From 2007.igem.org

Growth Control

Introduction

To prevent chance of infection or unwanted proliferation after hemoglobin production, we have engineered a genetic self-destruct mechanism whereby when induced, the bacterial cell will express a genetic material-degrading toxin which kills the cell, but leaves it physically intact.

Growth control in our system is established by the incorporation of a plasmid that can be triggered to translate a toxin. The toxins are endonucleases or RNAses that destroy the genetic material within a bacteria and thus prevent the bacteria from replicating. Throughout the summer, we worked on several different constructs of an inducible toxin and screened for the ones with the best phenotype.

 

An Inducible Toxin

Using a PBAD promoter, we constructed several variations of inducible toxins, including the colicin DNAse CeaB, endonucleases BamHI and BglII, and RNAse barnase. Additionally, ribosome binding site libraries were used in order to increase the likelihood of finding a construct that would exhibit no growth after being induced with arabinose, but normal growth when uninduced.

I716408C:

We screened libraries of potential hits with a Tecan growth assay, and in the end, the constructs that showed the desirable phenotype are the constructs shown below (I716408C and I716462). When uninduced, the cells show growth comparable to regular DH10B cells, but when induced, the growth plateaus as the cells lose their ability to replicate due to RNA and DNA destruction.

 

 

I716462:

 

 

 

 

Characterization of Growth Ability

 

Phenotype of dead cells