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<center><img src = "https://static.igem.org/mediawiki/2016/2/2b/T--MIT--EGSH-TP901_graph.jpg"; width = 50%></center> | <center><img src = "https://static.igem.org/mediawiki/2016/2/2b/T--MIT--EGSH-TP901_graph.jpg"; width = 50%></center> | ||
− | <i> <b> Figure. </b> Recombinase TP901 showed approximately a two-fold increase between the off and on states under PonA-inducible promoter EGSH. </i> | + | <center><i> <b> Figure. </b> Recombinase TP901 showed approximately a two-fold increase between the off and on states under PonA-inducible promoter EGSH. </i></center> |
<a href="https://2016.igem.org/Team:MIT/Experiments/EGSH_TP901_Experiment"><p style="font-family: Trebuchet MS;font-color:#7ECEFD; font-size:15px;"><i><b>Read more about recombinase experiments here</b></i></p></a> | <a href="https://2016.igem.org/Team:MIT/Experiments/EGSH_TP901_Experiment"><p style="font-family: Trebuchet MS;font-color:#7ECEFD; font-size:15px;"><i><b>Read more about recombinase experiments here</b></i></p></a> |
Revision as of 17:53, 19 October 2016
Recombinases:
Giving Memory to a Genetic Circuit
How can our circuit demonstrate temporal specificity?
Source: University of Rochester Introductory Biochemistry.
Endometriosis cells have distinct characteristics at different points in the menstrual cycle, presenting a major challenge in identifying diseased cells. Capturing chronological molecular traits is very important in the diagnosis of many diseases. For our project, we use DNA binding proteins called recombinases to achieve this temporal specificity.
Recombinases are enzymes that can recognize recombination sites, and can either cut out the DNA between these recognition sites or invert the DNA sequence. There are two main families of recombinases: serine recombinases (also sometimes called serine integrases) and tyrosine recombinases. Serine integrases invert sequences, while tyrosine recombinases can either cut or flip sequences depending on the orientation of recognition sites. Some recombinases exhibit unidirectionality, meaning that once they reverse or cut out the sequence, this action cannot be undone. This means that instead of behaving like a switch, capable of turning on or off, unidirectional recombinases behave as latches. Thus, unidirectional recombinases display higher efficacy in DNA modification than bidirectional recombinases. We can use recombinases as biological "latches" in our circuit to gain temporal specificity. Once the abnormal hormone level and the miRNA profile characteristic of a diseased cell have been identified during one phase of the menstrual cycle, the first recombinase can be activated to essentially “lock in” that information. When the second half of the circuit confirms the cell as being diseased in the second phase of the cycle, a second recombinase latch can be triggered, activating the overall circuit.
Do our recombinases work?
We investigated 2 models of recombinase for regulating gene expression:
- Using a unidirectional tyrosine recombinase (Cre or FLP) to excise a transcriptional stop signal, allowing a downstream gene to be expressed.
- Using a unidirectional serine recombinase (TP901) to flip gene from an off to an on orientation.
Our experimental data showed that:
- The flipped gene system (2nd model) successfully knocked down the expression of the gene, while the transcriptional stop signal (1st model) did not.
- The expression level of the flipped gene can be indirectly controlled by expression of the recombinases (TP901) under an inducible promoter.
Read more about recombinase experiments here
Challenges with High Efficiency of Recombinases
Recombinases are highly efficient enzymes. When combined with a high-basal-activity promoter, this presents a challenge. A few copies of the recombinase due to promoter's leaky expression, could lead to significant amount of undesired output gene expression. In order to effectively use of recombinases as biological latches, basal expression must be reduced as much as possible. A strong repression system must be used in order to reduce leaky expression.
Repressible Promoters
In order to gain tighter control of the recombinases, we paired them with repressible promoters that do not allow transcription of the recombinase to take place if a specific repressor protein is present. The three repressors we investigated included BM3R1, TAL14, and TAL21 because of their demonstrated success in literature.
Read more about repressor experiments here
Translational Regulation: L7Ae/kink-turn
We did a lot of research into effective high level repression systems, such as degradation tag RNA-based gene regulation systems. After talking to experts in the Weiss Lab, we decided to test the L7Ae k-turn system due to the availability of the system's components.
Read more about our L7Ae k-turn experiment here