iGEM 2025 — Team stony-brook

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* [IN-VITRO](#vitro)
* [IN-VIVO](#vivo)
* [DRY LAB](#dry)

# NOTEBOOK

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We separated our research journey into three over-arching notebooks: in-vitro, in-vivo, and dry lab. Look into each PDF to get more information about our process for each section!

## IN-VITRO

## IN-VIVO

## DRY LAB

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* [GENE CIRCUIT DESIGN](#genecircuit)
* [FUSION PROTEIN EXPRESSION](#fusionprotein)
* [MAMMALIAN CELL LAB:
  Lipofectamine Transfection Efficiency](#lipofectamine)
* [MAMMALIAN CELL LAB:
  Transfection of HEK293T Cells with FLP recombinase for CD4 expression](#transfect)
* [WESTERN BLOT ASSAY: CD4](#blot)

# ENGINEERING

---

### GENE CIRCUIT DESIGN

Iteration 1

DESIGN

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.

BUILD

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.

TEST

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.

LEARN

After running our two insertion methods, our results came back negative.

Iteration 2

BUILD

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.

TEST

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.

LEARN

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.

Iteration 3

BUILD

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.

TEST

We reran our HiFi and transformed into the two strains of bacteria, before plating and leaving overnight at 27C.

LEARN

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.

### FUSION PROTEIN EXPRESSION

Iteration 1

DESIGN

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.

BUILD

We expressed our fusion protein by first transforming three strains of e.coli typically used in protein expression: Rosetta, BL21 (DE3), and W3110.

TEST

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.

LEARN

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.

Iteration 2

BUILD

We wanted to test lower temperatures of protein induction, as well as differing IPTG concentrations as our expression still remained low.

TEST

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.

LEARN

We found that the lowest IPTG concentration of 0.1 mM was most effective, and switched to using that concentration for future expression testing.

Iteration 3

BUILD

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.

TEST

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.

LEARN

1% Glucose had negligible effect on background expression, and we continued with purification.

#### MAMMALIAN CELL LAB: Lipofectamine Transfection Efficiency

Iteration 1

DESIGN

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.

BUILD

Transfected equal concentrations of plasmid DNA into each well (3.5 ug per well).

TEST

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):

* 6 uL Lipofectamine + 3.5 ug EGFP plasmid
* 6 uL Lipofectamine + 3.5 ug null plasmid
* 9 uL Lipofectamine + 3.5 ug EGFP plasmid
* 9 uL Lipofectamine + 3.5 ug null plasmid
* 15 uL Lipofectamine + 3.5 ug EGFP plasmid
* 15 uL Lipofectamine + 3.5 ug null plasmid

Transfection efficiency was assessed by fluorescence microscopy to visualize EGFP expression.

LEARN

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.

![Fluorescence](https://static.igem.wiki/teams/5540/engineering/igem-duaa-bevis-1.webp)

Iteration 2

DESIGN

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.

BUILD

Transfected equal concentrations of plasmid DNA into each well (3.5 ug per well).

TEST

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):

* 6 uL Lipofectamine + 3.5 ug EGFP plasmid
* 6 uL Lipofectamine + 3.5 ug null plasmid
* 9 uL Lipofectamine + 3.5 ug EGFP plasmid
* 9 uL Lipofectamine + 3.5 ug null plasmid
* 15 uL Lipofectamine + 3.5 ug EGFP plasmid
* 15 uL Lipofectamine + 3.5 ug null plasmid

Transfection efficiency was assessed by fluorescence microscopy to visualize EGFP expression. Duaa’s 6-well plate did not display EGFP fluorescence again.

LEARN

After discussion with Dr. Gunn, she recommended vortexing the Lipofectamine solution to encourage lipid-DNA complex formation and improve transfection efficiency.

Iteration 3

DESIGN

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.

BUILD

Transfected equal concentrations of plasmid DNA into each well (3.5 ug per well).

TEST

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):

* 6 uL Lipofectamine + 3.5 ug EGFP plasmid
* 6 uL Lipofectamine + 3.5 ug null plasmid
* 9 uL Lipofectamine + 3.5 ug EGFP plasmid
* 9 uL Lipofectamine + 3.5 ug null plasmid
* 15 uL Lipofectamine + 3.5 ug EGFP plasmid
* 15 uL Lipofectamine + 3.5 ug null plasmid

Transfection efficiency was assessed by fluorescence microscopy to visualize EGFP expression.

