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Team:Brown/Project/Light pattern
From 2010.igem.org
Light Pattern Controlled Circuit
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Abstract
Biological manufacturing of complex compounds often requires the synthesis of many intermediate products. Production of these intermediates is currently triggered by inefficient methods, such as chemical inputs (tetracycline, estrogen-analogs, arabinose, etc) or drastic changes to the cellular environment (pH, oxygen levels, temperature, etc). On an industrial scale, this chemical induction requires large quantities of reagents and extensive purification, while environmental induction requires conditions that can adversely affect cell vitality and yield. To this end, we are engineering an E. coli genetic circuit that can pass through four stable states of protein production triggered solely by ON/OFF patterns of light. With this production method, we can link multiple synthesis steps to a single, clean and rapidly scalable input.
Overview
Our project this year attempts to tackle what we see as emerging issue in synthetic biology: the complexity of circuit inputs. There is a growing trend among iGEM projects towards intricate systems; the registry of standard parts is growing quickly and the tools at the disposal of a synthetic biologist are increasing rapidly. While many of these systems rely on an autonomous progression of events in the chasis - say, a cell encounters some environmental stimulus which triggers downstream responses - some require precise user control. This is especially true for projects in the manufacturing area, as often a product is achieved following a progression of steps within the cell. Functioning "behind the scenes" to achieve this sort of controllable progression is the genetic circuit, consisting of plasmids loaded with promotor and transcription factor pairs. Through a combination of regulators and activators, these circuits achieve distinct "states," or set of functions carried out by the cell. To move among states, a user provide some specific input to the cells. Whether chemical, heat, or some other environmental change, it is common practice that for each state, a unique input must be applied. With this approach, it is quite clear that an increasingly complicated circuit, with many different promotors, requires an increasingly intricate set of inputs.
We believe this to be a problem for a few reasons:
- Accessibility
- From an end user perspective, navigation across states could become nightmarish. Especially for a system targeted beyond the lab, perhaps at a non-technical setting, input/control must be accessible and easily attainable.
- Expense
- Chemical induction of individual components of a circuit, particularly on a large bioreactor scale as in the manufacturing of ____ , is expensive! For a full fledged, industrial bioreactor, an enormous amount of inducer must be introduced. One chemical input alone on this scale can vastly increase the cost of the end product; multiple chemical inputs drives costs extremely high.
- Interference
- Environmental changes, such as heat or pH, may remove a cell from optimal conditions for protein production. Again, from a manufacturing standpoint, a decrease in yield per reaction increases costs and decreases overal efficiency.
Where does this leave us? With room for improvement, of course! We sought to address the shortcomings detailed above in engineering of a four state circuit that is not controlled by multiple inputs. To avoid complexity in input, we created a kind of dependence of each logic component of the circuit on the presence or absence of light. We advanced past iGEM work on light control of circuits beyond a two state toggling of LIGHT OFF -> state 1 ; LIGHT ON -> state 2; LIGHT OFF -> state 1. Rather than returning to state 1 given a light change back to OFF, our circuit takes advantage of a memory component to effect a unique state 3. See graphic.
Workflow/Methods
Modeling
See our modeling page at: blah blah
Results
