Team:MIT/Experiments/Recombinases

• BACKGROUND
• EXPERIMENTS
  Promoters miRNA Recombinase Combining parts Collaborations
• FUTURE WORK & DESIGN
  Circuit Design Clinical Application
• HUMAN PRACTICES
  Accessibility Integrated Human Practices Public Outreach
• TEAM
  Team Acknowledgements
• NOTEBOOK
  Lab Notebook Basic Parts Parts collection Attributions Safety

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. Capturing chronological molecular traits is very important in diagnosis of many diseases. For our project, we use recombinases, DNA binding proteins, to achieve this temporal specificity.

A recombinase excises a segment of DNA.
Source: University of Rochester Introductory Biochemistry.

Recombinases are enzymes that can recognize target sequences, and depeding on their orientations, 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 out 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. Thus, unidirectional recombinases often display higher efficacy in DNA modification compared to bidirectional recombinases.

We can use recombinases as biological latches in our circuit to gain temporal specificity. Once a cell is identified as having an abnormal hormone level and the miRNA profile characteristic of a diseased cell, 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, a second recombinase latch can be triggered, activating the overall circuit.

Figure. Showing the mechanism of recombinase biological latches capturing temporal specificity during estrogen and progesterone cycle. 1) Disease-related biological traits during the estrogen high phase activate the inducible promter, leading to 2) expressiong of recombinase 1. Recombinase 1 would, then, 3) "lock-in" this information by irriversible gene modification. Similarly, 4) during the progesterone high phase, biomarkers associate with the disease would activate another promoter, leading to 5) expression of the second recombinase. Two irreviersible gene modification events performed by recombinase 1 and 2 at different time point form an AND gate, activaing expression of an output gene.

Do our recombinases work?

We investigated 2 models of recombinase for regulating gene expression:

1. Using a unidirectional tyrosine recombinase (CRE) to excise a transcriptional stop signal, allowing a downstream gene to be expressed.
2. Using a unidirectional serine recombinase (TP901) to flip gene from an off to an on orientation.

Figure. Regulating gene expression using recombinases models.

Our experimental data showed that:

1. The flipped gene system (2nd model) successfully knock down the expression of the gene, while the transcriptional stop signal (1st model) did not.
2. The expression level of the flipped gene can be indirectly controlled by expression of the recombinases (TP901) under an inducible promoter.

Read more about recombinases experiments here

Challenges with Recombinases

Recombinases are highly efficient enzymes. When combined with a high basal level of activity of the promoters, this presents a challenge. 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 for the transcription of the recombinase if the repressUsing a unidirectional serine recombinase (TP901) to flip gene from an off to an on orientation.or 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. After talking to experts in the lab, we decided to test the L7Ae k-turn system.

Read more about our L7Ae k-turn experiment here