iGEM 2023 — Team mit

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{% block title %}Wet lab notebook{% endblock %}
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# Notebook

Due to large file sizes, we have split our wet lab notebook into
3 parts, ordered chronologically.

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{% block title %}Engineering Success{% endblock %}
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# Iterations of our CAR constructs

### Introduction

Based on literature review and protein sequences available online, we designed and ordered plasmids from IDT
containing our CARs and our mCherry-IL6-3xFLAG fusion protein.
We then spent months experimenting with different ways to construct functional transcriptional units using
these type 3 CAR- or IL6-coding parts with other Mammalian Toolkit and Yeast Toolkit (MTK and YTK; El-Samad et al.) parts. In general, our engineering cycle here consisted of selecting MTK/YTK parts
for each component of a level 1 plasmid, constructing the plasmid via modular cloning, verifying correct construction via diagnostic
digest, then transfecting HEK293 cells and visualizing fluorescence.

### First iteration: learning from 0 transfection efficiency

In our first round of this cycle, we used the SV40 promoter and SV40 3' UTR as
our MTK type 2 and type 4 parts, respectively, and observed minimal fluorescence. In fact, our CAR transfections—which
were expected to fluoresce green due to the eGFP tag on the CARs—inexplicably showed significant red fluorescence.
Brainstorming with the team and mentors led us to believe that either (1) photoconversion of GFP to RFP occurred, perhaps due to spatial issues during fusion protein folding;
or (2) the cis-regulatory elements (CREs), including the SV40 promoter and 3' UTR, insufficiently stimulated translation. We were
concerned about (1) being a possibility because, if the sequence itself produced faulty proteins, we would need to start from scratch
and entirely redesign our parts. However, after replicating the transfection with identical conditions and comparing transfected cells with
control cells, we ruled out (1) by noticing that both cell populations exhibited red fluorescence; aided by literature review,
we determined that the red fluorescence was likely autofluorescence and was not cause for concern.

### Second iteration: achieving significant transfection efficiency

Having ruled out photoconversion, we moved on to the second round of this cycle by researching optimal CREs for transfection of HEK293 cells.
Literature review led us to select pEF1α for the promoter and retain SV40 for the 3' UTR. We again used modular cloning to construct plasmids with the same
coding sequences as in our first iteration, but now with a different promoter. Upon transfection of HEK293 cells with these new plasmids, we
observed significant fluorescent signal! For photos of the incredible difference in transfection efficiency between iterations 1 and 2, see our
[results page](https://2023.igem.wiki/mit/results/).

### Third iteration: attempting to optimize further

While we were content with the results of our second iteration, we did make an attempt to further optimize our constructs.
In particular, we tried changing the backbone in order to enable stable transfection of our plasmids. However, we experienced
difficulty with the modular cloning process due to the large number of parts we were attempting to clone together. Despite
multiple attempts, we were unsuccessful in constructing entirely correct plasmids. We ended up settling on the design from iteration 2.

# Chimeric Antigen Receptor Toolkit (CAR-TK)

### Introduction

While designing our chimeric antigen receptors (CARs), we noticed that CARs contain an inherently
modular structure: every CAR requires an extracellular domain to recognize a target,
a transmembrane domain to transduce signal into the cell, and an intracellular domain to
induce some reaction by the cell (e.g. phagocytosis or proliferation and cytotoxicity).
This realization inspired us to design CAR-TK, a chimeric antigen receptor toolkit that facilitates modular assembly of CARs.

To validate that our design and workflow produce the expected MTK-compatible parts, we used PCR to
extract components of CAR plasmids that we already ordered, and we verified results with diagnostic digests.

### Design and workflow

Either by ordering linear parts to be synthesized or by using PCR to extract linear
parts from plasmids, CAR-TK users must begin with linear gene blocks containing the sequence
of a CAR module. These gene blocks must also be flanked by specific overhangs that enable
the creation of an MTK-compatible Type 3 part via type IIS restriction enzyme digestion.

0. **Assembly of pL0D (level 0 domain) entry vector**

   To accommodate the fact that each gene block is a linear DNA sequence encoding a single component of a CAR,
   we designed a entry vector for these gene blocks to be cloned into, prior to assembly into a single level 0
   type 3 part. The pL0D entry vector—which we have termed *YTK000*—is identical to the pL0 entry vector
   from the yeast toolkit *(YTK001)* but with the BsmBI sites replaced with BbsI sites, and with the
   overhangs modified to avoid similarity to the BsmBI sites present during cloning into the pL1 entry vector.
   We synthesized *YTK000* by extracting parts of *YTK001* via PCR then using BsaI golden gate to
   ligate them to form *YTK000*; the primers that we used can be found as part of our primer software, detailed below.
1. **BbsI cloning into pL0D entry vector**

   Most distal from the CAR component sequence is a BbsI site. These overhangs are common to
   all CAR-TK gene blocks and enable cloning of each gene block into the *YTK000* plasmid
   for longer-term storage of individual CAR components.

   ![](https://static.igem.wiki/teams/4764/wiki/schematics/car-tk/3d-bbsi.png)

   Upon BbsI digestion followed by ligation, the GFP portion of *YTK000* drops out and
   the desired type 3D part replaces it to form a pL0D CAR component plasmid.

   ![](https://static.igem.wiki/teams/4764/wiki/schematics/car-tk/pl0d.png)
2. **BsmBI cloning into pL0 entry vector**

   Between each BbsI site and the CAR module sequence is a BsmBI site. These overhangs are specific to each
   type of CAR-TK part and enable cloning of four pL0D plasmids into the *YTK001* plasmid for
   assembly of an MTK-compatible type 3 part containing a fully functional CAR.

   ![](https://static.igem.wiki/teams/4764/wiki/schematics/car-tk/extra-bsmbi.png)
   ![](https://static.igem.wiki/teams/4764/wiki/schematics/car-tk/trans-bsmbi.png)

   ![](https://static.igem.wiki/teams/4764/wiki/schematics/car-tk/space-bsmbi.png)
   ![](https://static.igem.wiki/teams/4764/wiki/schematics/car-tk/intra-bsm.png)

   Upon BsmBI digestion followed by ligation, the GFP portion of *YTK001* drops out and the four
   CAR components enter to form a pL0 CAR plasmid that is MTK-compatible as a type 3 part.

   ![](https://static.igem.wiki/teams/4764/wiki/schematics/car-tk/pl0-yay.png)
3. **BsaI cloning into pL1**

   Between certain BsmBI sites and the CAR module sequence is a BsaI site. This is necessary so that,
   as an MTK-compatible type 3 part, the CAR pL0 can be used in typical BsaI Golden Gate assembly
   with other MTK or YTK pL0 parts to create a transcriptional unit that will express the CAR. In particular,
   the following overhangs are required in CAR-TK gene blocks:

   ![](https://static.igem.wiki/teams/4764/wiki/schematics/car-tk/extra-bsai.png)

   ![](https://static.igem.wiki/teams/4764/wiki/schematics/car-tk/intra-bsai.png)
4. **Decisions about restriction sites and overhangs**

   Our choices for how restriction sites are ordered and for the overhangs corresponding to each CAR-TK part
   type have been carefully thought out to maximize the chances of successful cloning.
   In each phase of cloning described above, the restriction site used is cut out of the final plasmid
   product; thus, any restriction sites needed for downstream cloning must lie more proximal to the CAR
   component sequence. This philosophy motivated the BbsI-BsmBI-BsaI framework present in our design.
   Additionally, the CAR-TK type-specific overhangs we chose for cloning into the pL0 entry vector are not
   random four-base sequences but rather overhangs taken from other MTK type-specific overhangs that have
   already been optimized for minimal off-target ligation.

