Overview / Rationale

While time delay might not be applicable in some situations, for example, containment of bacterial spill, it still has an implication on lab safety that must be met. Under a condition when modified bacteria secrete commercial products into a culture medium, this genetically engineered bacteria can be controlled to express within a constraint period of time before it is automatically knocked out. Keeping cell intact is crucial to prevent protein contamination in the media.

Therefore, this construct aims to set a timer for an inserted gene of interest to function for a controlled period of time before undergoing knock out by Cre recombinase. Knocking out of construct will lead to inactivation of the gene of interest, providing a secure and safe method for researchers to control the genetic modified organism (GMO).

Our Design

Time delay turns Cre recombinase either on or off dependent on the presence of AHL. All promoters in the Time delay module is a constitutively repressible promoter.

There are two scenarios of activity in Time Module Construct:

In the absence of AHL, only phlF molecules produce and repress its pphlF promoter. Then, the expression of downstream recombinase is rendered.

The second scenario is when sufficient AHL initiates cI protein production (BBa_C0051). The cI molecules will inhibit phlF expression by repressing pcI promoter (BBa_R0051). This turns on pphlF promoter, leading to expression of recombinase.

The length of time control is dependent on the strength of ribosomal binding site (RBS) downstream to pluxR promoter. The stronger the RBS strength, the higher recombinase expression.

Potential Issue

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Experiments & Results

In order to observe the change in GFP expression due to different ribosomal binding site strength, we select five RBS in this investigation: 0.01, 0.07, 0.6, 0.3, 1.0 respectively. RBS 1.0 will be the control in our experiment.

Additional

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References