Team:MIT/Experiments/Recombinases

• BACKGROUND
• EXPERIMENTS
  Synthetic Promoters microRNA Recombinase Proof of Concept Collaborations
• FUTURE WORK
  Circuit Design Clinical Applications
• HUMAN PRACTICES
  Parts Toolbox Accessible Protocols Integrated Human Practices Public Outreach Interlab Study
• NOTEBOOK
  Lab Notebook Parts Mammalian Parts Collection Attributions Safety
• AWARDS
  HP Silver HP Gold Integrated HP Basic Part
• TEAM

• Hormones Responsive Promoters
• miRNA Profile
• Recombinase Biological Latch

How does endometriosis respond to the menstrual cycle?

Endometriosis cells respond to the hormones associated with the menstrual cycle. Interestingly, the miRNA profile of these cells is different during the proliferative versus the secretory phase. TALK MORE ABOUT THIS STUFF. I DON’T KNOW ANYTHING :(

How can our circuit demonstrate temporal specificity?

Endometriosis cells have distinct characteristics at different points in the menstrual cycle, presenting a major challenge in identifying diseased cells. It is crucial that there is a way to achieve temporal specificity.

Recombinases are enzymes that can recognize target sequences, and depending on their orientation, can either cut out DNA between the 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 once they reverse or cut the sequence the action cannot be undone. This means that instead of behaving like a switch, capable of turning on or off, unidirectional recombinases behave as latches.

We can use recombinases as biological latches in our circuit to gain temporal specificity. Once a cell is identified as having the miRNA profile characteristic of a diseased cell, a recombinase can be activated to essentially “lock in” that information. When the second half of the circuit confirms the cell as being diseased, a second recombinase latch can be triggered, activating the overall circuit.

Do our recombinases work?

We used the serine integrase TP901 to flip an inverted eYFP gene. We first tested this system under an inducible promoter, EGSH.

Read more about recombinases experiments here

Challenges with Recombinases

Recombinases are highly efficient enzymes. When combined with a high basal level of activity, this presents a challenge. In order for the effective 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 for the transcription of the recombinase if the repressor protein is present. The three repressors we investigated included BM3R1, TAL14, and TAL21 because of their demonstrated success in the literature.

Read more about repressor experiments here

Translational Regulation: L7Ae - kink turn

Read more about our L7Ae k-turn experiment here