LEARN

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.

![Fluorescence](https://static.igem.wiki/teams/5540/engineering/igem-duaa-bevis-2.webp)

#### MAMMALIAN CELL LAB: Transfection of HEK293T Cells with FLP recombinase for CD4 expression

Iteration 1

DESIGN

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.

BUILD

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.

TEST

Cell Setup: One million cells per well were seeded in a 6-well plate the day prior to transfection and grown overnight.

Controls:

* EGFP (positive control)
* Null plasmid with no FLP (negative control)

Experimental Conditions (FLP:CD4): The remaining four wells were transfected with the following ratios of FLP:CD4:

* 5:2, 1.0 ug of CD4+2.5 ug of FlpO
* 2:5, 2.5 ug of CD4+1.0 ug of FlpO
* 1:6, 3.0 ug of CD4+0.5 ug of FlpO
* 1:1, 1.8 ug of CD4+1.8 ug of FlpO

All wells were transfected with 15 uL of Lipofectamine, and Lipofectamine was vortexed for approximately 15 seconds prior to DNA addition to promote complex formation. After the addition of the lipid-DNA complex, the cells were incubated with the transfection reagent overnight. After transfection, both Bevis and Duaa’s positive controls did not show any detectable EGFP expression, suggesting that the CD4 transfection may not have been successful. Despite this, the cells were placed under hygromycin selection in case integration had occurred without visible fluorescence. After one week of hygromycin pressure, none of the cells (which have hygromycin resistance) survived.

LEARN

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.

Iteration 2

DESIGN

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.

BUILD

* 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

TEST

Cell Setup: One million cells per well were seeded in a 6-well plate the day prior to transfection and grown overnight.

Controls:

* EGFP (positive control)
* Null plasmid with no FLP (negative control)

Experimental Conditions (FLP:CD4): The remaining four wells were transfected with the following ratios of FLP:CD4:

* 5:2, 1.0 ug of CD4+2.5 ug of FlpO
* 2:5, 2.5 ug of CD4+1.0 ug of FlpO
* 1:6, 3.0 ug of CD4+0.5 ug of FlpO
* 1:1, 1.8 ug of CD4+1.8 ug of FlpO

All wells (Duaa’s at a lower confluency, Bevis’ at a higher confluency) were transfected with 15 uL of Lipofectamine, and the transfection reagent was left on overnight. EGFP fluorescence was checked post-transfection, but no expression was detected once again. The cells were still transferred into T25 flasks and placed under 100 ug/mL hygromycin selection. After one week, no surviving cells were observed.

LEARN

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.

Iteration 3

DESIGN

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.

BUILD

* 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

TEST

Cell Setup: One million cells per well were seeded in a 6-well plate the day prior to transfection and grown overnight.

Controls:

* EGFP (positive control)
* Null plasmid with no FLP (negative control)

Experimental Conditions (FLP:CD4): The remaining four wells were transfected with the following ratios of FLP:CD4:

* 5:2, 1.0 ug of CD4+2.5 ug of FlpO
* 2:5, 2.5 ug of CD4+1.0 ug of FlpO
* 1:6, 3.0 ug of CD4+0.5 ug of FlpO
* 1:1, 1.8 ug of CD4+1.8 ug of FlpO

All wells were transfected with 15 uL of Lipofectamine, and the transfection reagent was left on overnight. EGFP fluorescence was checked post-transfection, but no expression was detected once again in Bevis’ 6-well plate. The cells were still transferred into T25 flasks and placed under 100 ug/mL hygromycin selection. After one week, no surviving cells were observed.

LEARN

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.

Iteration 3.5

DESIGN

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.

BUILD

* 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

TEST

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

* Two wells transfected with 3.5ug of EGFP (7.3 uL)
* Two wells transfected with 7.3 uL of water

All wells were transfected with 15 uL of Lipofectamine and 124 uL of 2M CaCl₂. Cells were incubated overnight with the transfection reagents. EGFP fluorescence was checked the following day by fluorescence microscopy, but no fluorescence was observed in either wells.