### CAR-TK primer design software

To facilitate design of primers for PCRing out CAR-TK-compatible parts from plasmids containing full CARs,
we have coded software that accepts annotated GenBank files and the desired CAR-TK part types corresponding to
annotated regions, and returns primer sequences with appropriate overhangs and homologous regions. Visit our
[software page](https://2023.igem.wiki/mit/software) to learn more and download the software!

### Our thought process behind our CAR-TK design

#### Initial design

At first, our toolkit was designed to accommodate three types of CAR parts:
extracellular receptor, transmembrane signal transduction domain, and intracellular
signaling domain. The overall workflow we proposed is cloning of linear CAR components
(either ordered online or PCR'ed out of a plasmid) into *YTK001*, the MoClo pL1 entry vector, to
create a normal type 3 part.
After ordering primers and extracting our desired test parts via PCR,
we constructed a pL1 CAR and verified its correct construction with a diagnostic digest gel.

 However, we soon realized that multiple aspects of our design could be improved
significantly. In particular, we felt it would be helpful if CAR-TK users
could first clone individual CAR components into a entry vector before cloning
those components into the pL1, because this would allow for creation of glycerol
stocks of the components rather than storage of linear parts. Additionally,
upon further literature review, we realized that enabling customizability of
the spacer between the extracellular and transmembrane parts is vital. These two
desired improvements motivated us to undergo another round of design to improve upon
our CAR-TK.

#### Second iteration

For our second iteration, our toolkit was designed to accommodate four types of CAR parts:
extracellular receptor, flexible spacer, transmembrane signal transduction domain, and intracellular
signaling domain. The overall workflow we proposed is depicted in the diagrams above.

 To assist with designing primers to PCR out the components of CAR plasmids we already
have, and to help future CAR-TK users design primers, we additionally created software to
automate CAR-TK primer design. To learn more and access the tool, view our [software page](https://2023.igem.wiki/mit/software).

#### Future steps

We are proud of our current CAR-TK design and believe it will serve as a useful tool
to significantly simplify researchers' CAR construction workflow. Some ideal additions
we would incorporate in future design iterations include the ability to customize new component types, for example an intracellular tag on the 3' end of the intracellular signaling domain. This could be useful for adding fluorescent tags, purification tags if needed for certain applications, extra signaling tags, etc.
We would also like to conduct more experimental validation of the design and potentially iterate upon it further by considering other Type IIS restriction enzymes that may improve the CAR-TK assembly success rate and prevent off-target effects.

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# Key protocols

### General bacterial workflow

#### Modular cloning

##### Notes

* The 2023 MIT iGEM team has successfully used this protocol to assemble Yeast Toolkit and Mammalian Toolkit plasmids, both pL1s (single transcriptional units) and pL2s (multiple transcriptional units).
* This protocol follows the NEB provided protocol exactly, although we always use the NEB thermocycler program designed for 5 to 10 inserts.
* Pipetting volumes less than 1 uL is unreliable. Create 75 ng/uL stock solutions of your part plasmid minipreps if their concentrations are above 100 ng/uL.

##### Materials

* NEBridge Golden Gate Assembly Kit
* BsaI-HFv2 (NEB #E1601) or BsmBI-v2 (NEB #E1602) depending on assembly
* 75 ng of each part plasmid (the 75 ng is considering the size of the whole plasmid, not just the fragment that is to be cloned together)
* 200 uL PCR tube strip

##### Protocol

1. Set up the assembly reaction in a 200 uL PCR tube in the following order:

* x uL nuclease-free water, with x = 17-N uL where N is the number of inserts. *This volume is calculated by taking the 20 uL total reaction volume and subtracting 2 uL for ligase buffer, 1 uL for NEB Golden Gate Assembly Mix, and N uL for total part plasmid volume, assuming 1 uL per part, i.e. miniprep concentration of 75 ng/uL*
* 1 uL each of 75 ng/uL part plasmid stock solutions
* 2 uL 10X T4 DNA ligase buffer
* 1 uL NEB Golden Gate Assembly Mix

2. Run the following thermocycler program:

1. Repeat 30 cycles: 37ºC for 1 minute (digestion) then 16ºC for 1 minute (ligation)
2. 60ºC for 5 minutes (final digestion and heat inactivation of enzymes)
3. 4ºC infinite hold

3. Transform immediately into competent E. coli or store at 4ºC overnight and use the next day.

#### Transformation

##### Materials

* 1 LB+antibiotic plate per transformation
* 1 LB+Amp plate for pUC19 control
* 50 µL competent cells per transformation
* ~50 ng plasmid to transform (stored at -20ºC)

##### Protocol

1. Fill heat block wells with dH2O and turn on the heat block to 42ºC.
2. Thaw a 50 µL tube of NEB 5-alpha or 10-beta competent cells for each reaction on ice for 10 minutes.
3. Use 10-beta cells for larger plasmids (>10kb), or plasmids with more repeats. Otherwise, 5-alpha cells should suffice.
4. Warm a LB+antibiotic agar plate for each transformation to room temperature. Prepare an LB+Amp agar plate for pUC19 transformation control.
5. Add the DNA you wish to transform into the bacteria. Be sure to gently mix your plasmid stock by pipetting up and down before adding it (otherwise the plasmid can settle to the bottom of your microfuge tube):
6. If transforming a plasmid: Add 1 µL of each plasmid to each cell aliquot. Gently mix by flicking the tube 4-5 times.
7. If transforming a MoClo reaction: Add 2 µL of MoClo reaction mix to each cell aliquot.
8. Incubate on ice for 30 minutes.
9. Heat shock at 42ºC for 30 seconds (exactly).
10. Quickly place the cells back on ice for 5 minutes.
11. Add 950 µL of room temperature growth media without antibiotics. Incubate at 37ºC for 60 minutes, shaking vigorously (250 rpm).
12. Use Stable Outgrowth Media if using NEB 10-beta or NEB Stable Cells.
13. Use Super Optimal broth with Catabolite repression (SOC) if using NEB 5-alpha cells.
14. Mix cells thoroughly by flicking the tube and inverting, then spread 50 µL using glass beads onto LB+antibiotic agar plate.
15. Spread a larger amount if transformation efficiency is expected to be low (e.g. many inserts, large plasmid).
16. Alternatively, perform a four-quadrant streak dilution on the LB+antibiotic agar plate.
17. Incubate the plate overnight at 37ºC. Keep the competent cell tube in 4ºC overnight.
18. Let the plate grow for no more than 16 hours. Parafilm and store in 4ºC.
19. Bleach remaining competent cell contents with 20% bleach in a 1:1 volume ratio and dispose.

#### Inoculation

##### Materials

* Glycerol stock of bacteria with desired plasmid OR plate containing colonies of bacteria transformed with desired plasmid
* Disposable culture tubes

##### Protocol

1. Per plate or glycerol stock, label a culture tube with identifying information for the plasmid.
2. Add 5 mL of LB media into each 14 mL tube.
3. Add 5 uL of appropriate 1000x antibiotic solution.
4. Make sure you pipet the antibiotic solution into the liquid and NOT the side of the tube. Also make sure you do not lower the tip too far below the surface of liquid to avoid contaminating the pipet.
5. If using plates, they may have a mix of colored and white colonies. Pick two colonies per plate to inoculate, determining the appropriate color to pick based on the construct (e.g. a GFP dropout plasmid will stay green if incorrectly constructed and will become white when correctly constructed).
6. For each tube: Using a P200, obtain a new tip, open the petri dish or glycerol stock, and dip the pipette tip into the colony.
7. For plates, it is important to avoid colony cross contamination — more important than getting a lot of bacteria on your pipette tip.
8. Take the tip and lower it into the liquid culture. Discard the tip into the biowaste bin.
9. Gather tubes and grow on a shaker for no more than 16 hours at 37ºC at 280 rpm.

