As a foundational project, we spent a considerable amount of time thinking about how to best build our platform as well as how to design the different parts to be introduced in a new system. Adapting a molecular system from one organism to another can be challenging especially within the timeframe of iGEM, therefore, we opted for a phased approach as described in our project description. The fact that we had to build all our own parts from scratch required many in silico engineering design cycles to get to our final in silico constructs. Throughout our project, we have made sure to document all of our design decisions, which can be found under the Experiments page of our wiki. More detailed general documentation on our engineering success can be found below.
PCR amplification issues with the EvolvR gene
One of the very first problems we faced in our project was that the EvolvR gene seemed to be difficult to amplify in a PCR reaction following our PCR protocol with NEB’s Q5 Master Mix. We first tried to amplify it with overhangs for Gibson Assembly into a Gateway entry vector destined for Golden Gate Assembly. However, the EvolvR coding sequence is very long (up to 7kb depending on the combination of Cas9 and DNA polymerase used). Later in the project, we faced similar issues when trying to clone EvolvR into the pALiCE01 vector for the cell-free experiment. Despite changing PCR conditions and gel electrophoresis conditions, as well as designing new primers, we consistently observed larger PCR fragments than expected. Therefore, in both of these situations, we decided to harness a specific molecular biology tool called Touchdown PCR (TD-PCR) . The basic concept of TD-PCR is that it allows for better specificity of the PCR by:
(1) Setting the primer annealing temperature 5°C higher than the estimated melting temperature for a specific primer pair.
(2) Dropping the annealing temperature in every subsequent cycle by 0.5°C until the calculated annealing temperature is reached.
(3) Running the rest of your PCR cycles at the calculated annealing temperature. TD-PCR compensates for potentially inaccurate primer melting temperature calculations as well as other factors that may affect PCR specificity by adjusting the first steps in the PCR. Running the first few cycles at a temperature higher than the estimated annealing temperature of the primers decreases primer specificity but increases the pool of single-stranded templates allowing them to melt more gradually. This method proved to be successful in the amplification of the EvolvR cassette, as described in our Experiments and Notebook pages.
Plasmid origin of replication compatibility problem
For the first phase of our project, we wanted to test EvolvR activity in E. coli. In order to achieve this, we had to introduce the EvolvR complex with a guide RNA on one plasmid and the target gene on a second plasmid. The EvolvR complex is carried in the pEvolvR plasmid, which is based on a pBR322 E. coli cloning vector with a pBR322 origin of replication (ori). We created our target gene, ΔsfGFP(Y93X), by introducing a point mutation (see Experiments page for more details). This gene was originally carried in a pET29 plasmid with the same pBR322 ori as pEvolvR. The origin of replication is based on two antisense RNA transcripts that can interact with each other and create a stable structure that can then bind to the transcription bubble and allow a DNA polymerase to start replication. If two plasmids with the same ori exist in one bacterial cell, RNA transcripts from their origins can interact with each other, which creates competition, and the slightest imbalance in the system can lead to a complete loss of one of the plasmids. Therefore, two plasmids with the same origin or origins with very similar sequences cannot coexist in the same cell and are called incompatible. To tackle this issue in our system, we had to clone our target ΔsfGFP into a different vector. We chose a pBAD vector since it has an ori compatible with the pBR322 ori in pEvolvR, allowing for both plasmids to be able to replicate in the same cell independently. More details of the cloning process can be found on the Experiments page.
Antibiotic resistance issue
We used the E. coli T7 express lysY/Iq strain for our phase 1 experiments. This strain is derived from a BL21 strain that allows for high levels of exogenous protein expression by T7 polymerase but carries a few genes introduced from the K12 line. Some of such genes are endonuclease genes that allow for high-quality DNA recovery, which is important for further sequencing of targeted mutations in our case. The LysY/Iq control system also allows for the expression of potentially toxic proteins, which can be beneficial for the expression of EvolvR that can be somewhat toxic as it contains an enCas9 nickase and an error-prone DNA-polymerase that can interfere with the DNA inside the cell. However, we quickly ran into a practical issue while using this strain as it already carries a chloramphenicol resistance gene, which we initially wanted to use for selection of our target pBAD-ΔsfGFP(Y93X) vector. To solve this problem, we cloned an ampicillin resistance cassette in place of the chloramphenicol (details can be found on the Experiments page). Changing the antibiotic resistance allowed us to successfully select transformants carrying the target vector in a chloramphenicol-resistant T7 express background.
