Problem Statement

Life science research utilizing cell cultures is very labor intensive, as it requires fixed time-points over the course of an experiment. The current solution to this problem is to assign graduate students experiments that often are very demanding and exhausting. Thus, life scientists performing tissue culture or bioreactor work could utilize new solutions to create time savings in their experiments.

Needs Analysis

Thus, to address this problem an autonomous, biocompatible solution must be implemented to deliver some molecular payload at a predetermined time interval to address this issue. It must be durable and reliable under the lengths of various experiments and the harsh conditions exposed in cell culture medium. It must not produce a graded response, rather, a strongly bistable system is highly preferable, as this would prevent overfeeding of the culture system. Lastly, it must be able to keep time in some fashion, so as to address the various timepoints necessary in typical culture experiments.

Solution

A solution utilizing an oscillating gene circuit coupled to a positive feedback loop looks to be a highly promising solution. This would be able to initially have a lag time, with a basal level of product production. However, after some period, enough of this product is made so that the system is able to rapidly increase, in a similar manner to a toggle-switch, which exists in an on/off state. In the following figure, the cI-SSrR is an unstable protein tagged for degradation.

However, it also activates Prm, which is a promoter that is able to produce cI coupled to a YFP (Yellow Fluorescent Protein) operon enabling the measurement of the stable cI gene product. Utilizing this positive feedback system in yeast cells enables it to be a biocompatible, timed system.

Goals and Experiences to be Gained

1. Construction of a functional gene circuit
2. Complete dynamic modeling of the gene circuit system
3. Measure the degradation rate of cI geneproduct
4. Understanding molecular biology techniques required to implement the solution