#### Glycerol stock preparation and miniprep

*Our team has prepared a miniprep walkthrough video [here!](https://www.youtube.com/watch?v=r0EhrlEW7QE)*

##### Materials

* QIAprep Spin Miniprep kit
* 1.5 mL microcentrifuge tubes
* Tabletop centrifuge
* Cryovials
* 40-50% glycerol

##### Protocol

1. If making a glycerol stock is desired (always the case unless the inoculation is already from a glycerol stock), add 900 uL of 40-50% glycerol and 900 uL culture to a cryovial. Label tubes with part ID, colony #, date, and miniprep number (if prepping more than one miniprep of the same colony). Transfer to -80ºC freezer.
2. Transfer 1 mL of bacterial overnight culture into a microcentrifuge tube. Centrifuge the culture at 6800g for 3 minutes. Discard the supernatant.
3. Obtain 31n µL of nfH2O in a microcentrifuge tube, where n is the number of minipreps you are performing.
4. Heat the nfH2O to 50ºC on a heat block as you complete the following steps.
5. Shake RNAse A + Buffer P1 vigorously to ensure all LyseBlue particles (if present) are dissolved. Resuspend pelleted bacterial cells in 250 µL of RNAse A + Buffer P1 and transfer the solution to a microcentrifuge tube. (After making glycerol stocks, it’s okay to pellet all remaining culture.)
6. No cell clumps should be visible after resuspension of the pellet.
7. Add 250 µL of Buffer P2 and mix thoroughly by gently inverting the tube 4–6 times. Continue inverting the tube until the solution becomes viscous and slightly translucent. Do not allow the lysis reaction to proceed for more than 5 min. Do not vortex the solution and do not pipette up and down aggressively, because this will result in shearing of genomic DNA and contamination of plasmid.
8. If LyseBlue has been added to Buffer P1, the cell suspension will turn blue after addition of Buffer P2.
9. Inverting the tube should result in a homogeneously colored mixture (blue if LyseBlue has been added).
10. Add 350 µL of Buffer N3. Mix immediately and thoroughly by inverting the tube 4–6 times. The solution should become cloudy, and small particles of “precipitate” should become visible.
11. Large culture volumes (>5 ml) may require inverting up to 10 times.
12. If LyseBlue reagent has been used, the suspension should be mixed until all traces of blue are gone and the suspension is colorless.
13. A homogeneous colorless suspension (translucent liquid with white, floating precipitate) indicates that the SDS has been effectively precipitated.
14. Centrifuge at 17,900g for 10 minutes in a table-top microcentrifuge. A white pellet will form spread along one face of the microfuge tube (the face that was most distal from the center of the centrifuge).
15. Transfer 800 µL of the supernatant from step 4 to the QIAprep 2.0 Spin Column. Avoid touching the pellet at all costs, even if it means not getting all 800 µL.
16. Transferring 750 µL works reliably in avoiding the pellet.
17. Centrifuge for 60 seconds at 17,900g. Discard the flow through.
18. If purifying a low-copy number plasmid OR using an endA+ strain of E. coli, wash the QIAprep 2.0 Spin Column by adding 500 µL Buffer PB and centrifuging for 60 seconds at 17,900g. Discard the flow through.
19. NEB 10-beta and NEB 5-alpha are both endA1 (endA deficient) and thus this step may be skipped.
20. Add 750 µL of Buffer PE to the QIAprep 2.0 Spin Column.
21. Centrifuge at 17,900g for 60 seconds. Discard the flow-through.
22. Centrifuge at 17,900g for an additional 60 seconds to remove residual wash buffer. Discard the flow-through.
23. Place the QIAprep 2.0 Spin Column in a clean 1.5 mL microcentrifuge tube. To elute the DNA off the column, add 50 µL of 50ºC-heated nfH2O to the center of each QIAprep 2.0 Spin Column.
24. Let stand for 1 min. This step is critical for releasing the plasmid DNA from the column.
25. Centrifuge at 17,900g for 60 seconds. Do not discard the flow-through. The elution in your clean 1.5 mL microcentrifuge tube is your miniprepped sample(s).
26. Quantify the plasmid concentration for each miniprepped sample via Nanodrop.
27. Keep the DNA elution product at -20ºC for long-term storage.

### HEK293 cell culture

#### Passaging HEK293 cells

*Our team has prepared a passaging walkthrough video [here!](https://www.youtube.com/watch?v=yEPGskCDgF4)*

##### Materials

* Complete media (DMEM + 10% FBS + 1x NEAA)
* PBS
* 0.05%–0.25% trypsin
* 10 cm dish (protocol can be modified to accommodate other sizes of dishes and flasks)

##### Protocol

1. Warm aliquots of complete media, PBS, and trypsin in a 37°C bead bath for 30 minutes.
2. Aspirate media from the 10 cm dish, being careful not to touch the monolayer of adherent cells.
3. Rinse the dish with 5 mL warmed PBS, then aspirate.
4. Add 1 mL trypsin to the dish and gently swirl.
5. Place the dish in the incubator for 3-5 minutes then examine under a microscope to see cells detached.
6. Add 9 mL of warmed complete media to the 10 cm dish. Pipette up and down several times in a "waterfall pattern" to ensure the cells are in single cell suspension.
7. Create dilutions (e.g. 1:5 and 1:20) and label tops of dishes with name, cell type, passage number, and date.
8. To create dilutions: for a 1:5 dilution in a 10 cm dish, place 8 ml prewarmed media and add 2 ml cells. Similarly, for a 1:20 dilution in a 10 cm dish, place 9.5 mL prewarmed media and add 0.5 mL cells.
9. Place the dish in the incubator.

##### Notes

* 1:5 dilution will typically allow you to passage every 2-3 days while 1:20 will allow you to passage every 5-7 days.
* Always make two dishes at two different dilutions! Ensures you have cells ready almost any day and if you make a mistake, don’t lose the cells.

#### Transfecting HEK293 cells

##### Materials

* Lipofectamine 2000
* Opti-MEM
* Complete media (DMEM + 10% FBS + 1x NEAA)
* PBS
* 0.05%–0.25% trypsin
* 24-well plate (this protocol can be modified for other sizes)
* 1.5 mL microcentrifuge tube

##### Protocol

###### Day 0 (seeding well plate)

1. Trypsinize the 10 cm plate and count cells. Passage as needed.
2. Seed cells at appropriate concentration based on ThermoFisher's [Useful Numbers for Cell Culture](https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/cell-culture-useful-numbers.html), e.g. 0.05x10^6 cells in 0.5 mL media without antibiotics for a 24-well plate.
3. After counting, pellet cells then resuspend in the appropriate amount of media and divide into wells. For example, if you are seeding 24 wells each with 0.5 mL media containing 10^5 cells, you need to pellet 26 \* 10^5 cells, and then add enough media to bring the concentration to 2 \* 10^5 cells/mL
4. Grow cells for 24 hours. Cells should be at 70-90% confluence on the day of transfection.
5. Prepare the plasmid DNA desired for transfection to concentrations of 50 ng/uL.

