iGEM 2022 — Team munich

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#### Notebook

On this page we wanted to give the viewer the opportunity to view all the work that has been done by the iGEM team munich in chronological order. For this we accumulated all events and put them into this interactive timeline. Because looking at all the event in parallel can be overwhelming, the viewer can choose which projects to view by toggling them in the menu below.on the right.

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#### Overview

![Overview of our engineering cycle.](https://static.igem.wiki/teams/4497/wiki/subpages/engineering/engineering1.svg)

**Figure 1: Overview of our engineering cycle.**

#### Reporter System

Our loop system design is based on three general components (Figure 2A):

* Secreted ligand
* Receptor recognizing the ligand, releasing a transcription factor
* Inducible gene activated by the transcription factor and expressing the ligand

![concept drawing of quorum sensing loop](https://static.igem.wiki/teams/4497/wiki/subpages/engineering/engineering2.svg)

**Figure 2: First Concepts of our Quorum Sensing Loop.** (A) In
the full loop system
the ligand 2xmCherry is secreted from the cells and binds to its corresponding receptor. The
receptor
binding releases a transcription factor further increasing ligand production. BFP can be linked
to the
transcription factor to check its expression (B) Our first tests employ EYFP as an inducible
reporter
for the transcription factor. The ligand 2xmCherry is added manually to the cells.

Before testing the full loop experimentally, we wanted to start by adding the ligand manually to
test
whether it could bind to the receptor and activate it. To test the activation of the transcription
factor, we used an inducible reporter different from the loop ligand (Figure 2B).

To investigate the individual processes, we developed a fluorescence reporter setup that enables
measuring several parameters parallely.
We initially decided on the following fluorescence signals to investigate our MESA system:

* Reporter: EYFP (Ex513/Em527)
* Loop Ligand: 2xmCherry (Ex587/Em610)
* Transcription Factor: BFP (Ex381/Em445)

These three fluorescent proteins are well distinguishable and allowed us to cover the whole
system:

Using EYFP we were able to read out our system activation caused by the release of the transcription
factor. 2xmCherry allowed us to track expression of the surface receptors as well as ligand binding
to
the receptor. Finally, BFP was used to correct for variations in expression levels of the receptors
that
might influence reporter activation.

As our system works through the transcription factor tTA, we ordered a tTA induceable plasmid
expressing
EYFP from Addgene (Plasmid #58855)
[[1]](https://2022.igem.wiki/munich/engineering#Refs).

![depiction of the three possible receptor ligand dimers](https://static.igem.wiki/teams/4497/wiki/subpages/engineering/engineering3.svg)

**Figure 3: Possible Receptor-Ligand Dimers of a Homodimeric
Ligand.** Besides the
active dimer created from a receptor dimer with both the TEV protease and the transcription factor,
combinations with two proteases or transcription factors are also possible. These combinations
are
not active, potentially affecting the efficiency of the receptor system.

Initially, the EYFP reporter worked well for our loop setup. We were able to investigate the
reporter
induction by the homodimeric ligand 2xmCherry binding to our MESA system (Figure 2). This setup,
however,
only allowed us to use a homodimeric ligand (2xmCherry). The receptor activation is dependent on
the
dimerization of the transcription factor (TF) and Protease (TEV) bearing complementing receptor halves. Using a
homodimer as ligand leads to four different receptor dimer configurations (Figure 3), of which some
are not
functional
[[2]](https://2022.igem.wiki/munich/engineering#Refs). To alleviate
this problem we also used a heterodimeric ligand (GFP-mCherry).
This
enabled us to split the receptor halves to recognize a unique antigen, in our case either GFP or mCherry.

This, however, led to the problem of fluorescence signal separation. As mEGFP (Ex488/Em507) and
EYFP
(Ex513/Em527) have a big overlap in excitation and emission, we could no longer separate ligand
binding
and reporter activation of our system (Figure 4, Figure 5B).

![two graphs of flurescence spectra of mEGFP & EYFP](https://static.igem.wiki/teams/4497/wiki/subpages/engineering/engineering4.png)

Figure 4: Fluorescence Spectra of mEGFP (A) and EYFP (B)
[[3]](https://2022.igem.wiki/munich/engineering#Refs).
Separation
of
mEGFP and EYFP fluorescence signals is not possible due to their overlap in both emission and
excitation
wavelengths.

To address this limitation, we decided to change the reporter protein from EYFP to miRFP680
(Ex661/Em680)
[[4]](https://2022.igem.wiki/munich/engineering#Refs), which allowed
for differentiation from the other three used fluorescent
proteins. We
assembled the new reporter using Gibson assembly and it worked as hoped (Figure 5). Using the
engineered
miRFP reporter system, the overlapping FITC signal is eliminated (Figure 5A-C). In its place,
reporter
activity is now determined through APC signal intensity (Figure 5D). We incorporated it into
all our
measurements for better comparison of our different MESA system setups
([See
Results](https://2022.igem.wiki/munich/results)).

![four flow cytometry histograms](https://static.igem.wiki/teams/4497/wiki/subpages/engineering/engineering5.png)

**Figure 5: Flow Cytometry Histograms comparing the usage of EYFP
and
miRFP in
our
loop system.** (A): Gating for FITC positive cells expressing either the miRFP or EYFP
reporter. Both
cell samples were also transfected with the heterodimeric MESA system G1TA & M1TEV. 500 ng/mL
GFP-mCherry were added to the samples. (B) Gated FITC Histogram of the EYFP reporter sample. It
is not
possible to differentiate the GFP-mCherry receptor binding from the induced reporter activation.
(C)
Gated FITC Histogram of the miRFP reporter sample. GFP binding is visible by the signal shift of
the
right wing of the curve. (D) Gated APC-A Histogram of the miRFP reporter sample. The reporter
signal is
visible in the APC-A channel separated from the FITC signal.

#### References

**[1]** Daringer, N. M., Dudek, R. M., Schwarz, K. A., & Leonard, J. N. (2014). Modular
Extracellular
Sensor Architecture for Engineering Mammalian Cell-based Devices.
<https://doi.org/10.1021/sb400128g>

**[2]** Schwarz, K. A., Daringer, N. M., Dolberg, T. B., & Leonard, J. N. (2017). Rewiring human
cellular
input-output using modular extracellular sensors. Nature Chemical Biology, 13(2),
202–209.
<https://doi.org/10.1038/nchembio.2253>

**[3]** Lambert, TJ (2019) FPbase: a community-editable fluorescent protein database. Nature
Methods. 16,
277–278.
<https://doi.org/10.1038/s41592-019-0352-8>

**[4]** Matlashov, M. E., Shcherbakova, D. M., Alvelid, J., Baloban, M., Pennacchietti, F.,
Shemetov, A.
A., Testa, I., & Verkhusha, V. v. (2020). A set of monomeric near-infrared fluorescent
proteins for
multicolor imaging across scales. Nature Communications, 11(1), 1–12.
<https://doi.org/10.1038/s41467-019-13897-6>

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To ensure that all our experiments and results are reproducible, we provide a collection of the standard laboratory protocols used, sequences of the PCR nd sequencing primers, as well as our laboratory journal entries in chronological order (grouped by subproject).

#### Standard protocols

brought to you by Till & Rosa

* Cell culture splitting
* Flow cytometry
* Gibson assembly
* Immunofluorescence labeling
* Transformation of *E. coli*
* Two step PCR

**Preps:**

* Maxiprep
* Miniprep

**Transfections:**

* Electroporation of COS Cells
* Transfection of HEK293T cells with Metafectene
* Transfection of ExpiCHO
* Transfection of Expi293

