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Revision as of 16:43, 8 October 2016
Clamp Down on Crosstalk
Alverno_CA iGEM
Clamping Down on Crosstalk:
How can we keep genes from interfering with each other in synthetic DNA circuits?
About this Project
leading to unpredictable behavior of synthetic biology systems. We will test if DNA clamps (made from DNA-binding proteins) placed between
genes can stop this interference. If this project succeeds, it will allow bioengineers to build more predictable genetic circuits.
What is the context of this research?
together on the same piece of DNA. Bioengineers have observed that
when two genes are placed next to each other, they often unexpectedly interfere with each other's
expression in an unexpectedly orientation-dependent manner. Nobody knows with certainty what causes this genetic crosstalk, but one promising
theory involves DNA supercoiling. The transcription of DNA into RNA, the transcription process introduces supercoils, similar to kinks in a tightly-wound phone
cord. Supercoils directly affect the expression of genes, turning them on or off depending on the direction of the supercoil.
What is the significance of this project?
This is particularly relevant when engineering metabolic pathways to produce chemicals
like methanol, insulin, or antibiotics, where circuits of many genes are routinely constructed. The physical layout of these circuits can unpredictably affect production of
the desired output by several orders of magnitude, so large engineered metabolic pathways must typically be hand-tuned or have many configurations screened for activity. By
making gene expression more predictable, our results could greatly improve the predictability (and, therefore, designability)
of large gene circuits for metabolic engineering.
What are the goals of the project?
demonstrate cross-talk between genes. These plasmids
will consist of genes for two different fluorescent proteins (green fluorescent protein and red fluorescent protein) next to each other, in different orientations.
We expect to see differences in the relative expression of the two genes depending on how they are arranged and oriented, and we will quantify this effect.
Next, we will try several strategies for removing these differences, including adding extra base pairs of spacing between the two genes and adding DNA "clamps"
made from DNA-binding repressor proteins between the two genes. We will again quantify the effects of cross-talk between genes,
which will hopefully be ameliorated by our additions.