###### Day 1 (transfection)

*Throughout this protocol, N refers to the number of wells being transfected.*

1. Warm aliquot of Opti-MEM in a 37°C bead bath for 30 minutes.
2. Create reaction mix A by combining, in a 1.5 mL microcentrifuge tube, N \* (50 uL Opti-MEM + 1.8 uL Lipofectamine 2000)
3. For each of the N wells being transfected, create reaction mix B in N 1.5 mL microcentrifuge tubes: 40 uL Opti-MEM + 12 uL plasmid DNA (diluted to 50 ng/uL)
4. Incubate reaction mix A and B at room temperature for 5 minutes.
5. Combine reaction mix A (51.8 uL) and B (52 uL), resulting in N microcentrifuge tubes with ~103.8 uL total volume.
6. Mix gently and incubate at room temperature for 20 minutes.
7. Add each microcentrifuge tube mix dropwise to appropriate corresponding well.
8. Place the well plate in the incubator.

#### Prepping cells for flow cytometry

##### Materials

* 5 mL polystyrene tube with cell strainer
* Complete media (DMEM + 10% FBS + 1x NEAA)
* PBS
* 0.05%–0.25% trypsin
* 15 mL conical tube

##### Protocol

1. Warm aliquots of complete media, PBS, and trypsin in a 37°C bead bath for 30 minutes.
2. Aspirate off media and wash cells with 500 uL PBS.
3. Add 500 uL trypsin to each well and incubate for 5 mins at 37°C until the cells come off.
4. Add 500 uL warmed complete media to each well
5. Transfer everything to a conical tube and centrifuge for 5 min at 250 g
6. Aspirate media and resuspend in 500 uL warmed complete media.
7. Pipet cells up and down to break up aggregates and create a single cell suspension.
8. Transfer cells to a 5 mL polystyrene tube; cells should be passed through a cell strainer to remove clumps.
9. Place cells on ice until ready to do flow cytometry.

### 3xFLAG-tagged IL-6 purification and incubation with CAR cells

#### Washing and purification

##### Materials

* ThermoFisher’s Pierce Anti-DYKDDDDK Magnetic Agarose beads
* PBS
* Milli-Q water
* 1.5 mL microcentrifuge tubes
* M-PER reagent
* End-over-end microcentrifuge tube spinner
* Magnetic stand that can hold microcentrifuge tubes

##### Protocol

1. Equilibrate magnetic agarose to room temperature and invert to mix.
2. Place 50 uL slurry into a 1.5 mL microcentrifuge tube. Add 450 uL of M-PER reagent and gently vortex to mix.
3. Place the tube into a magnetic stand to collect the beads against the side of the tube. Remove and discard the supernatant.
4. Add 500 uL of M-PER reagent to the tube, mix well, collect the beads with a magnetic stand, and remove the supernatant. Repeat this once more for a total of two washes.
5. Add the sample (at least 300 uL; dilute with M-PER if necessary to reach this volume) containing 3xFLAG-tagged protein to the washed magnetic agarose and gently vortex to mix.
6. Incubate the sample at room temperature with constant end-over-end mixing for 20 minutes.
7. Collect the beads with a magnetic stand, then remove the supernatant.
8. Add 500 uL PBS to the tube, mix well, collect the magnetic agarose with a magnetic stand, and remove the supernatant. Repeat this once more for a total of two washes.
9. Add 500 uL of milli-Q water to the tube, collect the beads with a magnetic stands, and remove the supernatant.
10. Elute with one of the following protocols below, acid elution or sample buffer elution (our team has successfully tried both protocols, with similar yield).

#### Acid elution

##### Materials

* 0.1 M glycine (pH 2.8)
* 1 M tris (pH 8.5)

##### Protocol

1. Add 100 uL of 0.1 M glycine (pH 2.8) to the tube, mix well, and incubate for 5 minutes at room temperature with frequent vortexing.
2. Collect the magnetic agarose with a magnetic stand and save the supernatant, which contains the eluted protein.
3. To neutralize the acid, add 15 uL 1 M tris (pH 8.5) per 100 uL eluate.

#### Laemmli sample buffer elution

##### Materials

* 1x Laemmli sample buffer

##### Protocol

1. Add 100 uL 1x Laemmli sample buffer to the tube, mix well, and incubate at 95C for 10 minutes.
2. Collect the magnetic agarose with a magnetic stand and save the supernatant, which contains the eluted protein.

#### Casting an SDS-PAGE gel

##### Materials

* Fume hood
* SDS-PAGE gel mold (glass plates, spacers, gasket, well comb
* Serological pipette gun and 10 mL pipettes
* 12% resolving gel
* 4% stacking gel
* 10% w/v ammonium persulfate (APS)
* TEMED
* Isopropyl alcohol
* Sharpie
* 15 mL conical tube

##### Protocol

1. Degas the resolving and stacking gel solutions in a vacuum desiccator for 15 minutes.
2. Assemble the SDS-PAGE gel mold following the manufacturer's instructions.
3. Mark with Sharpie the level that is 1 cm below the teeth of the well comb.
4. Place a comb into the empty gel mold and mark with Sharpie the locations of each of your wells.
5. Set the resolving gel: add 15 mL of 12% resolving gel solution, 75 uL of fresh 10% w/v APS, and 7.5 uL of fresh TEMED to a 15 mL conical tube to initiate polymerization.
6. Gently invert the tube multiple times to ensure the TEMED and APS are evenly mixed.
7. Use a serological pipette to quickly transfer the solution to the casting mold, filling up to the line you marked earlier (~1 cm below the wells).
8. Add a layer of isopropyl alcohol to prevent exposure to oxygen. Allow the gel to polymerize for 90 minutes.
9. After the gel has polymerized, use Kimwipes to soak up the layer of isopropyl alcohol.
10. Place the gel comb into the gel mold with the teeth at a 15º angle (this prevents air from becoming trapped under the comb while the acrylamide solution is being poured). Allow the gel to polymerize for 30 minutes.
11. Set the stacking gel: add 5 mL of 4% stacking gel solution, 25 uL of fresh 10% w/v APS, and 5 uL of fresh TEMED to a 15 mL conical tube to initiate polymerization.
12. Gently invert the tube multiple times.
13. Use a serological pipette to transfer the stacking gel solution until the casting mold is filled.
14. Align the comb in the gel mold. Allow the gel to polymerase for 90 minutes.
15. Remove the gasket from the gel.

#### Running an SDS-PAGE gel

##### Materials

- Tris-glycine running buffer
- Gel running box
- Stirbar and magnetic stir plate
- Ice packs
- 1x and 2x Laemmli sample buffer
- Protein ladder, like ThermoFisher's PageRuler Prestained Protein Ladder

##### Protocol

1. Assemble the gel box according to the manufacturer's instructions. If only running one gel, insert empty gel glass plates on the side that is not being used to seal the inner chamber.
2. Place a magnetic stir bar at the bottom of the outer chamber.
3. Remove the gaskets and spacers from the gel mold. Place the gel mold with the gel in it into the gel box, following the manufacturer's instructions. The plate with the indentation should face inward.
4. Fill the inner chamber with running buffer to the top of the gel, so that buffer fills the wells.
5. Remove the comb by pulling it straight up slowly and gently. If the wells appear to have pieces of broken polyacrylamide in them, pipette Milli-Q water into the wells to rinse them.
6. Add ice packs to the outer chamber. The goal is to maintain an outer chamber temperature of 15ºC.
7. Fill the outer chamber with running buffer until the buffer reaches just below the sample wells.
8. Load your samples. For a 14-well, 1.5 mm gel, the maximum recommended loading volume is 30 uL. A good rule of thumb is to load 2 ug of purified protein or 200 ug protein of cell lysate
9. Load your protein ladder on the far left, middle, and far right, based on the number of samples you are loading.
10. Load unused lanes with 5 uL of 1x Laemmli sample buffer
11. Turn on the stirrer (130 rpm).
12. Run at 80 V (constant voltage) until samples have fully entered the stacking gel (around 30 minutes) then 120V (constant voltage) until the dye is near the bottom of the gel, typically around 3 hours.