#### Primers

brought to you by Till & Rosa

| Name | Use | Used in PCR of | Label | Sequence | Initial Binding Region |
| --- | --- | --- | --- | --- | --- |
| V2-MESA-35F-M-tTA - 1,636 F | PCR | L2 tTA/TEV F | V2-tTA/TEV F | TAAGCAGAATTCGGAGGAGACTACAAGGACGATG | GGAGGAGACTACAAGGACGATG |
| V2-MESA-35F-M-tTA - 3,555 R | PCR | L1/L2 tTA R | V2-tTA R | TAAGCATCTAGATTACTTGTACAGCTCGTCCATG | TTACTTGTACAGCTCGTCCATG |
| V2-MESA-35F-Tev - 2,490 R | PCR | L1/L2 TEV R | V2-TEV R | TAAGCATCTAGAATTCATGAGTTGAGTCGCTTCC | ATTCATGAGTTGAGTCGCTTCC |
| pcDNA3.1-GBP-L2-MtTA-BFP - 1133 F | Sequencing |  | S01 | AGCATGCGTTGGTATCGTCA |  |
| pcDNA3.1-GBP-L2-MtTA-BFP - 1763 F | Sequencing |  | S02 | GAAGGGGAAAGCTGGCAAGA |  |
| pcDNA3.1-GBP-L2-MtTA-BFP - 2351 F | Sequencing |  | S03 | GACGAGCTCCACTTAGACGG |  |
| pcDNA3.1-GBP-L2-MtTA-BFP - 2945 F | Sequencing |  | S04 | CTAGTGAACCGCATCGAGCT |  |
| pcDNA3.1-GBP-L2-MtTA-BFP - 881 F | Sequencing |  | S05 | GGAGACCCAAGCTGGCTAG |  |
| pcDNA3.1-GBP-L2-TEV - 1232 F | Sequencing |  | S06 | GGCCGTTTTACCATTAGCCG |  |
| pcDNA3.1-GBP-L2-TEV - 1794 F | Sequencing |  | S07 | TGCCTAAGGATTTCCCACCA |  |
| pcDNA3.1-MBP-L2-MtTA-BFP - 1160 F | Sequencing |  | S08 | TGGTTCAGACAGGCCCCT |  |
| pcDNA3.1-SP-Flag HA-Tag-mCherrynb-gp130 - 1224 F | Sequencing |  | S09 | TGACCGATTACGCCGACAG |  |
| pcDNA3.1-SP-Flag HA-Tag-mCherrynb-gp130 - 1823 F | Sequencing |  | S10 | TCTTGTATGAGCAGCAGCCG |  |
| pcDNA3.1-spIL11R-cherry-TEV-Fc korrekt - 1070 F | Sequencing |  | S11 | TTCAAGGTGCACATGGAGGG |  |
| pcDNA3.1-spIL11R-GFP-Cherry-TEV-Fc - 1040 F | Sequencing |  | S12 | ACCATGGTGAGCAAGGGC |  |
| pcDNA3.1-spIL11R-GFP-Cherry-TEV-Fc - 1661 F | Sequencing |  | S13 | AAGCTGAGCAAAGACCCCAA |  |
| pcDNA3.1-spIL11R-GFP-Cherry-TEV-Fc - 2232 F | Sequencing |  | S14 | TGAAGGGCGAGATCAAGCAG |  |
| pcDNA3.1-spIL11R-GFP-Cherry-TEV-Fc - 2860 F | Sequencing |  | S15 | CTCCAAGGCTAAGGGACAGC |  |
| pcDNA3.1-spIL11R-GFP-Cherry-TEV-Fc - 749 F | Sequencing |  | S16 | ATGTCGTAACAACTCCGCCC |  |
| V2-MESA-35F-M-tTA - 1,717 F | PCR | L1 tTA F | L1-tTA F | TAAGCAGAATTCTCCGGCGGGAATCTGGTCGTGGTTGCTGGAG | CTGGTCGTGGTTGCTGGAG |
| V2-MESA-35F-Tev - 1,717 F | PCR | L1 TEV F | L1-TEV F | TAAGCAGAATTCTCCGGCGGGAATCTGGTCGTGGTTGCTGGAG | CTGGTCGTGGTTGCTGGAG |
| EYFP T2A - 4,369 F | PCR | EYFP T2A F | EYFP T2A F | TCTAGAACTAGGTCGACAAGCTTTTACTTGTACAGCTCGTCCATG | TTACTTGTACAGCTCGTCCATG |
| EYFP T2A - 5,094 R | PCR | EYFP T2A R | EYFP T2A R | GGTTCTGGAGAGGGCAGAGGCAGTCTGCTGACATGCGGTGACGTGGAAGAGAATCCCGGCCCTACTAGTATGGTGAGCAAGGGC | ACTAGTATGGTGAGCAAGGGC |
| IL11-GFP-mCherry - 923 F | PCR | GFP-mCherry F, mCherry A F | GFP-mCherry F | GCCCGGAATTCCTGCAGCGGCCGCGCCACCATGAGCAGCAGC | GCCACCATGAGCAGCAGC |
| IL11-GFP-mCherry - 2,461 R | PCR | GFP-mCherry R | GFP-mCherry R | TCTGCCCTCTCCAGAACCGAATTCCGCCAACTTGTACAGCTCG | CGCCAACTTGTACAGCTCG |
| mCherryA- 1,738 R | PCR | mCherry A R | mCherryA R | CATGGTGGCGACCGGACCAAGTTTGCCGCCTATCTTGTAC | AAGTTTGCCGCCTATCTTGTAC |
| mCherryB - 1,043 F | PCR | mCherry B F | mCherryB F | GGTCCGGTCGCCACCATGATGGCCATCATCAAGGAGTTCATG | ATGGCCATCATCAAGGAGTTCATG |
| mCherryB - 1,738 R | PCR | mCherry B R | mCherryB R | TCTGCCCTCTCCAGAACCGAATTCAAGTTTGCCGCCTATCTTGTACAG | AAGTTTGCCGCCTATCTTGTACAG |
| GFP-mCherry\_EYFP Reporter - 6,653 R | Sequencing |  | S25 | TCAGGGCTGAGCAGGGTC |  |
| GFP-mCherry\_EYFP Reporter - 6,373 R | Sequencing |  | S26 | TCGTGACCACCCTGACCTAC |  |
| GFP-mCherry\_EYFP Reporter - 6,355 F | Sequencing |  | S27 | TAGGTCAGGGTGGTCACGAG |  |
| GFP-mCherry\_EYFP Reporter - 5,686 F | Sequencing |  | S28 | TTGGTCACCTTCAGCTTGGC |  |
| GFP-mCherry\_EYFP Reporter - 5,400 R | Sequencing |  | S29 | CTCCTCCGAGCGGATGTAC |  |
| GFP-mCherry\_EYFP Reporter - 5,208 F | Sequencing |  | S30 | GGTGTAGTCCTCGTTGTGGG |  |
| GFP-mCherry\_EYFP Reporter - 4,884 R | Sequencing |  | S31 | CACCCTCGTGACCACCTTC |  |
| 2xmCherry\_EYFP Reporter - 6,560 F (S32) | Sequencing |  | S32 | AGGATCCCTTGTCATCGTCG |  |
| 2xmCherry\_EYFP Reporter - 5,864 R (S33) | Sequencing |  | S33 | ATAGGCGGCAAACTTGGTCC |  |
| 2xmCherry\_EYFP Reporter - 5,846 F (S34) | Sequencing |  | S34 | GACCAAGTTTGCCGCCTATC |  |
| 2xmCherry\_EYFP Reporter - 5,096 F (S35) | Sequencing |  | S35 | ATGTCAGCAGACTGCCTCTG |  |
| 2xmCherry\_EYFP Reporter - 4,736 F (S36) | Sequencing |  | S36 | TCTTGTAGTTGCCGTCGTCC |  |
| pBI-MCS-EYFP - 676 R (S37) | Sequencing |  | S37 | TCAAGGGTCCCCAAACTCAC |  |
| pBI-MCS-EYFP - 5,062 F (S38) | Sequencing |  | S38 | GAACAGCTCCTCGCCCTTG |  |
| pBI-MCS-EYFP - 125 R (S39) | Sequencing |  | S39 | TCGACCTATATAAGCAGAGCTCG |  |
| pBI-MCS-EYFP - 4,805 R (S40) | Sequencing |  | S40 | AGGAGCGCACCATCTTCTTC |  |
| pBI-MCS-EYFP - 4,637 F (S41) | Sequencing |  | S41 | ACGTTGTGGCTGTTGTAGTTG |  |
| Gibson miRFP Reverse | PCR | miRFP R | Gibson miRFP R | GCCCGGAATTCCTGCAGCAAGCTTGCCACCATGGCGGAAGGCTCCGTCGCC | ATGGCGGAAGGCTCCGTCGCC |
| Gibson miRFP Forward | PCR | miRFP F | Gibson miRFP F | TCTAGAACTAGGTCGACAGCGGCCGCTTACTCTTCCATCACGCCG | TTACTCTTCCATCACGCCG |

#### Quorum sensing

##### Lab protocol – 05.07

05.07.2022 - brought to you by Rosa

**Preparation for Maxiprep:** Pick clones and inoculate culture

##### Lab protocol – 06.07

06.07.2022 - brought to you by Rosa

**Preparation for Maxiprep:** Centrifuge cells

 → only with 0\_TEV, M\_tTA (have grown o/n, G\_tTA has not)

 →  *Preparation for Maxiprep:* pick clones and inoculate culture (G\_tTA)

##### Lab protocol – 07.07

07.07.2022 - brought to you by Rosa

**Preparation for Maxiprep:** Centrifuge cells (G\_tTA)

**Maxiprep:** M\_tTa and 0\_TEV cells frozen o/n, G\_tTA cells centrifuged right before

 → yield of G\_tTA really low (500 µL with 0.48 µg/µl), fine for now but backup cells would be good

 → Preparation for Maxiprep: pick clones and inoculate culture (G\_tTA)

**Preparation for Maxiprep:** Centrifuge cells (G\_tTA)

##### Lab protocol – 08.07

08.07.2022 - brought to you by Rosa

**Preparation for Maxiprep:** centrifuge cells (G\_tTA)

 → x2

 → stored in -20 °C freezer ready for Maxiprep when needed

##### Lab protocol – 13.07

13.07.2022 - brought to you by Lara

**Splitting** COS cells

##### Lab protocol – 03.08

03.08.2022 - brought to you by Till

**Preparation for Maxiprep:** pick clones and innoculate culture (eYFP, Tev, tTA\_BFP)

 → picked from stab culture into 5 mL LB + 1x Ampicillin, incubated at 37 °C for ~4 h

 → transferred to 0.5 L LB + Amp, incubated overnight

##### Lab protocol – 04.08

04.08.2022 - brought to you by Rosa

**Maxiprep (eYFP, TEV, tTA\_BFP):**

 → eYFP: 4 µg/µL

 → TEV: 2.56 µg/µL

 → tTA\_BFP: 2.58 µg/µL

##### Lab protocol – 08.08

08.08.2022 - brought to you by Rosa

**Splitting** COS for Electroporation

##### Lab protocol – 09.08

09.08.2022 - brought to you by Till

**Electroporation with dTomato MESA + Reporter:**

 → concentrations as described

 → 3 µL of 0.5 µg/µL DNA added

| Sample | C1 | C2 |
| --- | --- | --- |
| Description | Test sample, 3 Plasmids: dTomato-MtTA, dTomato-TEV, pBI-eYFP | Negative Control, Plasmids: dTomato-MtTA, pBI-eYFP |
|  | 1.5 µg each: 4.5 µg total DNA | 1.5 µg each: 3 µg total DNA |

**Next time:** empty plasmid to even out DNA amount/ plasmid count?

##### Lab protocol – 22.08

22.08.2022 - brought to you by Lara, Till & undefined

**Splitting CHO-cells:** for Transfection on 24.08.2022

 → 80 mL in 250 mL flask

 → 5.33 x 106 cells/mL 99% (5.04 x 106 cells/mL 99% & 5.62 x 106 cells/mL 99%)

 → 8.88 mL cells and 71.12 mL media

**Trafo**

##### Lab protocol – 23.08

23.08.2022 - brought to you by Till

**Inoculate cultures:** with GFP-GFP, mCherry-mCherry, GFP-mCherry, GBP, MBP

##### Lab protocol – 24.08

24.08.2022 - brought to you by Rosa, Lara & Till

**D-1 Splitting cells:** to density of 4 x 106 cells/mL for 75 mL in 250 mL flask

**Maxiprep:** with GFP-GFP, mCherry-mCherry, GFP-mCherry, GBP, MBP

**Cloning:**

 → Digest GBP & MBP Scheller plasmids with EcoRI, XbaI

 → PCR on V2-MESA tTA and V2-MeSA TEV for linker 1 and linker 2 variants

 → 4 samples total: 0.1 µg plasmid, 58 °C annealing, 4min elongation

 → gel purification of digest & PCR

 → Digest purified PCR EcoRI, XbaI

 → PCR purification

 → Ligation 15 min at RT 50 ng vector, 49 ng tTA or 22.8 ng TEV

 → 4 samples

 → WRONG! do ligation overnight next time (wrong protocol)

**Trafo:** of 5 µL ligation

##### Lab protocol – 25.08

25.08.2022 - brought to you by Lara & Till

**Transfection:** of ExpiCHO with mCherry Fc, GFP mCherry, and GFP Fc

 → 1.165 x 107 cells/mL 99% (1.33 x 107 cells/mL 99% & 1.00 x 107 cells/mL 99%)

 → 12.88 mL cells and 12.12 mL media (for 25 mL Transfection with cell density of 6 x 106 cells/mL in 125 mL flask)

| DNA construct | Density (µg/µl) | Required amount for 25 µg (µl) |
| --- | --- | --- |
| mCherry Fc | 1.7 | 14.71 |
| GFP mCherry | 0.85 | 29.41 |
| GFP Fc | 2.4 | 10.42 |

Plates don't look good

 → dephosphorylate remaining vector

 → ligate all 8 samples overnight at 4 °C + neg control (only vector)

##### Lab protocol – 30.08

30.08.2022 - brought to you by Till

**Ligation:** G1TA & M1TA again

**Miniprep:** G2TA, M2TA, M2TEV → sequencing

##### Lab protocol – 31.08

31.08.2022 - brought to you by Till

**Inoculate** clones 2(G2TEV), 9(M2TEV), 10(M2TA), 17(M1TEV) for maxi tomorrow

Repick G2TA, G1TEV 5x

##### Lab protocol – 01.09

01.09.2022 - brought to you by Till

Prepare protein A buffers, set pH at 4 °C!

