Mutatis Mutandis

An En-“lightened” tool for directed evolution

Mutatis Mutandis

Evolving 'LEAD'ing solutions for an en'LIGHT'ened change

Heard of directed evolution?

A recent hot topic and a powerful tool in the field of bioengineering, directed evolution deals with mimicking the process of natural selection to create user defined phenotypes. The process involves iterative cycles of mutagenesis to generate gene libraries, selection of the desired phenotype and further amplification till the target phenotype is achieved.

Mutagenic System

The mutator plasmid can be used to upregulate the mutation rate of E.coli and thus acts as an efficient tool for directed evolution.

The plasmid contains mutD5, a mutant gene coding for a faulty DNA polymerase resulting in error-prone DNA replication.This reflects in the mutation rate of the bacteria. The mutD5 gene is under a blue light-inducible system that regulates the protein expression to different levels based on the intensity of the input blue light. A negative feedback loop is incorporated in the construct to prevent the accumulation of the mutD5 protein and thus the bacterial system from crashing.

Lead Bioremediation System

The Lead bioremediator construct can be used to collect and sequester lead inside the cytoplasm of the cells. The construct codes for a transporter and sequestration protein that is specific to lead and will help reduce lead ions in solutions effectively.

The transporter gene (pbrT) and sequestration gene (pbrD) are from the pbr operon of C. metallidurans which is responsible for lead resistance. The transporter gene (pbrT) is downstream of the Repressor gene and is constitutively expressed which ensures that the lead influx is continuous. The sequestration protein is downstream of the lead responsive promoter. This ensures that sequestration rate is high when the lead concentration is high.

Lead Biosensor

The Lead biosensor biobrick (BBa_K1758333) was developed by the Bielefeld Team. It contains sfGFP as a reported under a promoter that is lead responsive. The part also contains a promoter repressing gene which is constitutively expressed. The lead biosensor can be a cheaper alternative to current methods of lead detection.

The Repressor gene (pbrR) and lead responsive promoter (pbrAP) are from the bacteria Cupriavidus metallidurans, which is a heavy metal resistant bacteria. The pbrR protein is bound to the promoter and the reporter sfGFP is silenced. In the presence of lead ions, the pbrR protein dissociates from the promoter and the sfGFP is expressed. Thus, fluorescence intensity can be used as a readout for lead ion concentration in water.

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