#### Staining an SDS-PAGE gel

##### Materials

* Coomassie blue staining solution
* Destaining solution
* Tupperware
* Large KimWipes or paper towels

##### Protocol

1. Rinse gel under tap water a few times to remove residual SDS.
2. Add 50 mL Coomassie blue staining solution to tupperware.
3. Place gel in the tupperware then microwave for 1 minute.
4. Place tupperware on shaking table for 10 minutes at moderate speed to constantly wash gel with staining solution.
5. Dispose of staining solution.
6. Rinse with Milli-Q water, used destaining solution. or used rinsing solution twice, each time pouring out the diluted solution into a bottle for rinsed solution.
7. Transfer the gel to a new tupperware and add 75 mL destaining solution.
8. Tear up a large KimWipse and surround gel with wipes, being careful not to allow wipes to lay over the gel (this causes uneven destaining).
9. Microwave for 1 minute or until solution boils.
10. Place on shaking table for 10 minutes at moderate speed.
11. Discard kimwipes and add another 25 mL destaining solution.
12. Prepare another 4-kimwipe masterpiece and surround gel with wipes, being careful not to allow wipes to lay over the gel.
13. Place on shaking table overnight at moderate speed.

#### Incubating CAR-positive HEK293 cells with IL-6

*This protocol is designed for a 12-well plate but can be appropriately adjusted to a plate of any other size.*

##### Materials

* Solution containing IL-6, whether that may be after protein purification or directly from a HEK293 whole cell freeze-thaw lysate
* 12-well plate

##### Protocol

1. Add 100-115 uL of the IL-6 solution or up to 1 mL of the whole cell lysate containing IL-6 to the well.
2. Let the cells incubate at 37 degrees Celsius and 5% CO2 for 6 hours.
3. Prepare the cell samples for flow cytometry and proceed with analysis.

# All protocols

Below is a PDF detailing every protocol we used over the course of our project, including
the protocols described above and many more.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block page\_content %}
Inspired by CAR-T cell therapy, we have developed a chimeric antigen receptor protein to treat cancer cachexia. We have
chosen to target the cytokine IL-6 because it is a main immunoinflammatory cytokine that triggers signaling pathways
that have been traced to the degradation of muscle and fat cells. We have successfully demonstrated expression of the
CAR protein in HEK293 cells and binding to our 3xFLAG-IL6-mCherry protein via flow cytometry.

# Overview of key achievements

* Constructed **2 anti-IL-6 CAR plasmids** and an **IL-6 fusion protein plasmid** using
  modular cloning, after iterations of optimizing cis-regulatory elements
* Transfected all plasmids into HEK293 cells and observed **significant expression of constructs** as
  determined by fluorescence levels
* Co-cultured CAR-expressing cells with IL-6 and observed **binding of IL-6 to anti-IL-6 CARs**

# Experimental design decisions

#### Plasmid sequence design

In order to plan for the experimentation of our CAR protein, we decided on creating a fluorescently tagged IL6
protein. We expected to employ mammalian cells to produce significant quantities of the fusion IL-6 protein, and
therefore planned to perform protein purification as well. Therefore, we designed a fusion IL-6 protein that had both
a
3xFLAG tag and mCherry reporter.

In our design process, we considered HA tags as well, but ultimately landed on 3xFLAG, which appeared to have more
protocols allowing for non-denaturing protein purification.
The selection of mCherry was due to our limitations of our fluorescence microscope, and wanting to differentiate from
eGFP and BFP (which we expected to use in other parts of our experiment).
Finally, before ordering the coding part from IDT, we codon optimized it for CHO (Chinese hamster ovary) cells and
added Mammalian Toolkit [1,2](#cite1) compatible ends, preparing this part as a Type 3 part.

Our CAR plasmids have the following components linked together with glycine/serine linkers (Parts BBa\_K4764005 and
BBa\_K4764006):

* extracellular: anti-Il-6 receptor
* transmembrane: CD8
* intracellular: Fcγ vs. Megf10
* reporter: eGFP

The extracellular component was taken directly from sirukumab, a monoclonal antibody for IL-6.
The transmembrane and intracellular components were inspired by Morrissey et al[1](#cite3). The transmembrane CD8 has typically
been used in other CAR proteins. For the intracellular component, we took note of the fact that the authors found Fcγ
and Megf10 to have the most significant phagocytosis when the CAR was expressed on J774A.1 murine macrophages.
Therefore, we aimed to create two CAR plasmids that only varied in their intracellular part, having either the Fcγ or
Megf10 protein domain.

#### Modular cloning and transfection

After receiving our plasmids from IDT, we cloned them into a plasmid backbone (YTK001) provided by the Yeast Toolkit
(YTK). The other components of the plasmid were directly taken from the YTK. Our designs are noted on our lab
notebook.

After verification of the plasmids via diagnostic digests, we cloned them again to include mammalian cis-regulatory
elements and to form a complete level 1 transcriptional unit plasmid. We initially started with the SV40 promoter and
SV40 3’ UTR, but saw nearly 0 transfection efficiency, meaning that extremely few cells per well in our 12 well plate
successfully transfected and fluoresced.

Representative photos of our first attempts are to the right.

![](https://static.igem.wiki/teams/4764/wiki/flow/red-bad.png)
![](https://static.igem.wiki/teams/4764/wiki/flow/green-bad.png)

***Figure 1: First attempt at transfecting
HEK293 cells with 3xFLAG-IL6-mCherry (left) and anti-IL6 Megf10 CAR***

However, we also ordered and used the CD22-Megf10 plasmid from Morrissey et
al. as a positive control; we saw that the CD22-Megf10 plasmid had extremely high transfection efficiency, so we
compared our existing promoter and 3’UTR to those used by Morrissey et al. Through many iterations of
cloning, detailed on our engineering page, we finally ended up using the pEF1α promoter and SV40 3’UTR.

After many iterations of plasmid construction, we ended up seeing substantial
fluorescence from all three of our constructs! Representative
photos from transfection of our finalized designs are to the right.

![](https://static.igem.wiki/teams/4764/wiki/flow/red-good.png)
![](https://static.igem.wiki/teams/4764/wiki/flow/green-good.png)

***Figure 2: Final transfection of HEK293
cells with 3xFLAG-IL6-mCherry (left) and anti-IL6 Megf10 CAR***

#### Mammalian cell line choice

We began learning and practicing good aseptic technique in the tissue culture room with NIH 3T3 murine
fibroblasts. We ran our pilot transfection protocol on 3T3 cells utilizing one vial of Lipofectamine 2000 which
had expired in 2017. We wanted to do a test run of the protocol ahead of running it on the HEK293 cells.

