flagellin production in B. subtilis. It is one of the strongest known constitutive promoters in the genome. It requires binding of the SigD transcription factor, which can be deactivated by a protein known as flgM. There are also two binding sites for an inhibitory protein.

Introduction

The Bacillus BioBrick Box designed by LMU-Munich iGEM 2012 was used extensively in the McGill 2022 project. The box contains three B. subtilis integrative vectors targeted to the amyE, thrC, and lacA sites, as well as constitutive and inducible promoters with varying strengths. We decided to design a fourth integrative vector and an accompanying promoter as our parts contribution.

Biology

Phag is the promoter of the hag gene, which encodes the flagellin protein. It has been shown to be one of the strongest contitutive promoters found in B. subtilis. In addition, the hag region of the genome, including flgM, is very well conserved. As such, the Phag promoter offers strong and stable protein expression.

Phag is inhibited by CsrA (“Carbon storage regulator A”), which binds at two sites of the promoter (denoted BS1 and BS2) and prevents RNA polymerase from transcribing the downstream gene. Our designed BioBrick incorporates a single base-pair mutation in both these sites, which prevents CsrA binding.

Active Phag requires binding of the SigD transcription factor at the -10 and -35 SigD sites. When flgM binds to SigD, it prevents binding to Phag and therefore inhibits protein expression. The flgM gene is disrupted when pBSChag integrates, thus achieving constitutive expression.

Sequence and Features