LEARN

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.

Iteration 4

DESIGN

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.

TEST

Cell Setup: 500,000 cells per well were seeded in a 6-well plate the day prior to transfection and grown overnight.

Controls:

* EGFP (positive control)
* Null plasmid with no FLP (negative control)

Experimental Conditions: Two wells were transfected with EGFP and two wells were transfected with water

* CaCl₂ transfection

+ 3.5 ug of CD4 plasmid
+ 3.5 ug of EGFP plasmid
+ 3.5 ug of null plasmid

* Lipofectamine transfection

+ 3.5 ug of CD4 plasmid
+ 3.5 ug of EGFP plasmid
+ 3.5 ug of null plasmid

All wells were transfected with 15 uL of Lipofectamine and 124 uL of 2M CaCl₂. Cells were incubated overnight with the transfection reagents. EGFP fluorescence was checked the following day by fluorescence microscopy, and small levels of fluorescence were detected. Since our positive control showed signs of fluorescence, this signified that our CD4 transfection possibly worked. CaCl₂ produced stronger and more visible EGFP expression compared to Lipofectamine. While faint fluorescence was observed in the Lipofectamine positive control, it was too weak to be captured by camera.

LEARN

Shown below is Duaa’s CaCl2 transfection:

![Fluorescence](https://static.igem.wiki/teams/5540/engineering/igem-duaa-bevis-3.webp)

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.

#### WESTERN BLOT ASSAY: CD4

Iteration 1

DESIGN

We aimed to determine the success of our CaCl2 and lipofectamine transfection of CD4 into HEK293T cells.

BUILD

* Two set of calcium chloride and lipofectamine transfection was performed
  + 2 positive control each, 2 negative control each, 2 CD4 each
* All cells in wells were pelleted
* Pelleted cells were lysed and proteins were ran through SDS-page
* Results under went Western Blot Assay

TEST

**Experimental Conditions (Lipofectamine wells):**

* 2x 15 uL Lipofectamine + 3.5 ug EGFP plasmid
* 2x 15 uL Lipofectamine + 3.5 ug null plasmid
* 2x 15 uL Lipofectamine + 3.5 ug CD4 plasmid

**Experimental Conditions (Calcium chloride wells):**

* 2x 3.5 ug EGFP plasmid
* 2x 3.5 ug null plasmid
* 2x 3.5 ug CD4 plasmid

The proteins were treated with 1:1000 dilution of primary CD4 antibodies for the western blot assay.

LEARN

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.

![Fluorescence](https://static.igem.wiki/teams/5540/homepage/igem-bevisduaa1.avif)

![Fluorescence](https://static.igem.wiki/teams/5540/homepage/screenshot-2025-10-08-at-9-56-21-am.avif)

Iteration 2

DESIGN

We aimed to re-run western blot to determine the success of our CaCl2 and lipofectamine transfection of CD4 into HEK293T cells.

BUILD

* 1 set of calcium chloride and lipofectamine transfection was performed in 6-well plate
  + 1 positive control each, 1 negative control each, 1 CD4 each
* 1 set of calcium chloride transfection was performed on T75
* All cells in wells were pelleted
* Pelleted cells were lysed and proteins were ran through SDS-page
* Results under went Western Blot Assay

TEST

**Experimental Conditions (Lipofectamine wells):**

* 15 uL Lipofectamine + 3.5 ug EGFP plasmid
* 15 uL Lipofectamine + 3.5 ug EGFP plasmid
* 15 uL Lipofectamine + 3.5 ug CD4 plasmid

**Experimental Conditions (Calcium chloride wells):**

* 3.5 ug EGFP plasmid
* 3.5 ug null plasmid
* 3.5 ug CD4 plasmid

**Experimental Conditions (Calcium chloride T75):**

* 16.86 ug CD4 plasmid

The proteins were treated with 1:500 dilution of primary CD4 antibodies for the western blot assay and 10 mL of 5% milk 1X TBST solution was used for blocking.

LEARN

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.