##### Lab protocol – 02.09

02.09.2022 - brought to you by Till

**Protein a purification of ligand-Fc samples:**

 → Filter samples before FPLC (25 mL per sample)

**Program:**

* wash 3CV buffer A
* load
* 5-10CV A (collect)
* 6CV B (collect + neutralize 1/10 of Volume with Buffer N) → add beforehand to tubes
* regenerate 5cV B
* wash 5CV A

 → GFP-Fc: A13-A18

 → GFP-mCherry: A13-18

 → mCherry: A41-A45

 → + 1x load & 1x flowthrough for SDS gel

 → run gels (see pictures)

**Combine:**

* GFP-Fc: A13-A18
* GFP-mCherry: A14-18
* mCherry: A41-A45

**Concentrate:**

 → final solutions:

* GFP-Fc: 194 µg/mL
* GFP-mCherry: 117 µg/mL
* mCherry: 100 µg/mL

##### Lab protocol – 05.09

05.09.2022 - brought to you by Till

**Miniprep:** G1TA, M1TA → sequencing

##### Lab protocol – 06.09

06.09.2022 - brought to you by Till

**Electroporate** 4 µg total:

| m2TEV | M2TA | Reporter |
| --- | --- | --- |
| 0.25 µg | 1.5 µg | 0.25 µg |
| 0.75 µg | 0.75 µg | 0.75 µg |
| 2 µg |  |  |
|  | 2 µg/td>  | |

See standard protocol

##### Lab protocol – 07.09

07.09.2022 - brought to you by Igor

**Immunostaining:** 16B12 & 05-724 + mouse & rabbit secondary Ab

##### Lab protocol – 10.09

10.09.2022 - brought to you by Till

**Electroporation for FACS:**

 → 0.5 µg/µL DNA, maxiprepped

 → 3-5 x 106 cells/mL, 200 µL

 → 3 x 104 cells/mL in 2 mL in 6 well plate

**FACS**

| Sample Name | Number | M2TA | M2TEV | EYFP | Transfection Control (GFP) | Empty Vector (pcDNA3.4) | mCherry | Usage |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Transfection Control | 1-1 |  |  |  | 0.5 µg | 3.5 µg |  | Check Transfection Efficiency |
| Reporter Control | 1-2 |  |  | 0.5 µg |  | 3.5 µg |  | Check Reporter Background? |
| M2TEV Control | 1-3 |  | 0.5 µg | 0.5 µg |  | 3 µg |  | ? |
| M2TA Control | 1-4 | 3 µg |  | 0.5 µg |  | 0.5 µg |  | Check BFP Expression |
| MESA Control | 1-5 | 3 µg | 0.5 µg | 0.5 µg |  |  |  | Test system background without mCherry ligand |
| MESA + Ligand500 | 1-6 | 3 µg | 0.5 µg | 0.5 µg |  |  | 500 ng/mL | Test system functionality with mCherry ligand |
| MESA + Ligand100 | 1-7 | 3 µg | 0.5 µg | 0.5 µg |  |  | 100 ng/mL | Test system functionality with mCherry ligand |

**Microscopy:** electroporate 100 µL x 106 cells/mL

| Sample Name | Position | M2TA | M2TEV | G2TEV | EYFP | Transfection Control (GFP) | mCherry | Usage |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Transfection Control | 1C, 1D |  |  |  |  | 2 µg |  |  |
| Reporter Control |  |  |  |  | 2 µg |  |  |  |
| G2TEV Control | 1E, 1F |  |  | 2 µg |  |  |  |  |
| M2TEV Control | 2A, 2B |  | 2 µg |  |  |  |  |  |
| M2TA Control | 2C, 2D | 2 µg |  |  |  |  |  |  |
| MESA 1 | 2E, 2F | 1.5 µg | 0.25 µg |  | 0.25 µg |  |  |  |

##### Lab protocol – 11.09

11.09.2022 - brought to you by Till

Add Ligand (mCherry-Fc) to FACS samples (see list from yesterday)

mCherry-Fc Stock: 100 µg/mL

 → MESA + Ligand500: 10 µL in 2 mL media

 → MESA + Ligand100: 2 µL in 2 mL media

Change buffer for all wells

Pick more M1TA, G2TA

##### Lab protocol – 12.09

12.09.2022 - brought to you by Till & Rosa

**FACS measurments:** transfection efficiency horrible (maybe solution too old)

**Mini-prep** + sequencing M1TA, G2TA

##### Lab protocol – 13.09

13.09.2022 - brought to you by Till

**Electroporation test** in 4 mm cuvettes (250 µL electroporation solution, 5 µg dna, 4.5 x 105 cells/mL)

##### Lab protocol – 15.09

15.09.2022 - brought to you by Till

**IF Staining** of cells

##### Lab protocol – 16.09

16.09.2022 - brought to you by undefined

**Fluorescence microscopy:** pretty much no difference between samples, better than before, but still pretty bad

##### Lab protocol – 19.09

19.09.2022 - brought to you by Till

**Cloning:** 2 x mCherry-t2A eYFP and GFP-mCherry-eYFP

 → digest eYFP reporter with NotI, HindIII

 → PCR: eYFP T2A, mCherry 1, mCherry 2, GFP-mCherry

 → 4 min elongation,  °C annealing

 → gel purify

##### Lab protocol – 20.09

20.09.2022 - brought to you by Till

**Electroporation for FACS:**

 → 0.5 µg/µL DNA, maxiprepped

 → 2 x 106 cells/mL, 250 µL (4 mm cuvettes)

 → 2.5 x 105 cells/mL in 2 mL in 6 well plate

 → 5 µg total DNA on cells

**FACS**

| Sample Name | Number | M2TA | M2TEV | EYFP | Transfection Control (GFP) | Empty Vector (pcDNA3.4) | mCherry | Usage |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Transfection Control | 1-1 |  |  |  | 0.625 µg 1.25 µL | 4.375 µg  8.75 µL |  | Check Transfection Efficiency |
| Reporter Control | 1-2 |  |  | 0.625 µg  1.25 µL |  | 4.375 µg  8.75 µL |  | Check Reporter Background? |
| M2TEV Control | 1-3 |  | 0.625 µg  1.25 µL | 0.625 µg  1.25 µL |  | 3.75 µg  7.5 µL |  | ? |
| M2TA Control | 1-4 | 3.75 µg  7.5 µL |  | 0.625 µg  1.25 µL |  | 0.625 µg  1.25 µL |  | Check BFP Expression |
| MESA Control A | 1-5 | 3.75 µg  7.5 µL | 0.625 µg  1.25 µL | 0.625 µg  1.25 µL |  |  |  | Test system background without mCherry ligand |
| MESA A + Ligand500 | 1-6 | 3.75 µg  7.5 µL | 0.625 µg  1.25 µL | 0.625 µg  1.25 µL |  |  | 500 ng/mL | Test system functionality with mCherry ligand |

Pick some more M1TA

**Gibson assembly:** 2xmCherry-t2A eYFP and GFP-mcherry-eYFP

A. mCherry2x

 → 134.7 ng BB

 → 26.52 ng mCH 1

 → 22.81 ng mCH 2

 → 25.09 ng EYFP T2A

B. GFP-mCherry

 → 100 ng BB

 → 72.78 ng GFP-mCherry

 → 37.24 ng eYFP T2A

##### Lab protocol – 21.09

21.09.2022 - brought to you by Till

Add ligand mCherry 500 ng/mL

Pick GFP mCherry & 2xmCherry

##### Lab protocol – 22.09

22.09.2022 - brought to you by Till

Prep & sequence:

 → M1TA

 → GFP mCherry, 2xmCherry

##### Lab protocol – 26.09

26.09.2022 - brought to you by Till & Rosa

**Transfection:** HEK293T with Metafectene as per protocol

 → 4 µg DNA total, 0.5 µg/µL plasmids

| Sample Name | Number | M2TA | M2TEV | EYFP | Transfection Control (GFP) | Empty Vector (pcDNA3.4) | mCherry | Usage |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Transfection Control | 1-1 |  |  |  | 0.5 µg **1 µL** | 3.5 µg  **7 µL** |  | Check Transfection Efficiency |
| Empty Control | 1-2 |  |  |  |  | 4 µg  **8 µL** |  |  |
| Reporter Control | 1-3 |  |  | 0.5 µg  **1 µL** |  | 3.5 µg  **7 µL** |  | Check Reporter Background? |
| M2TA Control | 1-4 | 3 µg  **6 µL** |  | 0.5 µg  **1 µL** |  | 0.5 µg  **1 µL** |  | Check BFP Expression |
| MESA 6xTC | 1-5 | 3 µg  **6 µL** | 0.5 µg  **1 µL** | 0.5 µg  **1 µL** |  |  |  | Test system background without mCherry ligand |
| MESA 6xTC + Ligand500 | 1-6 | 3 µg  **6 µL** | 0.5 µg  **1 µL** | 0.5 µg  **1 µL** |  |  | 500 ng/mL | Test system functionality with mCherry ligand |
| MESA 12xTC | 2-1 | 3 µg  **6 µL** | 0.25 µg  **0.5 µL** | 0.5 µg  **1 µL** |  | 0.25 µg  **0.5 µL** |  |  |
| MESA 6xTC + Ligand500 | 2-2 | 3 µg  **6 µL** | 0.25 µg  **0.5 µL** | 0.5 µg  **1 µL** |  | 0.25 µg  **0.5 µL** | 500 ng/mL |  |
| MESA 16xTC | 2-3 | 2 µg  **4 µL** | 0.125 µg  **0.25 µL** | 0.5 µg  **1 µL** |  | 1.375 µg  **2.75 µL** |  |  |
| MESA 16xTC + Ligand500 | 2-4 | 2 µg  **4 µL** | 0.125 µg  **0.25 µL** | 0.5 µg  **1 µL** |  | 1.375 µg  **2.75 µL** | 500 ng/mL |  |
| MESA 24xTC | 2-5 | 3 µg  **6 µL** | 0.125 µg  **0.25 µL** | 0.5 µg  **1 µL** |  | 0.375 µg  **0.75 µL** |  |  |
| MESA 24xTC + Ligand500 | 2-6 | 3 µg  **6 µL** | 0.125 µg  **0.25 µL** | 0.5 µg  **1 µL** |  | 0.375 µg  **0.75 µL** | 500 ng/mL |  |