The gracious Weiss Lab was kind enough to gift us 2 vials of frozen HEK293 cells. After thawing them and passaging
them a few times, we began to experiment with transfection. We first optimized our transfection protocols between
the possible transfection reagents that we procured (Lipofectamine 2000, FuGENE, and Viafect).

Our initial plan at the start of the summer was to use HEK293 cells to produce our IL-6 fusion protein, then to use
RAW264.7 murine macrophages to express our CAR protein. As the summer progressed, though, we ended up continuing
to use HEK293 cells for all of our experiments. Because of this, we were not able to detect if the CAR induced
phagocytosis; our proof of concept was limited to verifying the specificity and binding affinity of the receptor.

#### Stable versus transient transfection

In order to prepare for the later stages of our project, we considered cloning plasmids that could be stably transfected. Especially for the IL-6 fusion protein, we expected to harvest the protein over the course of a number of passages with the same cells. Therefore, we took time to resuspend and outgrow a plasmid that coded for the piggyBac transposase. In parallel, we also decided to clone our plasmids into Mammalian Toolkit level 2 vectors (pL2) with piggyBac transposons and hygromycin resistance embedded. When we started to optimize our stable transfection protocols, we simultaneously ran a hygromycin “kill curve” experiment on a 96 well plate of HEK293 cells. This was to identify the concentration of hygromycin that would weed out the cells without our desired plasmids but not harm the cells with hygromycin resistance.

Ultimately, we succeeded with observing relatively high transfection efficiency of control plasmids after the first 48 hours of stable transfection. However, due to the repeated adjustment of the cis-regulatory elements on our experimental plasmids, as well as the time-consuming process of cell sorting and regrowth along with the difficulty of cloning together multiple parts to form a pL2, we did not pursue the additional step of cloning them into pL2s. Our final experiments were completed with transient transfection.

# Validation of proof-of-concept

#### Purification of 3xFLAG-IL6-mCherry protein

A central component of our experiment rested on the assumption that we would be able to purify our 3xFLAG-IL6-mCherry fusion protein from HEK293 cell lysate with extremely high yield. First and foremost, we needed to optimize the transfection efficiency of our fusion protein. Figure 3 shows the transfection of HEK293 cells at passage 20 with 600 ng of plasmid DNA at a 48 hour timepoint.

![](https://static.igem.wiki/teams/4764/wiki/flow/p20mcherry.png)

***Figure 3: HEK293 cells transfected with our 3xFLAG-IL6-mCherry fusion protein***

At this point, we began to approach protein purification from a few different perspectives.

Initially we considered biotinylating our protein to fluorescent streptavidin silica beads.
This would knock out three birds with one stone:

1. We could create IL-6 clusters of variable, controlled sizes on a micron scale to surpass the size and signal threshold of macrophages.
2. We could isolate the IL-6 protein from the rest of the HEK293 cell lysate.
3. We could detect via fluorescence if the macrophages only bound to the IL-6 or actually phagocytosed the whole bead

However, we decided against the bead idea because it would require multiple rounds of purification, with unknown and likely decreasing protein yield rates. We valued the quantity of final protein above the existence of IL-6 aggregates for our proof-of-concept.
Although we had hypothesized that aggregates are necessary to induce phagocytosis, for the scope of one summer and with our limited resources, we decided it would be enlightening and worthwhile to study how soluble IL-6 interacts with our CARs. Even if we have the chance to move on to macrophages in the future, studying soluble IL-6 now will still provide insight into how macrophages may endocytose or pinocytose soluble IL-6 in addition to any aggregates we are able to form in the future.

We instead turned to simply running one round of anti-FLAG tag purification. We utilized Pierce anti-FLAG magnetic agarose beads to selectively isolate the 3xFLAG-tagged IL6. We optimized the protocol by varying over three elution buffers:

1. IgG Elution Buffer
2. 0.1 M glycine
3. SDS-PAGE sample loading buffer, namely 1x Laemmli buffer

In order to give us an idea of our protein yield rate, we turned to using SDS-PAGE gel electrophoresis, the Bradford assay, and the NanoDrop spectrophotometer. At first, we had hoped that the SDS-PAGE gels would be able to verify the existence of our protein (by length) and the relative concentration (by band color intensity). After dozens of attempts to optimize the SDS-PAGE equipment available to us, we concluded that it would be an unreliable form of measurement. Then, we looked to quantify our protein concentration via Bradford assay and absorbance comparisons to bovine serum albumin standards. We were getting readings extremely close to 0 Abs and even negative absorbances, and we verified this tragic data with the NanoDrop.

Regardless of this insanity check, we proceeded with attempting to incubate the so-called “purified protein elution” with our CAR-expressing HEK293 cells.
After concluding that there was no red fluorescence detected after incubation, we reconsidered our purification procedure. Noor, one of our mentors, brought up the idea of incubating the CAR-expressing HEK293 cells directly with the lysate of IL6-expressing HEK293 cells.
We examined the lysis and purification steps that we had been applying—so far, we had been lysing the IL6-expressing HEK293 cells with ThermoFisher M-PER reagent. Since M-PER was a detergent that would unintentionally lyse our CAR-expressing cells and could not be extracted from the final lysate solution, we turned to another form of mammalian cell lysis to accomplish our goal: freeze-thaw lysis. The final lysate was not purified; we directly added it to wells of CAR-expressing HEK293 cells to observe the resulting behavior. This way, we knew IL-6 was in the lysate and would no longer act as a limiting factor.

We followed the [2013 Calgary iGEM team’s protocol](https://2013.igem.org/Team%3ACalgary/Notebook/Protocols/FreezeThawProtocolforProteinSamples) but for a 10 cm dish of HEK293 cells. Ultimately, after viewing the fluorescence under our Keyence BZ-X810 microscope using the GFP and RFP filter cubes, we saw significantly more red fluorescence with freeze-thaw lysate than the purified protein eluted with Laemmli buffer and the purified protein eluted with IgG buffer.

![](https://static.igem.wiki/teams/4764/wiki/flow/coculture1.png)

***Figure 4: Incubating CAR HEK293 cells with 3xFLAG-IL6-mCherry freeze-thaw lysate***

![](https://static.igem.wiki/teams/4764/wiki/flow/cocultureigg.png)

***Figure 5: Incubating CAR HEK293 cells with IgG-eluted 3xFLAG-IL6-mCherry***

#### Incubating CAR-expressing (CAR+) HEK293 cells with 3xFLAG-IL6-mCherry

This phase of our project was full of the most unknowns. We would not be following any other lab’s protocols nor be able to reference multiple papers that cited running similar experiments.
Ultimately, we had three types of IL-6 protein solutions we wanted to try incubating with CAR-expressing HEK293 cells:

1. Protein eluted with 1x Laemmli buffer
2. Protein eluted with IgG buffer
3. IL6-expressing HEK293 freeze-thaw lysate

These three solutions corresponded to our intention of experimenting with multiple protein concentrations to determine the best concentration for binding efficiency. We had two types of CAR+ cells: those that expressed Fcγ intracellularly and those that expressed Megf10 intracellularly. These populations would hopefully determine which protein could be attributed to better binding efficiency. Since we were using free-floating IL-6 instead of aggregates, we were unable to mimic the experimental conditions that Morrissey et al. used in their experiments.

We had additionally thought about varying incubation time in order to observe if the net amount of binding between the CAR and the IL-6 would positively correlate with time. We ended up running one experiment with a 1 hour incubation period and one experiment with a 6 hour incubation period. We saw significantly better binding with the 6 hour incubation – the result could have also been attributed to the fact that we had used the freeze-thaw lysate in this longer experiment.

