Prior to our protein work we had to make several preparations. The purpose of the first engineering cycle was to design the dCasMINI and sgRNA, model them to make sure the binding activity was plausible and lastly integrated the designed protein sequences into the expression strain. For our build we will use the programs Geneious and PyMol for in silico design part. These tools are frequently used in academia for both visualization and design of protein sequences, granting us information about the proteins that would be difficult to acquire otherwise. Furthermore the protein we work with also is well characterized online so there are many resources available for us to use in combination with the tools. For the wet lab part we will use E.coli TOP10 cells as the cloning strain to make sure that the designed construct is correctly integrated. The designed construct will be assembled with constructs ordered from IDT and pASK-IBA2 vector donated by our supervisors. Through an agarose gel we can visualize the successful integration of the construct into the vector and if the bacteria have carry them. From this DBTL cycle we expect an expression strain with an integrated construct-plasmid with sizes as expected from the in silico design, aswell as proof that the binding of the dual casMINI complex is plausible with little steric hindrance.
Geneious was used for the in silico design of our dCasMINI variants and sgRNAs.
PyMol was used for the visualization of casMINI binding to make sure that there was no significance steric hindrance that may disrupt the dual binding action.
In this DBTL cycle it is quite easy to modify the build, depending on how large the changes are the team may have to redo the in silico design.
For the wetlab part, E. coli TOP10 cells were used to make two separate cultures and in these cultures a pASK-IBA2 vector with either HLC or CLS was inserted.
Therefore the wetlab lab work entails these two separate components that will be cloned.
In later cycles the plasmids will be transferred to the E. coli BL21 cells for expression of the proteins.
The DNA parts and reagents required for the wetlab were supplied by IDT and EvoGene.
The E. coli TOP10 cells have the CRISPR-CAS system endogenously which was one of the reason we used the casMINI system for our project as it is not toxic for the cells.
Therefore we don’t need to worry about stress responsed by the bacteria when expressing our genes.
In this build we used the ampicilin antibiotic to screen for the correct builds as the pASK-IBA2 vector includes ampicilin resistance.
Furthermore, during the lab we performed agarose gel to make sure we had bands of the expected size.
As a last checkpoint to make sure our construct was successfully integrated, we sent our plasmids to sanger sequencing by Eurofins Genomics.
Multiple tests were required for a successful engineering cycle, these tests include checking successful linearization of constructs and vector, ensuring correct integration of construct into the pASK-IBA2 vector and check transformation of the plasmid into the cloning strain E. coli TOP10.
These tests were ran simultaneously for both the HLC and CLS construct in multiple parts.
Most of these tests were done by running the construct, vector or plasmid through a agarose gel and compare the sizes to the expected size.
While throughout the experiment we also grew the colonies on cultures with antibiotics where we could observe correct transformation of the plasmid into the cloning strain as without the vector the TOP10 cells would not grow in the culture.
The results of these tests was qualitative in the form of pictures of the gels which you can access on our (link results) results.
Beside the gels there is a size ladder with known sizes that are used to compare the test to the standard sizes.
We need to make sure that the size are correct, as without the presence of these bands we cannot be sure that our experiment was successful.
From this engineering cycle we wanted to learn about the optimal design of the construct, and optimalize the protocol for cloning of the construct.
The system did work as anticipated and during this engineering cycle we did learn the optimal design of the construct and the protocol for the cloning of the construct seemed to be suitable.
As the first plasmid preparation was unsatisfactory we had to make some changes to our experiment which allowed us to improve it.
Our data is displayed on the wiki which allow objective reading of our results, furthermore multiple team members analyse the data to make sure we do not incorrectly interpret the data gathered.
The purpose of our second engineering cycle was to optimize our protein expression. Our engineered system would optimally produce a lot of our dCasMINI protein in a soluble fraction instead of an insoluble fraction. Proteins in the soluble fraction are easier to work with and do not require extensive lab work to use. For this engineering cycle we used E. coli BL21 (DE3) Gold cells as chassis for our expression strain. The expression strain was built from the construct-pASK-IBA2 plasmid and inserted into the BL21 (DE3) Gold cells. Furthermore, the dCasMINI expression function of the BL21 (DE3) Gold cells was tested in multiple conditions to check for the optimal condition. Our experimental data will show a large band at our protein size for atleast one of the conditions. Based on the literature we do expect this protein to be soluble.
The engineered system was created as two separate BL21 cultures growing the His-tag-casMINI and the Strep-tag-casMINI, both cultures were grown simultaneously as they required similar experiments during the engineering cycle. These two cultures are variants of each other as they both as based on the dCasMINI protein-cas12f but with some changes due to the presence of either a his or strep tag, for this reason it was important to keep these two separated as to not contaminate one culture with the other. In this build it would be easy to change the protein expression given that the cloned plasmids are avaliable, if they arent avaliable some additional time will be added as we would need to synthesize new plasmids from the start. As described in previous engineering cycle the CAS protein family is endogenous for the E. coli and by looking at avalible literture we ensured that the proteins were not toxic for the bacteria. The BL21 (DE3) Gold cells were grown in antibiotic nutrients, which kills the bacteria that do not have antibiotic resistance granted by the pASK-IBA2 vector, furthermore we made sure to run PCRs that would confirm the presence of the construct. This build was made with DNA produced by us in prior experiments and reagents from the EvoGene lab.
The most important aspects of this cycle was testing the optimal protein expression.
To determine the optimal protein expression we examined the amount of protein that was in the insoluble fraction and the soluble fraction.
After centrifugation the soluble fraction of the cells remain in the solution after lysis while the insoluble fraction form a pellet.
To further expand the soluble fraction remains soluble in the liquid and is easy to move afterwards while the insoluble fraction is hydrophobic and forms a pellet to avoid interaction with water making it difficult to work with in liquids.
The two soluble/insoluble ratio is affected by two factors, the temperature and time. E. coli has an optimal growth condition of 37 °C, however that does not mean that it is the optimal temperature for protein expression.
Therefore we test multiple temperatures to find the temperature that has the highest soluble to insoluble ratio. Furthermore the protein expression is induced by an inducer, the time spent expressing the protein also affects the ratio.
Based on this we had multiple conditions where we varied both the time and the temperature.
These tests were done with SDS-pages were we could visualize the amount of target protein in the soluble and insoluble fractions.
The results from the SDS-PAGE are qualitative. Images of the gels are periodically added to the results section as the tests are completed.
You can view them here (link to results).
To make sure that the system functions as expected we will analyse the gels and compare the postitive and negative controls with the the solube and insoluble fractions.
A large soluble fraction is the most crucial part of this engineering cycle as it allows advancement to the protein purification part.
From this engineering cycle we want to learn what conditions result in the largest amount of target protein in the soluble fraction compared to the other conditions. During the test phase of the cycle the system worked as expected and allowed detailed analysis of the optimal protein expression condtions. However even with optimal conditions there were significant presence of the proteins in the insoluble fractions, therefore to improve the system it may be required to modify the proteins to increase solubility. The protein was more insoluble than we expected based on the literature read in the design part. Our tests should be presented with the most optimal condition in focus as it is the most important take away from this engineering cycle.