**Digest:** dTomato\_TEV, eYFP → NotI, HindIII

**Digest:** GBP-gp130 → XbaI, AflII

##### Lab protocol – 27.09

27.09.2022 - brought to you by Till & Rosa

Addition of 500 ng/mL ligand according to table above

##### Lab protocol – 28.09

28.09.2022 - brought to you by Rosa & Till

**Cytometry Measurements**

##### Lab protocol – 02.10

02.10.2022 - brought to you by Till & Rosa

**Transfection:** HEK293T with Metafectene as per protocol

 → 4 µg DNA total, 0.5 µg/µL plasmids

| Sample Name | Number | M1TA | M1TEV | EYFP | Transfection Control (GFP) | Empty Vector (pcDNA3.4) | mCherry | Usage |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Empty Control | 1-1 |  |  |  |  | 4 µg **8 µL** |  |  |
| Reporter Control | 1-2 |  |  | 0.5 µg  **1 µL** |  | 3.5 µg  **7 µL** | 500 ng/mL | Check Reporter Background? |
| M1TA Control | 1-3 | 3 µg  **6 µL** |  | 0.5 µg  **1 µL** |  | 0.5 µg  **1 µL** | 500 ng/mL | Check BFP Expression |
| M1TEV Control | 1-4 |  | 0.125 µg  **0.25 µL** | 0.5 µg  **1 µL** |  | 3.625 µg  **6.75 µL** | 500 ng/mL |  |
| MESA 6xTC | 1-5 | 3 µg  **6 µL** | 0.5 µg  **1 µL** | 0.5 µg  **1 µL** |  |  |  | Test system background without mCherry ligand |
| MESA 6xTC + Ligand500 | 1-6 | 3 µg  **6 µL** | 0.5 µg  **1 µL** | 0.5 µg  **1 µL** |  |  | 500 ng/mL | Test system functionality with mCherry ligand |
| MESA 12xTC | 2-1 | 3 µg  **6 µL** | 0.25 µg  **0.5 µL** | 0.5 µg  **1 µL** |  | 0.25 µg  **0.5 µL** |  |  |
| MESA 6xTC + Ligand500 | 2-2 | 3 µg  **6 µL** | 0.25 µg  **0.5 µL** | 0.5 µg  **1 µL** |  | 0.25 µg  **0.5 µL** | 500 ng/mL |  |
| MESA 16xTC | 2-3 | 2 µg  **4 µL** | 0.125 µg  **0.25 µL** | 0.5 µg  **1 µL** |  | 1.375 µg  **2.75 µL** |  |  |
| MESA 16xTC + Ligand500 | 2-4 | 2 µg  **4 µL** | 0.125 µg  **0.25 µL** | 0.5 µg  **1 µL** |  | 1.375 µg  **2.75 µL** | 500 ng/mL |  |
| MESA 24xTC | 2-5 | 3 µg  **6 µL** | 0.125 µg  **0.25 µL** | 0.5 µg  **1 µL** |  | 0.375 µg  **0.75 µL** |  |  |
| MESA 24xTC + Ligand500 | 2-6 | 3 µg  **6 µL** | 0.125 µg  **0.25 µL** | 0.5 µg  **1 µL** |  | 0.375 µg  **0.75 µL** | 500 ng/mL |  |

##### Lab protocol – 03.10

03.10.2022 - brought to you by Till

Add ligand according to table above

##### Lab protocol – 04.10

04.10.2022 - brought to you by Rosa & Till

**Cytometry Measurements**

Redo PCR for 2xmCherry T2A EYFP and GfP-mCherry t2A eYFP

 → two temp annealing 10 cycles low, 23 high

 → gel purify, redo gibson like 20.9.