We also accounted for controls in our design. As a negative control we introduced purified IL-6 to untransfected HEK293 cells. We additionally intended to introduce untransfected HEK293 whole cell lysate to CAR-expressing HEK293 cells, but we were unable to due to time restraints. Thus we are unable to document whether or not the CAR is specific to IL-6, or possibly to simply any protein it is incubated with over time.

Ultimately, we had uncertainty approaching this phase of the project, but we were able to nail down certain experimental conditions that allowed us to test for our proof-of-concept.

We viewed our cells post-incubation in two ways: first, using our Keyence BZ-X810 fluorescence microscope, and second, using flow cytometry.
Figure 6 show one of our wells under the microscope, and below, you can read how we analyzed our data using flow cytometry.

Importantly, after the incubation period, the wells of cells that had been incubated with Laemmli-buffer-eluted IL-6 had fully lifted off the bottom of the wells. They were unable to be prepared for flow cytometry analysis – therefore, we were unable to quantify the frequency of double positive cells in those populations. We suspect this occurred because of the chemical nature of 1x Laemmli buffer, which is a reducing agent that may have denatured the proteins responsible for making the cells adherent.

![](https://static.igem.wiki/teams/4764/wiki/flow/coculture2.png)

***Figure 6: Incubating CAR HEK293 cells with 3xFLAG-IL6-mCherry freeze-thaw lysate***

#### Flow cytometry data

Ultimately, we have successfully achieved a proof-of-concept for the IL-6 binding component of our therapeutic
by demonstrating that IL-6 binds to CAR-expressing cells significantly more frequently than to control cells.

It took a few attempts to run everything smoothly, but our final protocol followed a precise timeline that
allowed us to see encouraging results!

*Day 0:* We transfected a 10cm dish of HEK293 cells with our 3xFLAG-IL6-mCherry fusion protein. mCherry
control wells were transfected too.
*Day 1:* We transfected a 12-well plate of HEK293 cells, half of the wells with our anti-IL6 Fcγ CAR and
half with our anti-IL6 Megf10 CAR. GFP control wells were transfected too.
*Day 2:* We extracted the cells of the IL6-transfected dish using a cell scraper and processed the cells
in 2 different ways.

1. Scraped cells were lysed according to a freeze-thaw protocol (detailed on our experiments page) then
   co-cultured with wells containing CAR-expressing cells (detailed in our lab notebook).
2. Scraped cells were lysed according to ThermoFisher's M-PER lysis protocol (detailed on our experiments page)
   then purified with IgG elution buffer (detailed on our experiments page). Purified IL6-mCherry fusion protein
   was added to wells containing CAR-expressing cells (detailed in our lab notebook).

After a day of incubation of CAR cells with IL-6, we washed the wells with PBS to remove free-floating IL-6
unbound to CARs then prepared
the cells for flow cytometry (detailed in our experiments page). We then ran flow with untransfected cells as a
negative control; mCherry-expressing cells and GFP-expressing cells as fluorescent controls; and
our CAR plus IL-6 experimental cells.

Due to our team's inexperience running flow cytometry, we struggled with establishing appropriate voltages and
determining settings for parameters such as forward scattering and back scattering.
Our post-flow analysis using the software FlowJo was challenging because of this, and we had to plot data with
unconventional methods such as switching from
linear to logarithmic axes to ensure all of our data points fit on graphs. However, with the help of mentors with
much more flow experience than us—Hayden Sandt and Noor Radde, in particular—we were able to
effectively and objectively re-format our data to better match academic standards.

We then gated our data to determine cell populations based on whether they fluoresced green (i.e., expressed CARs)
and/or whether they fluoresced red (i.e., bound to IL6-mCherry). For full
transparency, our backgating data and analysis is presented below. In each figure below,
the left graph illustrates gating for HEK293 cells; the middle graph illustrates
the subset of cells that are GFP positive (CAR+); and the right graph
illustrates the subset of CAR+ cells that are mCherry positive (CAR+IL6).

![](https://static.igem.wiki/teams/4764/wiki/flow/backgating-negative-control1.png)

***Figure 7: Negative control
backgating.***

First, we used negative control cells to separate cell debris from actual HEK293 cells in our data. This was
performed under the assumption that debris is significantly smaller in size than actual HEK293 cells. Thus,
we strictly gated for the area around the hotspot and above, but discarded the area that tailed underneath.
Retroactively, we applied our eGFP+ and mCherry+ gating to the negative control population to verify our
gating stringency; as expected, neither eGFP+ nor mCherry+ cell populations were observed in the negative
control HEK293 cells.

![](https://static.igem.wiki/teams/4764/wiki/flow/backgating-egfp1.png)

***Figure 8: eGFP control
backgating.***

In Figure 8, we next used eGFP-expressing cells as a positive control. We intended to gate for green
fluorescing cells (eGFP+) in this population, but due to the realization that we did not have a clear second
peak representing the eGFP+ population, we decided to use this population to gate for mCherry+ instead. We
knew these eGFP+ cells had no mCherry expression; therefore, our mCherry gate was decided where there was no
fluorescent signal on the histogram. In the same manner, we determined our eGFP+ gate by using the
mCherry-expressing population and selecting the portion with low to no mCherry signal. Retroactively, we
verified that the majority of the eGFP-expressing population was correctly gated for eGFP+, and similarly
for mCherry+.

Having gated for green expression, red expression, and no color, we then analyzed our CAR plus IL-6 experimental
cell populations.

![](https://static.igem.wiki/teams/4764/wiki/flow/backgating-megf10-igg1.png)

***Figure 9: Megf10 CAR with IgG-purified
IL-6 backgating.***

Figure 9 corresponds to cells transfected with our Megf10 CAR and incubated with IgG-purified IL-6.
We first subsetted the entire population to only HEK293 cells, removing debris. Next, we gated for cells
which were eGFP+, implying CAR expression. We then further subsetted this green population to cells which
also fluoresced red (doubly positive), implying that the CAR bound to our fusion IL6-mCherry protein. As
desired, a significant portion of the entire population fluoresces both green and red, indicating successful
binding.

![](https://static.igem.wiki/teams/4764/wiki/flow/backgating-megf10-ft1.png)

***Figure 10: Megf10 CAR with IL-6
freeze-thaw lysate backgating.***

Figure 10 corresponds to cells transfected with our Megf10 CAR and co-cultured with IL-6 freeze-thaw lysate.
We first subsetted the entire population to only HEK293 cells, removing debris. Next, we gated for cells
which were eGFP+, implying CAR expression. We then further subsetted this green population to cells which
also fluoresced red (doubly positive), implying that the CAR bound to our fusion IL6-mCherry protein. As
desired, a significant portion of the entire population fluoresces both green and red, indicating successful
binding.

![](https://static.igem.wiki/teams/4764/wiki/flow/backgating-fcgamma-igg1.png)

***Figure 11: Fcγ CAR with IgG-purified
IL-6 backgating.***

Figure 11 corresponds to cells transfected with our Fcγ CAR and incubated with IgG-purified IL-6.
We first subsetted the entire population to only HEK293 cells, removing debris. Next, we gated for cells
which were eGFP+, implying CAR expression. We then further subsetted this green population to cells which
also fluoresced red (doubly positive), implying that the CAR bound to our fusion IL6-mCherry protein. As
desired, a significant portion of the entire population fluoresces both green and red, indicating successful
binding.