![Fluorescence](https://static.igem.wiki/teams/5540/homepage/screenshot-2025-10-08-at-10-02-54-am.avif)

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* [BACTERIA LABS](#bact)
* [PROTEIN LAB](#protein)
* [MAMMALIAN CELL LAB](#mammal)

# EXPERIMENTS

---

For each experiment conducted throughout the course of our research journey, we have included a PDF of the protocol that we used. Click on different experiments to see how they were conducted!

## BACTERIA LABS

### Oligo Annealing & Ligation

### HiFi Assembly

### Transformation Into 10-Beta Cells

### Transformation Into Rosetta, BL21, W3110 Cells

### Plasmid Isolation (Miniprep)

### DNA Ligation For Cohesive Ends

### Post Enzymatic Reaction Cleanup (PCR Cleanup)

## PROTEIN LAB

### Cas13a Expression & Purification

### Cas13a-DTA-gp120 Assay

## MAMMALIAN CELL LAB

### Passaging

### Transfection: Calcium Chloride

### Transfection: Lipofectamine

### Establishing a Stable Cell Line

### Pelleting Cells

### Post-Transfection Identification of Fluorescence Under Microscope

### Western Blotting For CD4

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* [THE CAS13-DTA-gp120 SYSTEM](#system)
* [CLEAVAGE TESTING](#cleave)
* [CD4-gRNA & HIV MIMIC ssRNA STABLE CELL LINE](#cd4grna)

# RESULTS

---

## THE CAS13-DTA-gp120 SYSTEM

Originally, our team knew that we wanted to use Cas13a to target and cleave HIV RNA. However, as a therapeutic, we had to consider how Cas13a would be delivered into human body cells. To account for this, we first proposed a three-fold fusion protein design with the DNA that encodes for each protein formed into a plasmid. This original design, named Unit 0, intended for Cas13a to serve as the cargo being transported, Diphtheria Toxin (DTA) to serve as the facilitator for endocytosis, and CD4 monoclonal antibody (OKT4) to serve as our ligand that binds to the CD4 receptors on helper T cells. The OKT4 and DTA were on the C-terminus of the Cas13a. According to our understanding, the receptor-ligand binding of OKT4 and the CD4 receptor of the Helper T cell would trigger the endocytosis mechanism of the DTA, successfully transporting the Cas13a protein into the HIV-infected Helper T cell.

In experiments, we HiFi assembled the different parts of the fusion protein plasmid, using a plasmid backbone with Cas13a. This fully assembled plasmid with Cas13a, DTA, and OKT4 was transformed into Rosetta, BL21, and W3110 cells, and grown in triplicates to determine which cell variety was most efficient in growing the protein. Rosetta was determined to be the most efficient, but with one problem: the OKT4 was getting deleted from the transcription process, following assembly of our DTA-OKT4 insert into our Cas plasmid. Our theory as to why the OKT4 was not present when the uptaken plasmid was sequenced is due to the linkers in for the insert. The linkers were repeats of a nucleotide sequence, so the Rosetta cells could have possibly counted that as unnecessary scaffolding and cut out the OKT4 insert entirely.

Unfortunately, the Unit 0 design itself was also vastly flawed. It is more optimal for the Cas13a to be on the N terminus and the OKT4 on the C terminus, but due to the misaligned ORFs at the C terminus of the Cas13a as well as the issue of multiple STOP codons that interfered with assembly, we designed an insert to put OKT4 on the N terminus. This underlined the novelty of our work as a fusion protein had never been constructed with the targeting domain on the N terminus.

However, two issues arised. First, without binding, the OKT4 cannot work with the DTA to facilitate endocytosis, and the Cas13a protein will stay outside of the cell. Secondly, the OKT4 was not a ligand. It is a receptor, so it was not capable of serving as a ligand for CD4. The entire design would, effectively, not work, even if it was able to be fully expressed, which it was not. Only the Cas13a and DTA were translated into protein and frozen at -80C until used for in-vitro protein purification and testing purposes, since there was no mechanism to get it into a cell.