##### Lab protocol – 07.10

07.10.2022 - brought to you by Rosa

**Maxiprep** with miRFP reporter plasmid

**Miniprep** with MESA component plasmids G1TA G2TA und G1TEV

##### Lab protocol – 08.10

08.10.2022 - brought to you by Till & Lara

**Transfection:** HEK293T with Metafectene as per protocol

 → 4 µg DNA total, 0.5 µg/µL plasmids

| Name | Function | Well | ID | MESA 1 | MESA 2 | Reporter | Empty Vector | Protein |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Empty | just cells | 1-1 | 1 |  |  |  |  |  |
| miRFP Control | mCherry | 1-2 | 2 |  |  | miRFP 0.5 µg (1 µL) | 3.5 µg (7 µL) | 2xmCherry 500 ng/µL |
| miRFP Control | GFP | 1-3 | 3 |  |  | miRFP 0.5 µg (1 µL) | 3.5 µg (7 µL) | 2xGFP 500 ng/µL |
| miRFP Control | GFP-mCherry | 1-4 | 4 |  |  | miRFP 0.5 µg (1 µL) | 3.5 µg (7 µL) | GFPmCherry 500 ng/µL |
| M1TA Control |  | 1-5 | 5 | M1TA 3 µg (6 µL) |  | miRFP 0.5 µg (1 µL) | 0.5 µg (1 µL) | 2xmCherry 500 ng/µL |
| M1TEV Control |  | 1-6 | 6 |  | M1TEV 0.125 µg (0.25 µL) | miRFP 0.5 µg (1 µL) | 3.375 µg (6.75 µL) | 2xmCherry 500 ng/µL |
| M2TA Control |  | 2-1 | 7 | M2TA 3 µg (6 µL) |  | miRFP 0.5 µg (1 µL) | 0.5 µg (1 µL) | 2xmCherry 500 ng/µL |
| M2TEV Control |  | 2-2 | 8 |  | M2TEV 0.125 µg (0.25 µL) | miRFP 0.5 µg (1 µL) | 3.375 µg (6.75 µL) | 2xmCherry 500 ng/µL |
| G1TA Control |  | 2-3 |  | G1TA 3 µg (6 µL) |  | miRFP 0.5 µg (1 µL) | 0.5 µg (1 µL) | 2xGFP 500 ng/µL |
| G1TEV Control |  | 2-4 | 10 |  | G1TEV 0.125 µg (0.25 µL) | miRFP 0.5 µg (1 µL) | 3.375 µg (6.75 µL) | 2xGFP 500 ng/µL |
| G2TA Control |  | 2-5 | 11 | G2TA 3 µg (6 µL) |  | miRFP 0.5 µg (1 µL) | 0.5 µg (1 µL) | 2xGFP 500 ng/µL |
| G2TEV Control |  | 2-6 | 12 |  | G2TEV 0.125 µg (0.25 µL) | miRFP 0.5 µg (1 µL) | 3.375 µg (6.75 µL) | 2xGFP 500 ng/µL |
| MESA M1TA, M1TEV | 6x TA | 3-1 | 13 | M1TA  3 µg (6 µL) | M1TEV 0.5 µg (1 µL) | miRFP 0.5 µg (1 µL) |  |  |
| MESA M1TA, M1TEV | 6x TA | 3-2 | 14 | M1TA  3 µg (6 µL) | M1TEV 0.5 µg (1 µL) | miRFP 0.5 µg (1 µL) |  | 2xmCherry 500 ng/µL |
| MESA M1TA, M1TEV | 12x TA | 3-3 | 15 | M1TA  3 µg (6 µL) | M1TEV 0.25 µg (0.5 µL) | miRFP 0.5 µg (1 µL) | 0.25 µg (0.5 µL) |  |
| MESA M1TA, M1TEV | 12x TA | 3-4 | 16 | M1TA  3 µg (6 µL) | M1TEV 0.25 µg (0.5 µL) | miRFP 0.5 µg (1 µL) | 0.25 µg (0.5 µL) | 2xmCherry 500 ng/µL |
| MESA M1TA, M1TEV | 24x TA | 3-5 | 17 | M1TA  3 µg (6 µL) | M1TEV 0.125 µg (0.25 µL) | miRFP 0.5 µg (1 µL) | 0.375 µg (0.75 µL) |  |
| MESA M1TA, M1TEV | 24x TA | 3-6 | 18 | M1TA  3 µg (6 µL) | M1TEV 0.125 µg (0.25 µL) | miRFP 0.5 µg (1 µL) | 0.375 µg (0.75 µL) | 2xmCherry 500 ng/µL |
| MESA M2TA, M2TEV | 6x TA | 4-1 | 19 | M2TA  3 µg (6 µL) | M2TEV 0.5 µg (1 µL) | miRFP 0.5 µg (1 µL) |  |  |
| MESA M2TA, M2TEV | 6x TA | 4-2 | 20 | M2TA  3 µg (6 µL) | M2TEV 0.5 µg (1 µL) | miRFP 0.5 µg (1 µL) |  | 2xmCherry 500 ng/µL |
| MESA M2TA, M2TEV | 12x TA | 4-3 | 21 | M2TA  3 µg (6 µL) | M2TEV 0.25 µg (0.5 µL) | miRFP 0.5 µg (1 µL) | 0.25 µg (0.5 µL) |  |
| MESA M2TA, M2TEV | 12x TA | 4-4 | 22 | M2TA  3 µg (6 µL) | M2TEV 0.25 µg (0.5 µL) | miRFP 0.5 µg (1 µL) | 0.25 µg (0.5 µL) | 2xmCherry 500 ng/µL |
| MESA M2TA, M2TEV | 24x TA | 4-5 | 23 | M2TA  3 µg (6 µL) | M2TEV 0.125 µg (0.25 µL) | miRFP 0.5 µg (1 µL) | 0.375 µg (0.75 µL) |  |
| MESA M2TA, M2TEV | 24x TA | 4-6 | 24 | M2TA  3 µg (6 µL) | M2TEV 0.125 µg (0.25 µL) | miRFP 0.5 µg (1 µL) | 0.375 µg (0.75 µL) | 2xmCherry 500 ng/µL |
| MESA G1TA, G1TEV | 6x TA | 5-1 | 25 | G1TA 3 µg (6 µL) | G1TEV 0.5 µg (1 µL) | miRFP 0.5 µg (1 µL) |  |  |
| MESA G1TA, G1TEV | 6x TA | 5-2 | 26 | G1TA 3 µg (6 µL) | G1TEV 0.5 µg (1 µL) | miRFP 0.5 µg (1 µL) |  | 2xGFP 500 ng/µL |
| MESA G1TA, G1TEV | 12x TA | 5-3 | 27 | G1TA 3 µg (6 µL) | G1TEV 0.25 µg (0.5 µL) | miRFP 0.5 µg (1 µL) | 0.25 µg (0.5 µL) |  |
| MESA G1TA, G1TEV | 12x TA | 5-4 | 28 | G1TA 3 µg (6 µL) | G1TEV 0.25 µg (0.5 µL) | miRFP 0.5 µg (1 µL) | 0.25 µg (0.5 µL) | 2xGFP 500 ng/µL |
| MESA G1TA, G1TEV | 24x TA | 5-5 | 29 | G1TA 3 µg (6 µL) | G1TEV 0.125 µg (0.25 µL) | miRFP 0.5 µg (1 µL) | 0.375 µg (0.75 µL) |  |
| MESA G1TA, G1TEV | 24x TA | 5-6 | 30 | G1TA 3 µg (6 µL) | G1TEV 0.125 µg (0.25 µL) | miRFP 0.5 µg (1 µL) | 0.375 µg (0.75 µL) | 2xGFP 500 ng/µL |
| MESA G2TA, G2TEV | 6x TA | 6-1 | 31 | G2TA 3 µg (6 µL) | G2TEV 0.5 µg (1 µL) | miRFP 0.5 µg (1 µL) |  |  |
| MESA G2TA, G2TEV | 6x TA | 6-2 | 32 | G2TA 3 µg (6 µL) | G2TEV 0.5 µg (1 µL) | miRFP 0.5 µg (1 µL) |  | 2xGFP 500 ng/µL |
| MESA G2TA, G2TEV | 12x TA | 6-3 | 33 | G2TA 3 µg (6 µL) | G2TEV 0.25 µg (0.5 µL) | miRFP 0.5 µg (1 µL) | 0.25 µg (0.5 µL) |  |
| MESA G2TA, G2TEV | 12x TA | 6-4 | 34 | G2TA 3 µg (6 µL) | G2TEV 0.25 µg (0.5 µL) | miRFP 0.5 µg (1 µL) | 0.25 µg (0.5 µL) | 2xGFP 500 ng/µL |
| MESA G2TA, G2TEV | 24x TA | 6-5 | 35 | G2TA 3 µg (6 µL) | G2TEV 0.125 µg (0.25 µL) | miRFP 0.5 µg (1 µL) | 0.375 µg (0.75 µL) |  |
| MESA G2TA, G2TEV | 24x TA | 6-6 | 36 | G2TA 3 µg (6 µL) | G2TEV 0.125 µg (0.25 µL) | miRFP 0.5 µg (1 µL) | 0.375 µg (0.75 µL) | 2xGFP 500 ng/µL |
| M1TA, G1TEV | 6x TA | 7-1 | 37 | M1TA  3 µg (6 µL) | G1TEV 0.5 µg (1 µL) | miRFP 0.5 µg (1 µL) |  |  |
| M1TA, G1TEV | 6x TA | 7-2 | 38 | M1TA  3 µg (6 µL) | G1TEV 0.5 µg (1 µL) | miRFP 0.5 µg (1 µL) |  | GFPmCherry 500 ng/µL |
| M1TA, G1TEV | 12x TA | 7-3 | 39 | M1TA  3 µg (6 µL) | G1TEV 0.25 µg (0.5 µL) | miRFP 0.5 µg (1 µL) | 0.25 µg (0.5 µL) |  |
| M1TA, G1TEV | 12x TA | 7-4 | 40 | M1TA  3 µg (6 µL) | G1TEV 0.25 µg (0.5 µL) | miRFP 0.5 µg (1 µL) | 0.25 µg (0.5 µL) | GFPmCherry 500 ng/µL |
| M1TA, G1TEV | 24x TA | 7-5 | 41 | M1TA  3 µg (6 µL) | G1TEV 0.125 µg (0.25 µL) | miRFP 0.5 µg (1 µL) | 0.375 µg (0.75 µL) |  |
| M1TA, G1TEV | 24x TA | 7-6 | 42 | M1TA  3 µg (6 µL) | G1TEV 0.125 µg (0.25 µL) | miRFP 0.5 µg (1 µL) | 0.375 µg (0.75 µL) | GFPmCherry 500 ng/µL |
| G1TA, M1TEV | 6x TA | 8-1 | 43 | G1TA 3 µg (6 µL) | M1TEV 0.5 µg (1 µL) | miRFP 0.5 µg (1 µL) |  |  |
| G1TA, M1TEV | 6x TA | 8-2 | 44 | G1TA 3 µg (6 µL) | M1TEV 0.5 µg (1 µL) | miRFP 0.5 µg (1 µL) |  | GFPmCherry 500 ng/µL |
| G1TA, M1TEV | 12x TA | 8-3 | 45 | G1TA 3 µg (6 µL) | M1TEV 0.25 µg (0.5 µL) | miRFP 0.5 µg (1 µL) | 0.25 µg (0.5 µL) |  |
| G1TA, M1TEV | 12x TA | 8-4 | 46 | G1TA 3 µg (6 µL) | M1TEV 0.25 µg (0.5 µL) | miRFP 0.5 µg (1 µL) | 0.25 µg (0.5 µL) | GFPmCherry 500 ng/µL |
| G1TA, M1TEV | 24x TA | 8-5 | 47 | G1TA 3 µg (6 µL) | M1TEV 0.125 µg (0.25 µL) | miRFP 0.5 µg (1 µL) | 0.375 µg (0.75 µL) |  |
| G1TA, M1TEV | 24x TA | 8-6 | 48 | G1TA 3 µg (6 µL) | M1TEV 0.125 µg (0.25 µL) | miRFP 0.5 µg (1 µL) | 0.375 µg (0.75 µL) | GFPmCherry 500 ng/µL |
| M2TA, G2TEV | 6x TA | 9-1 | 49 | M2TA  3 µg (6 µL) | G2TEV 0.5 µg (1 µL) | miRFP 0.5 µg (1 µL) |  |  |
| M2TA, G2TEV | 6x TA | 9-2 | 50 | M2TA  3 µg (6 µL) | G2TEV 0.5 µg (1 µL) | miRFP 0.5 µg (1 µL) |  | GFPmCherry 500 ng/µL |
| M2TA, G2TEV | 12x TA | 9-3 | 51 | M2TA  3 µg (6 µL) | G2TEV 0.25 µg (0.5 µL) | miRFP 0.5 µg (1 µL) | 0.25 µg (0.5 µL) |  |
| M2TA, G2TEV | 12x TA | 9-4 | 52 | M2TA  3 µg (6 µL) | G2TEV 0.25 µg (0.5 µL) | miRFP 0.5 µg (1 µL) | 0.25 µg (0.5 µL) | GFPmCherry 500 ng/µL |
| M2TA, G2TEV | 24x TA | 9-5 | 53 | M2TA  3 µg (6 µL) | G2TEV 0.125 µg (0.25 µL) | miRFP 0.5 µg (1 µL) | 0.375 µg (0.75 µL) |  |
| M2TA, G2TEV | 24x TA | 9-6 | 54 | M2TA  3 µg (6 µL) | G2TEV 0.125 µg (0.25 µL) | miRFP 0.5 µg (1 µL) | 0.375 µg (0.75 µL) | GFPmCherry 500 ng/µL |
| G2TA, M2TEV | 6x TA | 10-1 | 55 | G2TA 3 µg (6 µL) | M2TEV 0.5 µg (1 µL) | miRFP 0.5 µg (1 µL) |  |  |
| G2TA, M2TEV | 6x TA | 10-2 | 56 | G2TA 3 µg (6 µL) | M2TEV 0.5 µg (1 µL) | miRFP 0.5 µg (1 µL) |  | GFPmCherry 500 ng/µL |
| G2TA, M2TEV | 12x TA | 10-3 | 57 | G2TA 3 µg (6 µL) | M2TEV 0.25 µg (0.5 µL) | miRFP 0.5 µg (1 µL) | 0.25 µg (0.5 µL) |  |
| G2TA, M2TEV | 12x TA | 10-4 | 58 | G2TA 3 µg (6 µL) | M2TEV 0.25 µg (0.5 µL) | miRFP 0.5 µg (1 µL) | 0.25 µg (0.5 µL) | GFPmCherry 500 ng/µL |
| G2TA, M2TEV | 24x TA | 10-5 | 59 | G2TA 3 µg (6 µL) | M2TEV 0.125 µg (0.25 µL) | miRFP 0.5 µg (1 µL) | 0.375 µg (0.75 µL) |  |
| G2TA, M2TEV | 24x TA | 10-6 | 60 | G2TA 3 µg (6 µL) | M2TEV 0.125 µg (0.25 µL) | miRFP 0.5 µg (1 µL) | 0.375 µg (0.75 µL) | GFPmCherry 500 ng/µL |
| EYFP Control |  | 11-1 | 61 |  |  | EYFP 0.5 µg (1 µL) | 3.5 µg (7 µL) | 2xmCherry 50 ng/µL |
| M1TA Control |  | 11-2 | 62 | M1TA 3 µg (6 µL) |  | EYFP 0.5 µg (1 µL) | 0.5 µg (1 µL) | 2xmCherry 50 ng/µL |
| M1TEV Control |  | 11-3 | 63 |  | M1TEV 0.125 µg (0.25 µL) | EYFP 0.5 µg (1 µL) | 3.375 µg (6.75 µL) | 2xmCherry 50 ng/µL |
| M2TA Control |  | 11-4 | 64 | M2TA  3 µg (6 µL) |  | EYFP 0.5 µg (1 µL) | 0.5 µg (1 µL) | 2xmCherry 50 ng/µL |
| M2TEV Control |  | 11-5 | 65 |  | M2TEV 0.125 µg (0.25 µL) | EYFP 0.5 µg (1 µL) | 3.375 µg (6.75 µL) | 2xmCherry 50 ng/µL |
| MESA M1TA, M1TEV | 6x TA | 11-6 | 66 | M1TA  3 µg (6 µL) | M1TEV 0.5 µg (1 µL) | EYFP 0.5 µg (1 µL) |  | 2xmCherry 50 ng/µL |
| Loop M1TA, M1TEV | 6x TA | 12-1 | 67 | M1TA  3 µg (6 µL) | M1TEV 0.5 µg (1 µL) | 2xmC-EYFP 0.5 µg (1 µL) |  |  |
| Loop M1TA, M1TEV | 6xTA | 12-2 | 68 | M1TA  3 µg (6 µL) | M1TEV 0.5 µg (1 µL) | 2xmC-EYFP 0.5 µg (1 µL) |  | 2xmCherry 50 ng/µL |
| MESA M1TA, M1TEV | 24x TA | 12-3 | 69 | M1TA  3 µg (6 µL) | M1TEV 0.125 µg (0.25 µL) | EYFP 0.5 µg (1 µL) | 0.375 µg (0.75 µL) | 2xmCherry 50 ng/µL |
| M1TA, M1TEV | 24x TA | 12-4 | 70 | M1TA  3 µg (6 µL) | M1TEV 0.125 µg (0.25 µL) | 2xmC-EYFP 0.5 µg (1 µL) | 0.375 µg (0.75 µL) |  |
| M1TA, M1TEV | 24x TA | 12-5 | 71 | M1TA  3 µg (6 µL) | M1TEV 0.125 µg (0.25 µL) | 2xmC-EYFP 0.5 µg (1 µL) | 0.375 µg (0.75 µL) | 2xmCherry 50 ng/µL |
| MESA M2TA, M2TEV | 6x TA | 12-6 | 72 | M2TA  3 µg (6 µL) | M2TEV 0.5 µg (1 µL) | EYFP 0.5 µg (1 µL) |  | 2xmCherry 50 ng/µL |
| M2TA, M2TEV | 6x TA | 13-1 | 73 | M2TA  3 µg (6 µL) | M2TEV 0.5 µg (1 µL) | 2xmC-EYFP 0.5 µg (1 µL) |  |  |
| M2TA, M2TEV | 6xTA | 13-2 | 74 | M2TA  3 µg (6 µL) | M2TEV 0.5 µg (1 µL) | 2xmC-EYFP 0.5 µg (1 µL) |  | 2xmCherry 50 ng/µL |
| MESA M2TA, M2TEV | 24x TA | 13-3 | 75 | M2TA  3 µg (6 µL) | M2TEV 0.125 µg (0.25 µL) | EYFP 0.5 µg (1 µL) | 0.375 µg (0.75 µL) | 2xmCherry 50 ng/µL |
| M2TA, M2TEV | 24x TA | 13-4 | 76 | M2TA  3 µg (6 µL) | M2TEV 0.125 µg (0.25 µL) | 2xmC-EYFP 0.5 µg (1 µL) | 0.375 µg (0.75 µL) |  |
| M2TA, M2TEV | 24x TA | 13-5 | 77 | M2TA  3 µg (6 µL) | M2TEV 0.125 µg (0.25 µL) | 2xmC-EYFP 0.5 µg (1 µL) | 0.375 µg (0.75 µL) | 2xmCherry 50 ng/µL |
| M1TA, G1TEV but with EYFP | 6x | 13-6 | 78 | M1TA  3 µg (6 µL) | G1TEV 0.5 µg (1 µL) | EYFP 0.5 µg (1 µL) |  | GFPmCherry 500 ng/µL |

