We based our project off of Tian, et al by applying their design of a fusion protein to deliver and use Cas13 to treat SARS-CoV-2. By applying their fusion protein construct to the context of HIV, we decided on an initial plasmid that we could express within 10-beta e.coli to produce our fusion protein.
The primary part of our fusion protein is that of a LshCas13a, which serves as the primary function of our fusion protein, to cleave HIV within a cell. LshCas13a is derived from a specific strain of e.coli found in the oral cavity and promotes the production of lactic acid, and is one of the primary forms of Cas13a used in microbiology. Our LshCas13a sequence was pulled directly from the reference paper.
Additionally, we included a diphtheria toxin in our fusion protein, which serves as the vehicle for our LshCas13a. This toxin causes the disease diphtheria by inhibiting protein synthesis within the cytoplasm. In the context of microbiology, diphtheria toxin is modified to use its functional protein domains to transport a desired enzyme to the cell. For our project, this enzyme is Cas13a.
The last part of our fusion protein is an OKT4 region. OKT4 is an antibody that binds to the CD4 receptor present in T-Helper cells. The CD4 receptor plays a critical role in T-cell function and development, making it a common target in HIV research. By attaching an OKT4 region to our protein, we can bind our entire fusion protein to said CD4 surface receptor and allow our Cas13a to enter the cell.
Our initial plasmid was pulled from the source paper, encoding just a LshCas13a. However, we still need to insert our two other protein regions into our plasmid to create our final fusion protein. Hence, we developed two methods. The first method was via insertion on the N-terminus of the Cas13a, performed via designing corresponding HiFi inserts to a restriction site in front of the Cas13a region. The second method was via insertion on the C-terminus of the Cas13a. Firstly, a PCR would be performed to remove a sequence of STOP codons at the end of the Cas13a in the original plasmid, followed by a ligation to insert our desired proteins.
We ordered our Cas13a plasmid, which arrived as a bacteria stab. We streaked the stab and picked colonies to grow in LB before miniprepping. From the miniprepped colonies, we proceeded to proceed with the two methods of inserting our proteins of interest.
After running our two insertion methods, our results came back negative.
We decided to test higher ratios of HiFi insert relative to our Cas13a plasmid, hoping it would increase the efficiency of HiFi for N-terminus. Regarding the C-terminus, we decided to drop the idea as it was hard to troubleshoot and was significantly more complex than N-terminus.
We ran HiFi ratios with the higher ratios. Rather than just 1:2, we ran 1:5, 1:10, 1:20, 1:50, 1:80, and 1:100.
We discovered that the HiFi ratios that were on the higher end had success compared to the 1:2 ratios. After plating, miniprep, and sequencing, we had a successful fusion protein within our plasmid.
Additionally, we wanted to try cloning our fusion protein within SC e.coli, another strain used in bacterial-based plasmid cloning. By trying SC in addition to 10-beta, we wanted to compare their efficiencies.
We reran our HiFi and transformed into the two strains of bacteria, before plating and leaving overnight at 27C.
We found that 10-beta were significantly more effective than Stable Competent in cloning our fusion protein, as we had little success with SC in terms of colonies growing on the plate.
We based our protein expression of our fusion protein based off of IPTG induction. IPTG is a chemical inducer of the lac operon by binding to the lac repressor gene, changing its shape and allowing for transcription to occur. We ordered our IPTG in powder form and hydrated the salt in order to be used, before diluting it to the appropriate concentration for expression. We added the IPTG at the mid-log phase at OD600 ~0.6 to maximize expression.
We expressed our fusion protein by first transforming three strains of e.coli typically used in protein expression: Rosetta, BL21 (DE3), and W3110.
We grew starter culture overnight in LB before inoculating main cultures with each bacterial strain and growing at 37C to OD600 ~0.6. We separated a pre-induction sample, and then added IPTG and incubated overnight at 18C to create the post-induction sample. We then ran all the samples on an SDS-page to determine protein expression. By comparing the band intensities of the proteins following SDS, we examined which strain had the most optimal expression.
We found that Rosetta had the most optimal expression of our fusion protein at the desired protein size, and switched to using Rosetta exclusively for future expression testing.
We wanted to test lower temperatures of protein induction, as well as differing IPTG concentrations as our expression still remained low.
We repeated our expression testing with lower IPTG values, ranging from 0.1 mM to 1.6 mM. Again, we repeated the SDS and ran the same sample with different concentrations together on the gel to compare.
We found that the lowest IPTG concentration of 0.1 mM was most effective, and switched to using that concentration for future expression testing.
