Team:Carnegie Mellon/Bio-Overview

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Revision as of 19:49, 29 September 2012

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Promoter variants

The BioBricks are T7/Lac promoters that are mutated from both the T7 promoter and in the lac operator, which offers unique functionality in cells. This year, we are submitting a total of 4 BioBricks to the Registry of Standard Biological Parts. See our method of analysis

T7

The T7 promoter comes from the T7 phage and is a class of very strong promoters. T7 promoters can only be expressed in strains of bacteria with the gene that codes for T7 RNA polymerase (the unique polymerase that binds to the T7 promoter). The T7 promoters come in different classes, and each class corresponds to a different level of expression. The commonly used T7 promoter is the class I variant because of its ability to produce a high amount of protein. The T7 promoter has 3 distinct regions: the recognition site, the melting box and the initiation site. The recognition site is the specific region that the T7 promoter binds to. The melting box is highly conserved, consisting of TATA. The melting box allows the T7 RNAP to open the two strands of DNA and start adding NTPs to build mRNA. The last region is called the initiation site; this is where the first nucleotide of the mRNA is added. Most prokaryotic RNAPs favor the addition of adenine to start transcription but T7 RNAP differs in that it favors the addition of guanine. As a result, most T7 promoters have a poly-G region of 3-5 nucleotides to increase the chance of initiation. BBa_K613007 is a classic example of a T7 lac promoter but let's analyze the T7 region.

BBa_K613007:TAATACGACTCACTATAGGGAGAGGAATTGTGAGCGGATAACAA
T7 region:
TAATACGACTCAC - Recognize
TATA - Melt
GGGAGA - Initiate Transcription

LacO/LacI

The Lac operator is a DNA sequence upstream of the gene of interest that binds to the lacI repressor. The lacI repressor is found in the lac operon in E. coli. The lacI repressor prevents transcription from occurring by forming a "hairpin" like structure that prevents RNAPs from traveling along the DNA. The lacI repressor releases the DNA when lactose is present and binds to it. In E. coli, the gene that codes for ß-galactosidase is turned on when lactose is present. This allows for E coli to have an alternative carbon source but conserve its energy when it needs to. This property can be exploited to prevent expression of a certain gene unless lactose or a lactose analog is added to the cells. Lactose is consumed in E. coli because of the presence of the lac operon. Instead, researchers found an analog (called IPTG) that binds to the lacI repressor but is not consumed by the ß-galactosidase enzyme. The wild type lac operator is nearly-symmetrical and has its own binding properties, similar to that of the T7 promoter. In our constructs, we characterized a symmetrical lac operator and measured its leaky expression (the amount of expression without the inducer present).

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