**Maxiprep** with G1TA G2TA und G1TEV

##### Lab protocol – 09.10

09.10.2022 - brought to you by Till

Addition of ligand according to table above

##### Lab protocol – 10.10

10.10.2022 - brought to you by Rosa & Till

**Cytometry measurements**

#### pH sensitivity

##### Lab protocol – 20.06

20.06.2022 - brought to you by Till

**Splitting** of ExpiCHO & Expi293

##### Lab protocol – 21.06

21.06.2022 - brought to you by Lara

**Preparation for Maxiprep:** transformation of E.coli with pH plasmid

##### Lab protocol – 22.06

22.06.2022 - brought to you by Lara & Rosa

Splitting of ExpiCHO & Expi293

**Preparation for Maxiprep:** pick clones and inoculate culture

##### Lab protocol – 23.06

23.06.2022 - brought to you by Rosa

**Preparation for Maxiprep:** centrifuge cells, freeze for tomorrow

##### Lab protocol – 24.06

24.06.2022 - brought to you by Rosa & Lara

**Maxiprep** with pH plasmid

##### Lab protocol – 27.06

27.06.2022 - brought to you by Lara

**Transfection:** of ExpiCHO & Expi293 with pH plasmid (T0)

##### Lab protocol – 28.06

28.06.2022 - brought to you by Rosa

Feeding ExpiCHO & Expi293 (T1)

##### Lab protocol – 29.06

29.06.2022 - brought to you by Rosa

Take sample of ExpiCHO & Expi293 (T2)

 → **CHO:** 93% live, 1.34 x 107 cells/mL & 94% live, 1.35 x 107 cells/mL

 → **293:** 75% live, 5.33 x 106 cells/mL & 77% live, 6.11 x 106 cells/mL

##### Lab protocol – 30.06

30.06.2022 - brought to you by Rosa

Take sample of ExpiCHO & Expi293 (T3)

 → **CHO:** 90% live, 1.36 x 107 cells/mL & 89% live, 1.35 x 107 cells/mL

 → **293:** 75% live, 5.33 x 106 cells/mL & 77% live, 6.11 x 106 cells/mL

##### Lab protocol – 01.07

01.07.2022 - brought to you by Rosa

Take sample of ExpiCHO & Expi293 (T4)

 → **CHO:** 94% live 1.32 x 107 cells/mL & 94% live 1.27 x 107 cells/mL

 → **293:** 74% live 8.16 x 106 cells/mL & 67% live 7.88 x 106 cells/mL

##### Lab protocol – 04.07

04.07.2022 - brought to you by Lara

Take sample of ExpiCHO & Expi293 (T7)

 → **CHO:** 91% live 1.57 x 107 cells/mL & 91% live 1.17 x 107 cells/mL

 → **293:** 23% live 3.06 x 106 cells/mL & 17% live 3.07 x 106 cells/mL

##### Lab protocol – 05.07

05.07.2022 - brought to you by Rosa & Lara

Take sample of ExpiCHO & Expi293 (T8)

 → **CHO:** 92% live 1.33 x 107 cells/mL & 89% live 9.65 x 106 cells/mL

 → **293:** 7% live 1.0 x 106 cells/mL & 57% live 7.27 x 105 cells/mL

##### Lab protocol – 06.07

06.07.2022 - brought to you by Lara

Take sample of ExpiCHO & Expi293 (T9)

 → **CHO:** 90% live 1.23 x 107 cells/mL & 88% live 1.09 x 107 cells/mL

 → **293:** 5% live 8.21 x 105 cells/mL & 6% live 1.21 x 106 cells/mL

##### Lab protocol – 07.07

07.07.2022 - brought to you by Till & Lara

**Prepare SDS-Gels for Expression Analysis**

| 12% SDS Gel (20 mL) | Stacking Gel (10 mL) |
| --- | --- |
| 8.6 mL H2O | 7.4 mL H2O |
| 5 mL 1.5 M Tris/HCl pH 8.8 | 1.3 mL 1.0 M Tris pH 6.8 |
| 6 mL 40% Acrylamide | 1.1 mL 40% Acrylamide |
| 200 µL 10% SDS | 100 µL 10% SDS |
| 200 µL APS | 100 µL APS |
| 16 µL TEMED | 10 µL TEMED |

Take sample of ExpiCHO & Expi293 (10)

 → **CHO:** 88% live 1.30 x 107 cells/mL & 85% live 1.56 x 107 cells/mL

 → **293:** 2% live 4.57 x 105 cells/mL & 2% live 4.05 x 105 cells/mL

##### Lab protocol – 08.07

08.07.2022 - brought to you by Till

Take sample of ExpiCHO & Expi293 (11)

 → **CHO:** 84% live 7.68 x 106 cells/mL & 82% live 8.13 x 106 cells/mL

 → **293:** 3% live 4.11 x 105 cells/mL & 5% live 6.57 x 105 cells/mL

**Load SDS-Gels with Expression Samples**

 → 100 µL mixed with 25 µL 5x reducing Lämmli

 → 4 Gels: 293: SDS & Western Blot (WB), CHO: SDS & WB

 → Samples: T2, 3, 4, 7, 8, 9, 10, 11 on each gel

 → 3.5 µL of marker (PageRuler Plus Prestained) "M"

 → Unused Wells filled with 1x Lämmli (1xL)

**Gel 1: 293 SDS**

(accidentally inverted sample application)

| Lanes | **1** | **2** | **3** | **4** | **5** | **6** | **7** | **8** | **9** | **10** | **11** | **12** | **13** | **14** | **15** |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Samples | 1xL | 1xL | 1xL | 1xL | 1xL | M | T11 | T10 | T9 | T8 | T7 | T4 | T3 | T2 | M |
| Volume (µL) | 25 | 25 | 25 | 25 | 25 | 3.5 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 3.5 |

**Gel 2: 293 WN**

| Lanes | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Samples | M | T2 | T3 | T4 | T7 | T8 | T9 | T10 | T11 | M | 1xL | 1xL | 1xL | 1xL | 1xL |
| Volume (µL) | 3.5 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 3.5 | 10 | 10 | 10 | 10 | 10 |

**Gel 3: CHO SDS**

(accidentally inverted sample application)

| Lanes | **1** | **2** | **3** | **4** | **5** | **6** | **7** | **8** | **9** | **10** | **11** | **12** | **13** | **14** | **15** |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Samples | 1xL | 1xL | 1xL | 1xL | 1xL | M | T11 | T10 | T9 | T8 | T7 | T4 | T3 | T2 | M |
| Volume (µL) | 25 | 25 | 25 | 25 | 25 | 3.5 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 3.5 |

**Gel 4: CHO WB**

| Lanes | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Samples | M | T2 | T3 | T4 | T7 | T8 | T9 | T10 | T11 | M | 1xL | 1xL | 1xL | 1xL | 1xL |
| Volume (µL) | 3.5 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 3.5 | 10 | 10 | 10 | 10 | 10 |

Run at 150V for 1.5 h

##### Lab protocol – 18.07

18.07.2022 - brought to you by Rosa

**Transfection** of ExpiCHO (D1)

##### Lab protocol – 19.07

19.07.2022 - brought to you by Lara

**Transfection** of ExpiCHO (D0), pH linker

##### Lab protocol – 20.07

20.07.2022 - brought to you by Lara

Feed CHO cells (D1)

##### Lab protocol – 25.07

25.07.2022 - brought to you by Rosa

**Harvest cells** (D6)

 → centrifuge at 4 °C in 6 x 50 mL Falcons 5000 x g, 30 min

 → take supernatant, centrifuge at 4 °C for 15 min 20000 x g

**Dialysis o/n**

 → Protein in dialysis tube, fixated with white clamps

 → in 5 L 20 mM Tris/HCl pH 7.5 at 4 °C

**Prepare SDS-Gels:** 12 % gels x4

**Prepare IMAC**

 → Binding buffer: 20 mM Tris/HCl, pH 7.5 at 4 °C (2 L prepared)

 → Elution buffer: 20 mM Tris/HCl, pH 7.5, 500 mM imidazole at 4 °C (500 mL prepared)

**Prepare Gefi**

 → 200 pg column

 → 20 mM Tris/HCl pH 7.5 100 mM NaCl

##### Lab protocol – 26.07

26.07.2022 - brought to you by Rosa

**IMAC**

**SDS-PAGE**

 → Fractiones from IMAC were selected with the chromatogramm (detection at 280 nm)

 → 40 µL samples + 10 µL 5x Laemmli + 10% β-Mercaptoethanol, heated at 95 °C

 → 3.5 µL of marker (PageRuler Plus Prestained) "M"

| Lanes | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Samples | M | 31 | 32 | 33 | 34 | 35 | 36 | 37 | 38 | M | X |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| volumes (µL) | 3.5 |  |  |  |  |  |  |  |  | 3.5 |  |

**Concentrate Protein**

****Gefi o/n****

##### Lab protocol – 28.07

28.07.2022 - brought to you by Till

**Buffer preparation** for pH-sensitivity experiment:

 → leave in Tris buffer for pH 7.5 for one sample (dilute as others)

 → pH 7, 6.8, 6.5, 6.3, 6: make 500 mL 100 mM NaH2PO4 + 100 mM NaCL, set pH using NaOH in 50 mL falcons

 → dilute pH protein in tube in 0.5 mL by factor 24 (to 0.2 µg/µL)

 → immediately take one sds sample from each tube (2 µg) add Lämmli, heat shock, freeze for tomorrow

 → leave samples overnight at 37 °C.

##### Lab protocol – 29.07

29.07.2022 - brought to you by Till

**SDS Gel:** from overnight sample, load 2 µg of protein from each sample

| Lanes | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | M |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Samples | 1xL | M | pH7.5 0 | pH 7.5 ON | pH7.5 0 | pH 7.5 ON | pH7 0 | pH 7 ON | pH6.8 0 | pH 6.8 ON | pH6.5 0 | pH 6.5 ON | pH6 0 | pH 6 ON |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| volumes (µL) |  | 3.5 |  |  |  |  |  |  |  |  |  |  |  | 15 | 3.5 |

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block image %}{% endblock %}
{% block page\_content %}

#### pH-sensitive linker cleavage

##### Results & Discussion

brought to you by
Till &
Rosa

Our approach in creating a pH-sensitive CAR was through the usage of an pH-induced auto-proteolytic
linker. We chose the linker sequence (EAAAK)5, which was shown to cleave around the tumor relevant value
of pH 6.5 by
 [Wu et al. [1]](https://2022.igem.wiki/munich/results#Refs). Before
testing this
functionality on a CAR, we decided to design and create a
simple fusion protein linked by (EAAAK)5. The fusion protein is made of a secretion signaling sequence
and two times two Kappa light constant chain domains linked together with the pH-linker (Figure 1). For
purification, we added a C-terminal 6x His-Tag.

![visual representation of our test gene for the ph linker concept](https://static.igem.wiki/teams/4497/wiki/subpages/results/res1.svg)

**Figure 1: Schematic Structure of the pH-sensitive Linker Protein.** The
IL27alpha
signaling sequence for secretion is followed by two Kappa CL domains, the pH-sensitive linker (EAAAK)5,
two
Kappa CL domains and a 6x His-Tag for purification.