![](https://static.igem.wiki/teams/4764/wiki/flow/backgating-fcgamma-ft1.png)

***Figure 12: Fcγ CAR with IL-6
freeze-thaw lysate backgating.***

Figure 12 corresponds to cells transfected with our Fcγ CAR and co-cultured with IL-6 freeze-thaw lysate.
We first subsetted the entire population to only HEK293 cells, removing debris. Next, we gated for cells
which were eGFP+, implying CAR expression. We then further subsetted this green population to cells which
also fluoresced red (doubly positive), implying that the CAR bound to our fusion IL6-mCherry protein. As
desired, a significant portion of the entire population fluoresces both green and red, indicating successful
binding.

Finally, we aggregated the above data and concluded with exciting results: **a significant percentage of
cells expressed CARs *and* bound IL6-mCherry!!**

![](https://static.igem.wiki/teams/4764/wiki/flow/finalhist.png)
![](https://static.igem.wiki/teams/4764/wiki/flow/finalfreqs.png)

***Figure 13: Control and experimental
cell populations positive for both green and red fluorescence. Being positive for both implies
successful CAR expression *and* ability to bind IL-6.***

From top to bottom, the populations are:

1. Negative control: as expected, cells not transfected with CARs bound no IL-6.
2. anti-IL6 Megf10 CAR (IgG method): 10.4% of cells transfected with our Megf10 CAR and incubated with
   IgG-purified IL-6 were double positive.
3. anti-IL6 Megf10 CAR (freeze-thaw method): 12.0% of cells transfected with our Megf10 CAR and co-cultured
   with IL-6 freeze-thaw lysate were double positive.
4. anti-IL6 Fcγ CAR (IgG method): 11.0% of cells transfected with our Fcγ CAR and incubated with
   IgG-purified IL-6 were double positive.
5. anti-IL6 Fcγ CAR (freeze-thaw method): 16.2% of cells transfected with our Fcγ CAR and co-cultured with
   IL-6 freeze-thaw lysate were double positive.
6. eGFP control: as expected, cells transfected only with an eGFP plasmid did not appear to fluoresce red.

#### Analysis of results

From the final chart comparing our different variables against each other, we can conclude that the Fcgamma CAR
incubated with IL-6 freeze-thaw lysate had the greatest double positive frequency of the parent population, but
the other three samples were significant as well and not too different from each other. We are pleased to find
that the incubations with freeze-thaw lysate were both higher than the incubations with IgG-eluted IL-6. This
supports our suspicion that the protein purification protocol had annihilated the concentration of IL-6 in those
solutions. We suspect that the manyfold greater concentration of IL-6 in the freeze-thaw lysate improved the
probability of CAR binding events.

Additionally, we were eager to compare the behavior of the Fcgamma vs. Megf10 intracellular component. Although
it is important to note that we were not able to compare its impact on the phagocytotic behavior of a
macrophage, we theorize that the intracellular component had an impact on the folding of the protein or even the
binding affinity of the protein. Due to the limited scope of the experiments we ran, we cannot conclude which is
more optimal than the other; we determine that they are both great candidates to move forward with and perform
macrophage experiments with. Based on phagocytic activity, it may become more apparent which CAR is preferable.

# Replicating our experiments

The precise protocols that we followed are detailed on this page and on
our experiments page and lab notebook. We have made every effort to thoroughly document our experiments
and ensure other researchers can reproduce our results, and we welcome questions or clarifications
addressed to igem-2023-students@mit.edu

For replication of our experiments, we recommend preparing all of the possible controls to account for all variables of the project. Namely, to test for binding specificity, we suggest incubating CAR+ cells with whole cell lysate (ideally with some sort of fluorescent reporter) for 6 hours and comparing the double positive data with the data we have achieved.

Moreover, it is unclear why we observed significant loss of protein yield during the two-step M-PER lysis protocol and anti-FLAG protein purification protocol. We urge future researchers to test anti-FLAG purification of the freeze-thaw lysate, with the goal of understanding where yield is lost. It is possible that the loss of protein lies in the M-PER lysis protocol. Additionally, if it is found that the FLAG tag is the issue, we urge the creation of a similar IL-6 protein tagged with an amino acid chain that can assist in a different method of non-denaturing purification.

# Future plans

We are extremely proud of having achieved a proof-of-concept that HEK293 cells expressing our CARs
bind IL-6 significantly more strongly than cells lacking our CARs. Due to constraints on our time and resources,
we were unable to
progress further than this level of achievement. However, we have planned potential next steps that
can be taken to achieve the loftier goal of developing a real, practical cachexia therapy.

1. **Transfecting macrophages:**
   We wish to be able to test the transfection of our plasmids on macrophages such as RAW264.7 cells. We are curious to observe the transfection efficiency and the temperament of epitopes on the macrophages, especially as we experiment on them. We anticipate that macrophages may prove to be more difficult to engineer because, as immune cells, they have a proclivity for immune activation and changes in morphology.
2. **Inducing IL-6 aggregation (if needed):**
   We wish to consider the need for meeting the size and signal threshold for macrophages at the experimental level. This could mean engineering a different plasmid that codes for a secreted aggregation protein that is specific to IL-6.
3. **Incorporating modulation via genetic circuitry:**
   We wish to specify our therapy’s directed travel to the tumor micro-environment. This could be informed by chemotaxis, especially because the tumor micro-environment is notably characterized. Furthermore, we would ideally produce a therapy that modulates IL-6 levels rather than depleting it without a sense for if levels become dangerously low. We hope that next steps could include a sense and response genetic circuit.
4. **Designing a delivery system:**
   We wish to further consider the clinical delivery of the therapeutic, whether that involves engineering cells in vivo through lentiviral or CRISPR-based systems, or directly engineering cells after extracting them from patients, or something entirely different. Although the therapeutic is largely inspired by CAR T-cell therapy, we understand that our therapeutic would work in tandem with other cancer therapy. Therefore, we wish to answer the question of how administration of the therapy could coexist with other cancer treatment.
5. **Quantifying pharmacokinetic/pharmacodynamic parameters:** Due to a lack of time and resources, we were unable to thoroughly characterize many aspects of our therapeutic. We wish to undergo more stringent calculation of PK/PD parameters, such as association/dissociation constants of our CAR cells binding to IL-6. This well help us compare our therapeutic to related therapies, such as monoclonal antibodies, and enable computational modeling and simulation to advise further improvementss.
6. **Testing in animal models of cachexia:** Prior to even considering advancing testing to humans, we wish to utilize animal models of cachexia to get a feel for the efficacy of our therapy. Some of the experts we consulted, in particular Dr. Ishan Roy, have offered our team the resources to complete this; if we are able to progress that far, we would love to take this opportunity to begin in vivo studies.
7. **Continuing to consider social and ethical implications:**
   As mentioned in our human practices tab regarding the various conversations we held with experts, we wish to be better informed about how our therapy could affect people of different demographics. We understand that our therapy is currently limited to being informed by data that has a heavy male-bias. How can we make our therapy more inclusive, or identify the populations that would benefit the most from our therapy? Additionally, how can we make the education and treatment of cancer cachexia culturally-sensitive and knowledgeable? We wish to continue raising awareness about cancer cachexia so more patients can receive appropriate care.

# References

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2. Assembly of Genetic Circuits with the Mammalian ToolKit. Fonseca JP, Bonny AR, Town J, El-Samad H. Bio Protoc. 2020 Mar 5;10(5):e3547. doi: 10.21769/BioProtoc.3547. PubMed: PMID: 33659521.
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