Proof-of-concept experiment issues
In our proof-of-concept experiment with E. coli, we noticed that samples containing both EvolvR with targeted guide RNA and target plasmid were dying very rapidly after the expression of EvolvR was induced with anhydrotetracycline. This observation suggested that the presence of the whole system may be very toxic to the host bacteria, and perhaps the targeted activity of the EvolvR interferes with the target plasmid and reduces its copy number, leading to an insufficient amount of antibiotic resistance gene being expressed in the cell. To test this hypothesis and see if this is a general issue or if it is caused by EvolvR being targeted to a specific region in the plasmid, we designed two troubleshooting methods.
Firstly, incubation of the cell culture at a lower temperature after EvolvR induction would slow down protein synthesis, expression, and accumulation in the cell, leading to a potential reduction in EvolvR activity after expression, which could potentially reduce its toxicity.
Secondly, to test whether the issue lies in EvolvR being targeting to a specific region of a target DNA via gRNA, we cloned 3 different protospacers that target the mutation in the ΔsfGFP(Y93X) gene from varying distances. Testing multiple protospacers in parallel would allow us to see if targeting a different region in the plasmid would be as damaging to the cells as when we tested the original protospacer. These new protospacers were previously designed for the ‘molecular ruler’ experiment, and correspond to protospacers #1, #4 or #8 from that experiment. We chose these protospacers because they are spaced throughout the entire ΔsfGFP(Y93X) gene— if all of them were to show a similar level of toxicity, it would suggest that the issue stems from general toxicity of EvolvR and not with the originally targeted region. On the other hand, if the level of toxicity would differ between the conditions with different protospacers, it would suggest that the issue lies in the region that is being targeted, not so much EvolvR activity.
Method for plant cell recovery after transformation with EvolvR
The common procedure during transformation of a BY-2 suspension cell culture is to incubate BY-2 cells with Agrobacterium tumefaciens for up to two days and then plate them on solid medium to let them grow into calli, which can take up to three weeks to become visible. This method is typically used as it allows one to isolate a single mutant and start a new transgenic culture. However, waiting for sufficient callus growth is time-consuming and negates the advantage of speed in a continuous directed evolution experiment with EvolvR. Therefore, we redesigned our experiment to not include the callus growth phase at all. Based on previous findings that inserted genes are already expressed when cells are still in suspension , we attempted to grow transformed cells for up to 5 days in liquid medium instead while killing the remaining Agrobacterium tumefaciens cells using an array of antibiotics. At this stage we were not interested in starting a transgenic BY-2 cell line and we only wanted to observe whether EvolvR is functional and capable of inducing mutations in a user-defined genomic target region. Therefore, all we required was the recovery of the BY-2 genomic DNA and for it to be sent for sequencing to identify potential mutations. In the future, we would like to optimize the liquid Agrobacterium-mediated transformation protocol to accelerate continuous directed evolution on plant cells. Bioreactors like the CellED (see Hardware page) could automize this method, getting rid of the time-consuming callus growth step.
 Korbie, D. J., & Mattick, J. S. (2008). Touchdown PCR for increased specificity and sensitivity in PCR amplification. Nature Protocols, 3(9), 1452–1456. https://doi.org/10.1038/nprot.2008.133
 Narasimhulu, S. B., Deng, X. ., Sarria, R., & Gelvin, S.(1996). Early transcription of Agrobacterium T-DNA genes in tobacco and maize. The Plant Cell, 8(5), 873–886. https://doi.org/10.1105/tpc.8.5.873.