After designing and ordering the whole protein sequence as a gene in pcDNA3.4, we used the ExpiCHO
expression system to obtain the protein over one week. Purification was performed using an Immobilized
Metal
Affinity Chromatography (IMAC) followed by size-exclusion chromatography. Fractions were collected and
pooled based on an SDS-Gel (Figure 2).

The calculated molecular size of our protein lies at 58 kDa. The gel bands at approximately 58 kDa are
therefore likely our protein of interest. It can be seen that all fractions contain the linker protein,
while fractions 33 to 36 show the thickest bands, hence highest amount of protein.

![a sds gel](https://static.igem.wiki/teams/4497/wiki/subpages/results/res2.png)

**Figure 2: 12 % SDS-Gel of pH linker purification.** The pH linker
was
purified with
Immobilized Metal Affinity Chromatography (IMAC) and size-exclusion chromatography. Fractions 31 to
38
were
determined with IMAC chromatograms at n = 280 nm, reduced samples were loaded (red. 31 to 38).
Marker
(M)
‘PageRuler Plus Prestained’ was used.

While the purification of our ph linker protein worked well, the protein showed no pH sensitive
auto-cleavage properties. We tested its stability at pH values from 7.5 to 6 at 37 °C overnight (Figure
3).
No
cleavage products from our 58 kDa protein are visible. Given the position of the linker, we would
have
expected protein fragments ranging from 23 to 25 kDa.

![another sds gel](https://static.igem.wiki/teams/4497/wiki/subpages/results/res3.png)

**Figure 3: SDS Gel of pH linker cleavage experiment.** pH linker was
loaded right
after
buffer change (0) and after incubation overnight at 37 °C (1) at buffer pH values 7.5, 7.0, 6.8,
6.5, 6.2 and 6.0. (M) is the Marker ‘PageRuler Plus Prestained’.

There could be different reasons for the (EAAAK)5 linker not showing any cleavage activity. For one,
the
cleavage activity might not only be related to the linker itself but also parts of the connected
proteins
used by Wang et al. Furthermore, this auto-catalytic cleavage might be dependent on other outside
factors
not shown in the paper. Overall, the available characterization of the cleavage function is quite
limited
and has not been properly reproduced in other literature. If there are certain other conditions
needed
for
the auto-catalytic cleavage to occur, the usability in cell culture/ in vivo conditions is likely
low.

##### Outlook

During our literature research, we did not find other pH-sensitive proteolytic
peptide sequences to mimic the protease induced cleavage of the masked CAR from
[Han et al. [2]](https://2022.igem.wiki/munich/results#Refs). There
might be other
ways to achieve pH-specificity, like through pH-induced conformational changes of, for example, the CAR
scFv binding epitopes.

#### Quorum Sensing

##### Results & Discussion

The goal of our first experiments was to determine whether we could use a synthetic bioorthogonal
ligand
dimer to induce a receptor system, which in turn would induce a reporter. For the ligand, we chose mCherry as
well
as
GFP, both as homo- and heterodimer: 2xmCherry, 2xGFP and GFP-mCherry. This way, we were hoping
to
observe our ligands through fluorescence. Furthermore, the corresponding nanobodies(GBP
[[3]](https://2022.igem.wiki/munich/results#Refs) & LaM-2
[[4]](https://2022.igem.wiki/munich/results#Refs))
that
we used for the receptor system are well established and characterized. The transmembrane and
intracellular
parts of our receptor system were taken from the Modular Extracellular Sensor Architecture (MESA)
from
[Daringer et al. [5]](https://2022.igem.wiki/munich/results#Refs).
For
the reporter system, we chose EYFP, which Daringer et al. have used before. We
later
changed the reporter to miRFP680 by cloning (as described on the [Engineering
page](https://2022.igem.wiki/munich/Engineering)). The MESA system has been
used
to
induce expression through transcription factor release induced by Vascular Endothelial Growth Factor
(VEGF)
[[6]](https://2022.igem.wiki/munich/results#Refs). We aimed to
achieve
the same functionality for functionally orthogonal ligands (mCherry & GFP),
before
turning the system into a loop by replacing the induced reporter with the ligand itself.

As our first testing system was not designed to produce a ligand by itself, we had to purify the
relevant
proteins. Prof. Scheller generously provided us with plasmids for the expression and purification of
mCherry-Fc, GFP-Fc and GFP-mCherry-Fc from
[Mossner et al. 2020
[7]](https://2022.igem.wiki/munich/results#Refs). Using a protocol adapted from their
publication (see Experiments), we were able to obtain all three proteins in one week using ExpiCHO
and
Protein A column purification. Given the reduced expression time and modified methodology compared
to
the
publication, the purification worked surprisingly well for us (Figure 4). While some additional band impurities
are
visible,
the main bands around 54 kDa (mCherry & GFP) and 80 kDa (GFP-mCherry) are well defined. Most of the
lower
bands correspond to degradation products that are also visible in Mossner et al. As the dimerization
of
mCherry- and GFP-Fc is based on disulfide bonds, we also checked the native redox state using
N-ethylmaleimide (NEM) containing Lämmli. Given the acceptable purification, we used the three
purified
proteins for further experiments.

![three sds gels](https://static.igem.wiki/teams/4497/wiki/subpages/results/res4.png)

**Figure 4: SDS Gels of ligand protein purification.** Ligands
are
2xmCherry, 2xGFP
and
GFP-mCherry. Proteins were purified with Protein A columns. Loading samples (L), waste samples
(W)
and
fractions were loaded. The fractions were chosen with chromatography at λ = 280 nm. Reduced
(reducing)
samples using β-Mercaptoethanol and non reduced (oxidizing) protein samples using NEM were
loaded.
Marker
(M)
‘PageRuler Plus Prestained’ was used.

Using GFP and mCherry as homo- and heterodimeric ligands allowed us to design different receptor
configurations for our MESA (Figure 5). We combined either the mCherry or GFP nanobody with the
transcription
factor (TF) or Tobacco Etch Virus (TEV) protease intracellular domain.
[Schwarz et al. [6]](https://2022.igem.wiki/munich/results#Refs) showed
that the
distance between extracellular receptor domain and transmembrane domain can affect the activation
efficiency
of the MESA system. Therefore, we designed all constructs with two differently sized domains linking
nanobody to the transmembrane domain, called linker 1 and 2 in the following descriptions.

![MESA combinations](https://static.igem.wiki/teams/4497/wiki/subpages/results/res5.svg)

**Figure 5: Overview of possible combinations of MESA System.**
(A): Our designs
incorporate two different nanobodies against mCherry (M) and GFP (G). Two different linker
lengths to
the
transmembrane domain were created 1 (SEFSGGN) and 2 (SEFGGDYKDDDDNGGSGGSGGSGGSGGGTG). Finally,
two
different
internal functions can be used: Protease (TEV), Transcription Factor tTA (TA). This leads to 8
different
combinations in total: M1TA, M1TEV, M2TA, M2TEV, G1TA, G1TEV, G2TA, G2TEV. (B) Two examples for
possible
combinations - left: homodimeric combination M1TA & M1TEV - right: heterodimeric combination M1TA
&
G1TEV.

Each half of the built MESA system is encoded on a separate plasmid. To express our starting system
in
HEK293T, we transfected two corresponding MESA plasmids as well as the reporter (see Experiments for
details). We then investigated the functionality of the MESA system by manually adding purified
ligand
24 h
after transfection. 48 h after transfection, the samples were investigated by flow cytometry. The
obtained
cytometry data was first gated to obtain only single cells. The used gating strategy can be seen in
Figure
6.

![presentation of facs data](https://static.igem.wiki/teams/4497/wiki/subpages/results/res6.jpg)

**Figure 6: Gating strategy for FACS data.** Forward scatter
(FSC-A) vs.
sideward
scatter (SSC-A) was plotted to distinguish cells from cell debris, width and height of FSC and
SSC
was
plotted to obtain only single cells. The remaining single cells were then gated according to the
used
fluorescence signals. FITC filter was used for EYFP or GFP signal, DAPI filter for
gating
BFP
signal, Texas Red filter for gating mCherry signal and APC filter for gating
miRFP680
signal.
The histograms show examples of gating FITC, DAPI and TexasRed.

First, we wanted to test whether our MESA components are expressed and whether they are capable of
ligand
binding. We therefore transfected HEK293T cells with only one MESA part. Cells without any MESA part
with
and without added ligand were used as a control. To all MESA component cell samples ligand was
added. As
shown in Figure 7, Cells containing a MESA part show a significantly higher signal than those who do
not.
This indicates a low to no retention of ligand on the cell surface in the absence of receptors. On
transfection of the receptors, the corresponding fluorescence signals increase by a factor of at
least
1.5 indicating both the expression on the cell surface and ligand binding. The exception here is
GFP-Linker
1-TEV MESA showing no significant binding.

![flourescence signals of our two proteins displayed as bar charts](https://static.igem.wiki/teams/4497/wiki/subpages/results/res7.png)

**Figure 7: GFP and mCherry fluorescence signals of mCherry (A) and GFP
MESA (B)
components in HEK293T cells.** Forward scatter (FSC-A) vs. sideward scatter (SSC-A) was plotted to
distinguish cells from cell debris, width and height of FSC and SSC was plotted to obtain only
single
cells.
The remaining single cells were then gated according to the used fluorescence signals. FITC filter
was
used
for EYFP or GFP signal, DAPI filter for gating BFP signal, Texas Red filter for
gating
mCherry signal and APC filter for gating miRFP signal. The histograms show examples of
gating
FITC,
DAPI and TexasRed.

Our fluorescence microscopy results visualize these findings. Figure 8 shows immunostaining images
of
COS7
cells that were transfected with M2TA or M2TEV. DAPI stain was used to image the cell nuclei (blue).
The
MESA components were stained using immunofluorescence labeling against the HA-tag (red). For both
samples
red cell outlines can be seen, indicating surface expression of the MESA components.

Combining the results of the cytometry data analysis and immunostaining, it can be said that the
MESA
components are expressed on the cell surface and that generally ligand binding takes place for both
mCherry
and GFP system components.

![6 pictures from fluorescence microscopy](https://static.igem.wiki/teams/4497/wiki/subpages/results/res8.png)

**Figure 8: Fluorescence microscopy images of immunostained COS7
cells.** Cells
were
transfected with mCherry nanobody bound TF combined with linker 2 (M2TA) and mCherry nanobody bound
TEV
combined with linker 2 (M2TEV). For immunostaining DAPI (blue) and Texas Red (red) was used.

