Difference between revisions of "Team:BostonU/Proof"

Phase 1 Results

The first phase of our project was to develop a library of digital parts. Our journey began with finding the guide RNA sequences we would use in our work. We generated over one thousand 20 base guide RNA sequences in silico using a random sequence generator.

It was critical these guide RNA sequences be orthogonal to the human genome because we wanted to test our system in HEK293FT cells and prevent off target activation in the host genome. To test for orthogonality, we entered the sequences into the CRISPR Optimized Design tool developed by the Feng Zhang lab. We selected the top 18 sequences, which had an orthogonality score of 98% or higher, to synthesize and use in our research.

We synthesized these guide RNA sequences through IDT and cloned each one into guide RNA expressing vectors and guide RNA operator pairs. To act as controls for our experiments, we also cloned the guide RNA sequences Tre and UAS into guide RNA expressing vectors and guide RNA operator pairs. These 20 initial operators expressed an iRFP gene.

We transiently transfected and ran through a flow cytometer our paired gRNA expression vectors and gRNA operator reporters and dCas9-VPR to test their expression behaviors. We wanted to see high activated states coupled with low basal expression, a true digital system. The screen of this can be seen below.

Based on this screen, we decided to proceed with guide 1, guide 3, guide 8, and guide 13 for all future experiments. Next, we needed to prove that our parts could drive a diverse library of genes of interest. To complete this, we replaced the iRFP in our guide RNA operator reporters with a GFP, a BFP, and an mRuby protein. The results from these test can be seen in the matrix below.

In all cases, there was low basal expression and strong activation, proving that our system not only behaves digitally, but that it could also do so over multiple genes.

Finally, our system needed to be mutually orthogonal to prevent significant undesired crosstalk. If there was off target gene activation between operators, then we could not transfect multiple operator-expression pairs and obtain predictable results. We performed an orthogonality transfection with the GFP driven operators from our library. The experiment was designed such that every guide RNA expressing vector would be paired with each guide RNA operator reporter vector. We predicted significant GFP fluorescence when the guide RNA expressing vector has the same guide RNA sequence as the guide RNA operator reporter vector, and no significant expression elsewhere. The results of the experiment can be seen in the matrix below.

The diagonal down the matrix demonstrates that there is mutual orthogonality between the guides in our system. With this matrix, we proved that we developed a digital parts library.

Our final test was to compare the relative strength of our parts to a CMV, a strong mammalian promoter, to determine the relative strength of our system. This would also allow for a better interpretation of where our parts fit into the grand scheme of synthetic biology. The graph below shows the results from this experiment.

While our parts expressed well, they did not have a higher expression than the CMV. This was not particularly surprising. But it did raise the question, would we ever be able to show higher activity with a minimal CMV in our system than with a full CMV?

Our next step was to expand to a functioning analog library...

Phase 2 Results

The next step in developing our library was to expand into the analog domain. We are more specifically referring to an array of quantized levels of expression that on a macroscopic scale appear analog, similar to a Riemann Sum of an analog signal.

To create this “quantized-analog” pattern, we had two driving hypotheses: multimerize the operators to increase expression and mutate the operators to decrease expression.

The conceptualization behind multimerizing, or adding multiple binding sites for the dCas9, harkens to previous work done with TAL effectors and Zinc Finger motifs. By adding additional binding sites, we expect to recruit more dCas-VPR transactivators to the operator vector, increasing expression and producing a synergistic effect. Additionally, we varied the space between binding sites. The motivation behind this was that we wanted space for the dCas9-VPR to bind effectively. The results for these experiments for each of the four guides in our library can be seen in the graphs below.

In general, increasing the number of binding sites also increased the expression of the operator reporter. Additionally, with only minor variations from this trend, increasing the space between binding sites also increased expression. This set of experiments proves that multimerization is an effective technique to increase gene activity.

We also compared the relative strength a CMV from the Registry to one of our multimerized operators, consisting of three binding sites for guide RNA 13. CMVs are known among mammalian synthetic biologists as being a strong constitutive promoter. The data from this comparison can be seen below.

As the graph demonstrates, our triple multimerized operator containing a minimal CMV had greater expression than the full CMV. This proves that our system produces additional varied levels of gene activation as well as have some temporal inducibility.

After we completed cloning and assaying our multimerized operator reporters we sequentially mutated every base in our twenty base guide RNA sequences, beginning at the five prime end. Every adenosine was mutated to a cytosine and vice versa and every guanine was mutated to a thymine and vice versa. This produced a complete shift from purines to pyrimidines of the opposite DNA base pair. In addition to these single base mutations, we also clustered 2 and 3 point mutations around base 1 and base 11 in the guide. The results of this screen can be seen below.

Though the data did not produce the decreasing step function we had expected, there were some results of extreme interest. The mutations on bases 4 through base 7 produced a distinguishable downward trend. Moreover, mutations at base 10 and base 11 produced nearly a fivefold decrease in expression when compared to the non-mutated operator. From our multi point mutations, the data did not represent any significant or controllable changes in expression. Therefore, we have decided to not move forward with multi-point mutations.

With this experiment we have proven that we have developed an analog parts library. We have a system than can range from five times below normal expression to five times above normal expression under a minimal promoter, out competing a strong full promoter. Now, what can we accomplish with our library of both digital and analog parts?

An answer lies in Genetic Logic Circuits...

Phase 3 Results

Now that we could achieve a variety of expression levels, we began integrating our system into recombination based circuitry. The circuit we used could express a different gRNA for every combination of drugs introduced. Before integrating our multimerized plasmids, we first tested the circuit by making each gRNA correspond to a different fluorescent reporter. The data for this experiment can be found below. There were four possible combinations of two drugs, and each combination yielded a relatively high expression of its corresponding fluorescent gene.

Results

Results