Difference between revisions of "Part:BBa K3634012"

Revision as of 10:28, 7 August 2020

Glucose-Mediated Death Sensor

The lac operon found in E.coli consists of the three lactose metabolising genes lacZ, lacY and lacA which when expressed, allow the bacteria to use the sugar as a source of energy. The initial regulatory mechanisms in the pathway were outlined by Jacob and Monod in 1961, where the topic of inducible and repressible enzyme systems was discussed. In this system, the transcriptional repressor is a protein known as Lac I which binds to DNA at various operator sequences (termed O1, O2 and O3) which exist both upstream and downstream of the transcriptional start site (TSS). Interaction between the Lac I and operator sequences reduces transcription of the downstream lactose metabolising genes unless relieved by the lactose isomer allolactose. In the absence of Lac I, transcription is constitutive and can be further activated by the catabolite activator protein (CAP), with binding site upstream of the promoter sequence.

Oehler et al. (1990) mutated each individual operator sequence respectively and then determined the effect of repression by Lac I to which they found mutation in O1 (downstream of the promoter) sufficient to lose almost all total repression. Mutation of O2 and O3 further decreased repression by Lac I 70 fold. Here, the St Andrews iGEM team 2020 aimed to utilise these findings to create a regulatory region solely under the control of glucose concentration to allow expression of the toxin ccdB.

The ccdAB toxin-antitoxin (TA) module is a type II TA module where the toxic ccdB protein, poisons the enzyme DNA gyrase, required for negative supercoiling of DNA (Bernard and Couturier, 1992). Through ccdB-gyrase complex formation, DNA cleavage results as well as inhibition of transcription by the formation of RNA polymerase roadblocks. The activity of the unstable ccdA antitoxin separates the ccdB-gyrase complex if present (Vandervelde et al, 2017).

The 'glucose-mediated death sensor' is a vital part of the St Andrews iGEM 2020 kill switch as when glucose is absent, greater expression of ccdB will overcome intracellular ccdA concentrations causing transcriptional inhibition and DNA cleavage as previously discussed. Expression of ccdB will also relieve the associated ccdAB promoter from ccdA binding which as a consequence, will allow for cviJI endonuclease expression to chew up the integrated plasmid constructs and target restriction sites within the genome. Sequence and Features