In an attempt to ameliorate the lack of a ligand in plasmid design, we looked at the mechanism of action of HIV, which shows that HIV itself actually uses a ligand to bind to CD4 receptors on Helper T cells, called gp120, a glycoprotein. We recreated our plasmid design to now include Cas13a, DTA, and gp120. Multiple attempts to ligate the gp120 dna sequence to the Cas13a C terminus failed, so we instead used the N terminus, which worked. We transformed the now formed Cas13a + DTA + gp120 plasmid into competent Rosetta cells, and incubated them overnight at 18 degrees Celsius with 0.1 mM IPTG, 100 µg/mL Ampicilin, and 20% glucose diluted to 0.2%, all in LB Broth at 250 RPM. Due to the nature of bacteria, they are unable to synthesize glycoproteins, so the resulting protein was Cas13a linked to DTA, meaning that another attempt to synthesize the full protein complex for in vivo testing was unsuccessful. We utilized the resulting Cas13a+DTA protein for in vitro testing to evaluate if our novel fusion protein can successfully cleave HIV ssRNA, and aimed to use a cell-free system to fully create our Cas13a+DTA+gp120 fusion protein without the restrictions of prokaryotic cells or maintenance of mammalian cell synthesis.

Our main takeaways for future teams to consider are that:

* 10-beta and Rosetta are the best strains of e.coli for cloning and expression, respectively
* Consider running HiFi assemblies at higher ratios (1:5+) for higher efficiency
* 0.2% glucose and 0.1mM IPTG are the most optimal parameters for expression

Similarly, some major issues that stunted our progress that future teams should take into account, as well as potential solutions that we suggest trying are:

* Degradation of Cas13-based fusion protein within expression.
* Potentially codon optimization and linker redesign could improve protein stability
* Cleavage of Cas13-based fusion protein within cloning
* Linker redesign of repetitive sequences that may be cleaved out as redundant
* Inability of e.coli to glycosylate proteins such as gp120
* Use alternative expression systems, such as cell-free or mammalian systems

Future iGEM teams can adapt these findings for not just Cas13a-based constructs but other fusion protein systems. Consider tweaking expression, stability, and post-translational modification parameters.

## CLEAVAGE TESTING

In the works… stay tuned for Paris!

## CD4-gRNA & HIV MIMIC ssRNA STABLE CELL LINE

Our goal was to generate a stable HEK293T (Human Embryonic Kidney Cell) landing pad cell line expressing both the CD4 receptor with gRNA attachment cassette and HIV-mimic cassette using FLP recombinase integration. Despite multiple transfection attempts under varying conditions, stable integration was not achieved.

We initially tested a range of FLP:CD4 plasmid ratios (5:2, 2:5, 1:6, and 1:1) to determine optimal efficiency and whether CD4 will be transfected and integrated into the landing pad by flip recombinase. However, no EGFP fluorescence was observed in the positive control wells, suggesting transfection failure. Even after optimizing Lipofectamine conditions, utilizing a lower concentration of antibiotic selection pressure, transferring the cells to a bigger surface area, and adjusting cell transfection confluency; all cells failed to survive hygromycin selection.

To assess CD4 expression independent of FLP recombination, we performed a transient transfection, utilizing both Lipofectamine and CaCl₂ transfection, of the CD4 cassette without FLP recombinase. Cells were cultured for 48 hours post-transfection, lysed, and analyzed via Western blot to verify CD4 protein expression.

Our first Western blot did not show detectable CD4 bands, likely due to low transfection efficiency (the fluorescence in the positive controls were very minimal) and/or a low amount of protein being loaded into the SDS-Page gel. After troubleshooting with optimizing our lysis buffer and re-running transfection, we repeated the experiment.

The second Western blot successfully showed bands, however location of bands were significantly higher than the expected ~55 kDa, exact band size can’t be determined. Possible explanations for this can be non-specific antibody binding and/or incomplete denaturation. Further testing, such as using a denaturing control or performing an antibody validation experiment, will be necessary to confirm whether the bands correspond to CD4 or another product.

![Sensitiveity Analysis](https://static.igem.wiki/teams/5540/wetlab/igem-sam-1.webp)

**Figure 1a**: Rapid scan 1.

![Sensitiveity Analysis](https://static.igem.wiki/teams/5540/wetlab/igem-sam-2.webp)

**Figure 1b**: Optimal results.

![Sensitiveity Analysis](https://static.igem.wiki/teams/5540/wetlab/igem-sam-3.webp)

**Figure 2**: Rapid scan 2.

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