When running our SDS gels, we noticed significant background expression. This is due to the T7 promoter, which although very strong in promoting protein expression, can often “leak” and express uninduced genes.
We repeated our expression experiments, but with adding 1% glucose to our starter cultures before conducting expression testing. This is likely due to the basal levels of T7 Polymerase naturally existing inside e.coli, leading to transcription even in the absence of IPTG.
1% Glucose had negligible effect on background expression, and we continued with purification.
We aimed to determine which volume of Lipofectamine per well yields the highest transfection efficiency. To test this, we compared three different volumes of Lipofectamine: 6 uL, 9 uL, and 15 uL. An EGFP plasmid was used to visualize transfection, while a null plasmid served as the negative control. Results from this experiment will guide us in selecting the optimal Lipofectamine amount for our CD4 transfection. Duaa and Bevis worked in parallel, with both conducting their own lipofectamine transfection efficiency tests.
Transfected equal concentrations of plasmid DNA into each well (3.5 ug per well).
Cell Setup: One million cells per well were seeded in a 6-well plate the day prior to transfection and grown overnight.
Controls: Null plasmid with no gene of interest (negative control)
Experimental Conditions (Lipofectamine Volumes):
Bevis’ 6-well plate displayed EFGP fluorescence, suggesting a successful transfection. Duaa’s 6-well plate did not display EGFP fluorescence, suggesting a non-successful transfection. The cause of the transfection not working was unknown, but attributed to just the first time ever conducting transfection. Among Bevis’ results (shown below), 15 uL of Lipofectamine produced the highest transfection efficiency, (illustrated by the greater amount of cells with EGFP fluorescence). Therefore, 15 uL of Lipofectamine can be utilized for future transfections involving our CD4 and HIV mimic cassettes.
Duaa aimed to re-run transfection and determine which volume of Lipofectamine per well yields the highest transfection efficiency. Similarly to Iteration 1, three different volumes of Lipofectamine: 6 uL, 9 uL, and 15 uL were tested. An EGFP plasmid was used to visualize transfection, while a null plasmid served as the negative control. Results from this experiment will guide us in selecting the optimal Lipofectamine amount for our CD4 transfection.
Transfected equal concentrations of plasmid DNA into each well (3.5 ug per well).
Cell Setup: One million cells per well were seeded in a 6-well plate the day prior to transfection and grown overnight.
Controls: Null plasmid with no gene of interest (negative control)
Experimental Conditions (Lipofectamine Volumes):
After discussion with Dr. Gunn, she recommended vortexing the Lipofectamine solution to encourage lipid-DNA complex formation and improve transfection efficiency.
Duaa aimed to re-run transfection and determine which volume of Lipofectamine per well yields the highest transfection efficiency, this time vortexing the Lipofectamine solution to encourage lipid-DNA complex formation. Similarly to Iteration 1 and 2, three different volumes of Lipofectamine: 6 uL, 9 uL, and 15 uL were tested. An EGFP plasmid was used to visualize transfection, while a null plasmid served as the negative control. Results from this experiment will guide us in selecting the optimal Lipofectamine amount for our CD4 transfection.
Transfected equal concentrations of plasmid DNA into each well (3.5 ug per well).
Cell Setup: One million cells per well were seeded in a 6-well plate the day prior to transfection and grown overnight.
Controls: Null plasmid with no gene of interest (negative control)
Experimental Conditions (Lipofectamine Volumes):
Duaa’s 6-well plate displayed EGFP fluorescence, indicating a successful transfection. Among both Bevis’ results (shown in Iteration 1) and Duaa’s results (shown below), 15 uL of Lipofectamine produced the highest transfection efficiency. This is illustrated by the greater amount of cells with EGFP fluorescence in the well with 15 uL of Lipofectamine. Therefore, 15 uL of Lipofectamine will be utilized for future transfections involving our CD4 and HIV mimic cassettes.
We aimed to use HEK293T landing pad cells (engineered with FRT sites) to stably integrate our genetic cassettes. This system would be utilized to insert both the CD4 receptor cassette and the non-infectious HIV mimic cassette. We utilized EGFP as a control to confirm that the FLP recombinase can correctly insert the DNA at the landing pad site. Duaa and Bevis worked in parallel, with both creating their own stable cell lines.
Co-transfected landing pad cells with the FLP recombinase plasmid and one of the cassettes (EGFP for positive control, CD4 for experiment); varied plasmid ratios to optimize integration efficiency.