In the next step, we tested whether ligand binding induces reporter expression in our system.
[Schwarz et al. [6]](https://2022.igem.wiki/munich/results#Refs)
showed that, depending on the ratio of the two transfected MESA parts, ligand induction changes, in
some
cases even decreasing with ligand addition. Therefore, we screened every combination of MESA parts
for
reporter activation with different transfection ratios of the TF and TEV component. An increase in
Reporter signaling is visible in all samples compared to the controls with only the reporter transfected
([Supplementary Figure 1](#Supplementary)). The obtained
fluorescence signals for the miRFP reporter without and with ligand addition can be seen in Figure 9.
Overall ligand addition results in, if any at all, only slight signal differences. The highest one being
+
22%
for the G1TA M1TEV combination at a 24x ratio of TF to TEV upon GFP-mCherry addition (Figure 7F).
As
previously shown
[[6]](https://2022.igem.wiki/munich/results#Refs), the ratio of
transfected TF to TEV plasmid affects the reporter background
signaling.
Interestingly, the overall corrected APC MFI seems to be dependent on the transfected TF MESA
component,
with G2TA showing the lowest signaling induction. While we are not sure of the reason, we
theorize
that
the extracellular receptor might have an influence on the intracellular domain of the MESA
component,
affecting TF release.

Unfortunately, none of the screened MESA combinations show a significant increase in reporter
signaling
on
ligand addition.
[Schwarz et al. [6]](https://2022.igem.wiki/munich/results#Refs)
showed reporter induction of 2x and more for their best MESA
combinations. It might be that our chosen incubation times, both before adding ligand and before
measuring,
are not optimal. Since miRFPs have a longer maturation time compared to other fluorescence proteins [[8]](https://2022.igem.wiki/munich/results#Refs), it is possible that
our miRFP reporter does not yet show a fluorescence signal after 24 h. Additionally, we might not have
screened the optimal plasmid ratios.
Overrepresentation of the TF half might also be a possible reason. Both cases would lead to a high
background signal that is not influenced by the ligand binding. Additionally, there are more linker
lengths that might need to be considered.
A broader screen with more constructs might reveal an optimal
condition. It has to be added that the shown cytometry analysis results only resemble one
transfection
sample, so one biological replicate. While this is not enough for obtaining significant results, we
were
hoping to find a promising MESA component and ligand combination in this scanning experiment.

![8 bar charts representing different reporter activation upon ligand binding](https://static.igem.wiki/teams/4497/wiki/subpages/results/res9.png)

**Figure 9: Reporter activation of the MESA system upon ligand binding
in HEK293T
cells.** Different MESA component combinations were tested without (blue) and with (red) ligand
addition,
M1TA M1TEV (A), M2TA M2TEV (B), G1TA G1TEV (C), G2TA G2TEV (D), M1TA G1TEV (E), G1TA M1TEV (F), M2TA
G2TEV
(G), G2TA M2TEV (H). For each combination, three transfection ratios of TF to TEV were tested: 6x,
12x
and
24x. The APC signal was corrected by dividing APC MFI (Median) by the BFP MFI (Median) of each
sample.
Ligand was added 24 h after transfection, cytometer measurements were performed 48 h after
transfection.
Bar
plots show the median of one sample with a total of at least 3000 APC positive gated events counted.

##### Outlook

Further establishing a MESA system for a possible application in quorum sensing of CAR T-cells
definitely
requires a lot more experiments. Since the tested combinations of MESA components, linkers,
and
ratios could not show any promising reporter induction, several screening tests might be expedient:
Varying the linker
length,
testing more than only two, varying times after transfection as well as ligand addition and
measurements.
Establishing a dose response curve might help in finding the amount of protein that has successfully
induced
the loop. Finally, testing the MESA system itself and the loop induction with different cell
concentrations
should lead to more insight about its functionality.
If those tests turn out well, as a next step, the whole loop functionality has to be examined. This
can
be
tested through repeating the transfection and FACS experiment, but instead of using the miRFP
reporter,
the
ligand is expressed.

#### Key Findings

**pH sensitive CAR**

* pH linker (EAAAK)5 cleavage at around the tumor relevant value of pH 6.5 did not occur for
  our
  construct

**Proximity activation**

* MESA system was successfully integrated into the cell

+ MESA receptor components were successfully expressed on cell surfaces
+ Ligand binding to the MESA receptor occurs
+ Our system induces reporter signaling

* For the initially tested system conditions, no promisingly high reporter induction could
  be shown. Further experimental optimization is needed to find a productive system setup.

#### Supplementary

![visual representation of our test gene for the ph linker concept](https://static.igem.wiki/teams/4497/wiki/subpages/results/res.svg)

**Figure 10. Histograms of APC Fluorescence comparing the reporter
control to one of the samples shown in Figure 9.** (A) APC Histogram from the single gated cells.
(B) APC Histogram of APC+ gated cells. The median of this sample is around 4 times higher than the
reporter background.

Table 1: MF (Median)I Values for APC and DAPI used for Bar Plot in Figure 9. The APC values
correspond to the miRFP reporter readout. The DAPI readout was used to correct for differences
in MESA receptor expression between samples.

| Sample | MFI APC (Median) | MFI DAPI (Median) |
| --- | --- | --- |
| Reporter Only Control | 5676 | - |
| M1TA , M1TEV , 6x Ratio , - | 21768 | 2951 |
| M1TA , M1TEV , 6x Ratio , + | 22395 | 2905 |
| M1TA , M1TEV , 12x Ratio , - | 23206 | 3132 |
| M1TA , M1TEV , 12x Ratio , + | 22502 | 3420 |
| M1TA , M1TEV , 24x Ratio , - | 21210 | 3332 |
| M1TA , M1TEV , 24x Ratio , + | 22502 | 3266 |
| M2TA , M2TEV , 6x Ratio , - | 19576 | 2350 |
| M2TA , M2TEV , 6x Ratio , + | 19347 | 2350 |
| M2TA , M2TEV , 12x Ratio , - | 16691 | 2433 |
| M2TA , M2TEV , 12x Ratio , + | 15967 | 2466 |
| M2TA , M2TEV , 24x Ratio , - | 14483 | 2588 |
| M2TA , M2TEV , 24x Ratio , + | 14618 | 2604 |
| G1TA , G1TEV , 6x Ratio , - | 21820 | 4024 |
| G1TA , G1TEV , 6x Ratio , + | 21512 | 4091 |
| G1TA , G1TEV , 12x Ratio , - | 20862 | 4955 |
| G1TA , G1TEV , 12x Ratio , + | 19854 | 4692 |
| G1TA , G1TEV , 24x Ratio , - | 19211 | 4575 |
| G1TA , G1TEV , 24x Ratio , + | 20280 | 4934 |
| G2TA , G2TEV , 6x Ratio , - | 15455 | 5556 |
| G2TA , G2TEV , 6x Ratio , + | 13296 | 5235 |
| G2TA , G2TEV , 12x Ratio , - | 11693 | 5125 |
| G2TA , G2TEV , 12x Ratio , + | 12698 | 5050 |
| G2TA , G2TEV , 24x Ratio , - | 11377 | 5580 |
| G2TA , G2TEV , 24x Ratio , + | 9685 | 5114 |
| M1TA , G1TEV , 6x Ratio , - | 22502 | 3359 |
| M1TA , G1TEV , 6x Ratio , + | 23371 | 3016 |
| M1TA , G1TEV , 12x Ratio , - | 22555 | 3332 |
| M1TA , G1TEV , 12x Ratio , + | 19438 | 2826 |
| M1TA , G1TEV , 24x Ratio , - | 19901 | 3539 |
| M1TA , G1TEV , 24x Ratio , + | 21310 | 3332 |
| G1TA , M1TEV , 6x Ratio , - | 20424 | 3754 |
| G1TA , M1TEV , 6x Ratio , + | 19529 | 3831 |
| G1TA , M1TEV , 12x Ratio , - | 19576 | 4407 |
| G1TA , M1TEV , 12x Ratio , + | 19948 | 4308 |
| G1TA , M1TEV , 24x Ratio , - | 17904 | 4537 |
| G1TA , M1TEV , 24x Ratio , + | 21310 | 4453 |
| M2TA , G2TEV , 6x Ratio , - | 17286 | 2821 |
| M2TA , G2TEV , 6x Ratio , + | 18502 | 2568 |
| M2TA , G2TEV , 12x Ratio , - | 15672 | 2665 |
| M2TA , G2TEV , 12x Ratio , + | 15563 | 2718 |
| M2TA , G2TEV , 24x Ratio , - | 13669 | 2723 |
| M2TA , G2TEV , 24x Ratio , + | 14720 | 2476 |
| G2TA , M2TEV , 6x Ratio , - | 15419 | 4761 |
| G2TA , M2TEV , 6x Ratio , + | 14961 | 4791 |
| G2TA , M2TEV , 12x Ratio , - | 12757 | 5393 |
| G2TA , M2TEV , 12x Ratio , + | 13481 | 5180 |
| G2TA , M2TEV , 24x Ratio , - | 12524 | 5486 |
| G2TA , M2TEV , 24x Ratio , + | 12381 | 5849 |

#### References

**[1]** Wu, Y. J., Fan, C. Y., & Li, Y. K. (2009). Protein purification involving a unique
auto-cleavage
feature of a repeated EAAAK peptide. Journal of Chromatography B, 877(31), 4015–4021.
<https://doi.org/10.1016/J.JCHROMB.2009.10.009>

**[2]** Han, X., Bryson, P. D., Zhao, Y., Cinay, G. E., Li, S., Guo, Y., Siriwon, N., & Wang, P.
(2017).
Masked Chimeric Antigen Receptor for Tumor-Specific Activation. Molecular Therapy, 25(1),
274–284.
<https://doi.org/10.1016/J.YMTHE.2016.10.011>

**[3]** Rothbauer, U., Zolghadr, K., Muyldermans, S., Schepers, A., Cardoso, M. C., & Leonhardt,
H.
(2008). A
versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins.
Molecular
and Cellular Proteomics, 7(2), 282–289.
<https://doi.org/10.1074/mcp.M700342-MCP200>

**[4]** Fridy, P. C., Li, Y., Keegan, S., Thompson, M. K., Nudelman, I., Scheid, J. F., Oeffinger,
M.,
Nussenzweig, M. C., Fenyö, D., Chait, B. T., & Rout, M. P. (2014). A robust pipeline for rapid
production of versatile nanobody repertoires. Nature Methods 2014 11:12, 11(12),
1253–1260.
<https://doi.org/10.1038/nmeth.3170>

**[5]** Daringer, N. M., Dudek, R. M., Schwarz, K. A., & Leonard, J. N. (2014). Modular
Extracellular
Sensor
Architecture for Engineering Mammalian Cell-based Devices.
<https://doi.org/10.1021/sb400128g>

**[6]** Schwarz, K. A., Daringer, N. M., Dolberg, T. B., & Leonard, J. N. (2017). Rewiring human
cellular
input-output using modular extracellular sensors. Nature Chemical Biology, 13(2), 202–209.
<https://doi.org/10.1038/nchembio.2253>

**[7]** Mossner, S., Phan, H. T., Triller, S., Moll, J. M., Conrad, U., & Scheller, J. (2020).
Multimerization strategies for efficient production and purification of highly active synthetic
cytokine
receptor ligands. PLOS ONE, 15(4), e0230804.
<https://doi.org/10.1371/JOURNAL.PONE.0230804>

**[8]** Shcherbakova, D. M., Baloban, M., Emelyanov, A. V., Brenowitz, M., Guo, P., & Verkhusha,
V. V. (2016). Bright monomeric near-infrared fluorescent proteins as tags and biosensors for
multiscale imaging. Nature communications, 7(1), 1-12.
<https://doi.org/10.1038/ncomms12405>

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