Cell Setup: One million cells per well were seeded in a 6-well plate the day prior to transfection and grown overnight.
Controls:
The advisors recommended reducing the initial hygromycin concentration to allow the cells more time to recover from both transfection and passaging before selection pressure. In future trials, hygromycin concentration will be lowered from 200 ug/mL to 100 ug/mL. Additionally, it was suggested to transfer the cells to T25 flasks instead of T12 flasks post-transfection. This can provide the cells with more space and nutrients for recovery before applying selection pressure.
We aimed to repeat the transfection, this time reducing the hygromycin concentration from 200 ug/mL to 100 ug/mL to allow the cells more recovery time post-transfection and transferring the cells to T25 flasks instead of T12 flasks. Duaa’s confluency of her 6-well plate was a bit lower than expected, but Dr. Gunn said that is fine and actually may be beneficial for DNA uptake.
Cell Setup: One million cells per well were seeded in a 6-well plate the day prior to transfection and grown overnight.
Controls:
Dr. Gunn suggested that the cell confluency at the time of CD4 transfection may have impacted efficiency. High confluency (~90%) can reduce transfection efficiency, as the cells have limited surface contact with the Lipofectamine. It was recommended that future transfections be performed at approximately 50% confluency by seeding 500,000 cells per well instead of 1 million.
Since Duaa already attempted the transfection at a lower confluency in Iteration 2, only Bevis aimed to repeat the CD4 transfection to try a lower confluency (~50%), possibly improving DNA uptake. Based on advisor feedback, 500,000 cells were seeded per well instead of one million to provide more surface area for interaction with the Lipofectamine.
Cell Setup: One million cells per well were seeded in a 6-well plate the day prior to transfection and grown overnight.
Controls:
Dr. Gunn suggested that the Lipofectamine reagent may have been contaminated or degraded, which could explain the repeated lack of transfection efficiency. It was recommended to test an alternative transfection method, calcium chloride transfection, to test whether the previous hypothesis was correct.
We aimed to test whether the repeated transfection failures were due to the contamination or degradation of the Lipofectamine. To evaluate this, a simple transfection was performed using both Lipofectamine and CaCl₂ with an EGFP plasmid and water (negative control) to compare transfection outcomes.
Cell Setup: 500,000 cells per well were seeded in a 6-well plate the day prior to transfection and grown overnight.
Controls: Water (negative control)
Experimental Conditions: Two wells were transfected with EGFP and two wells were transfected with water
The cause of transfection failure remained undetermined. To eliminate all possible sources of error, all significant materials were replaced: the cell line, Lipofectamine reagent, and we re-made plasmids. The next transfection will be performed under Dr. Gunn’s supervision to help identify any source of failure, possibly in our methodology. Due to time constraints, the team decided to shift focus from generating a stable cell line and using FLP recombinase to performing transient transfections with just CD4 for future experiments.
We aimed to re-run transfection but now under the supervision of Dr. Gunn to troubleshoot previous transfection failures and directly compare CaCl₂ transfection with Lipofectamine. The goal was to determine which method provided higher transfection efficiency and expression of CD4 and EGFP in landing pad HEK293T cells.
Cell Setup: 500,000 cells per well were seeded in a 6-well plate the day prior to transfection and grown overnight.
Controls:
Shown below is Duaa’s CaCl2 transfection:
Following successful expression in the positive controls, the cells in the wells transfected with CD4 were pelleted and stored at -20 degrees Celsius to be utilized in Western Blot.
We aimed to determine the success of our CaCl2 and lipofectamine transfection of CD4 into HEK293T cells.
Experimental Conditions (Lipofectamine wells):
The resulting images from the assay contained no visible bands and a lot of noise. This noise could be attributed to poor blocking from the 5% milk 1X TBST solution, 5 mL of solution was not enough and 10 mL would be used. Signs of no bands can be attributed to the antibodies being weak, thus the dilution needs to be decreased from 1:1000 to 1:500. Furthermore, it could be due to poor transfection that yielded little protein, transfection will be rerun with more cells.
We aimed to re-run western blot to determine the success of our CaCl2 and lipofectamine transfection of CD4 into HEK293T cells.
Experimental Conditions (Lipofectamine wells):
The results showed bright bands, however the location of the bands were too high. It was expected to be around 55 kD, but results show the bands significantly larger than 55 kD. The reasoning is unknown, but the protein product has a high chance of not being CD4. Further testing, such as using a denaturing control or performing an antibody validation experiment, will be necessary to confirm our hypothesis.
