Design:

First, we reproduced MESA from a previous paper¹ to demonstrate the concept of the Detection unit.

The extracellular part of the chimeric receptor uses the extracellular domain of FKBP (FK506-binding protein) and FRB (FKBP-rapamycin binding protein), which dimerize in response to rapamycin,the transmembrane domain is derived from CD28, The intracellular part uses TEV protease, and its Cleavage Site, and tTA respectively. In this unit, the extracellular parts approach each other in response to rapamycin, causing the protease to cleave the Cleavage Site and release tTA into the cell line. Later, TRE3G-ZsGreen in the genome reacts with tTA, expressing green fluorescence (ZsGreen). CD4signal was added upstream of the extracellular part to be transported to the cell membrane.

Build:

Three plasmids were transfected into HEK293A: the PC (Protease Chain) part and TC (Target Chain) part of MESA from a previous paper, and YT82, which is a plasmid encoding coordinated expression of the green fluorescent protein along with tTA.

Plasmid 1 encoding CD4signal-FKBP-CD28TMD-TEVprotease. Plasmid 2 encoding CD4signal-FRB-CD28TMD-TEVcs-tTA. Plasmid 3 encodingTRE-ZsGreen. The respective complete plasmids are shown below:

Figure 2: Completed Plasmid of Plasmid 1

Figure 3: Completed Plasmid of Plasmid 2

Figure 4: Completed Plasmids of Plasmid 3

DNA fragments and vectors were transformed into E. coli by InFusion, followed by colony PCR, and only those with bands at the correct positions were cultured for Miniprep. The extracted plasmids were determined for sequencing by the Sanger sequencing (ordered to Eurofins Genomics) and compared with the sequence of the plasmid of interest. Only E. coli that yielded the correct plasmids were further cultured for Midiprep to extract a high concentration of the plasmid. The resulting plasmids were transfected into human cell lines together with transposase.

Test:

Three days after transfection, DW only, rapamycin 0.1nM, 1nM, 10nM and 100nM were added and incubated at 37°C for 24h.

FACS fluorescence intensity was measured using FACS melody. Results are as follows.

Figure 5: Percentage of cell lines with fluorescence stronger than the maximum fluorescence intensity of non-transfected cell lines (HEK293A) for each concentration of rapamycin at 24 h after addition of rapamycin (n=4401~5275)

(a) (b) Figure 6 a&b: (a)Mean fluorescence intensity for each concentration of rapamycin at 24 h after addition of rapamycin.
Error bar indicates standard error (n=4077~4908).
(b)The mean of fluorescence intensity.
In actual applications using SWIFT or the MESA system, cell lines that show the desired results are sorted and the clones are used. Therefore, to ensure that only cell lines expressing ZsGreen are selected, cell lines showing fluorescence greater than the maximum fluorescence intensity of 293A were extracted and the average fluorescence intensity was shown on the graph.

Figure 7: Histogram of fluorescence intensity for each concentration of rapamycin at 24 h after addition of rapamycin; Normalize represents relative value with mode as 100
Orange →NC, Blue →rapamycin 0 nM, Red → rapamycin 10 nM

Learn:

We tested that the MESA system in the previous paper worked, showing that the MESA system can work as an artificial chimeric receptor and supporting the concept of modularity of SWIFT. The results are also comparable to our Detection unit.

In addition, as described in detail in the engineering cycle, we found that a GS linker between the TM and the intracellular part in the MESA system had a lot of leakage and mistakenly operated without a ligand. Therefore, we decided not to use a GS linker in the MESA system.

Moreover, we had used a CAG promoter to express each part, and we found that if the number of copies incorporated into the genome was large, the expression level would be too high, crowding the system at the cell membrane and causing a lot of leakage. Also, when the number of copies incorporated into the genome is low, there are few receptors that function as the MESA system in response to rapamycin, resulting in little or no fluorescence. In this study, we changed the concentration at which transfection was performed and succeeded in finding the appropriate concentration.

Design:

Next, we created a MESA system that operates in response to IFN-γ to show that MESA is a modular, recombinable system and that our own designed MESA works.

The Ectodomain of the chimeric receptor is the extracellular domain of IFNGR (IFNGRex, IFN-γ receptor extracellular domain), which dimerizes in response to IFN-γ, and the transmembrane domain is derived from CD28, The intracellular part uses TEV protease, and its Cleavage Site, and tTA, respectively. In this system, the Ectodomain approach each other in response to IFN-γ, causing the protease to cleave the Cleavage Site and release tTA into the cell line. Later, TRE3G-ZsGreen in the genome reacts with tTA, expressing green fluorescence (ZsGreen). CD4signal was added upstream of the extracellular part to be transported to the cell membrane.

Build:

Three plasmids were transfected: the PC and TC parts of MESA with IFN-γ as the ligand, and plasmid 3, which is a plasmid encoding coordinated expression of the green fluorescent protein along with tTA.

Plasmid 4 encoding CD4signal-IFNGRex-CD28TMD-TEVprotease.

Plasmid 5 encoding CD4signal-IFNGRex-CD28TMD-TEVcs-tTA.

Plasmid 3 is described in M1.

Figure 8: Completed plasmids of plasmid 4

Figure 9 Completed plasmids of plasmid 5

DNA fragments and vectors were transformed into E. coli by InFusion, followed by colony PCR, and only those with bands at the correct positions were cultured for Miniprep. The extracted plasmids were determined for sequencing by the Sanger sequencing (ordered to Eurofins Genomics) and compared with the sequence of the plasmid of interest. Only E. coli that yielded the correct plasmids were further cultured for Midiprep to extract a high concentration of the plasmid. The resulting plasmids were transfected into human cell lines together with transposase.

Test:

Three days after transfection, DW only, IFN-γ 1pM, 1nM, 10nM and 100nM were added and incubated at 37°C for 24 h. FACS fluorescence intensity was measured using melody. Results are as follows.

Figure 10: Percentage of cell lines with fluorescence stronger than the maximum fluorescence intensity of non-transfected cell lines (HEK293A) for each IFN-γ concentration at 24h after IFN-γ addition (n=3043-4320)

Figure 11: Mean fluorescence intensity for each IFN-γ concentration at 24h after IFN-γ addition.
Error bar indicates standard error (n=3165-4532).

Figure 12: Histogram of fluorescence intensity by IFN-γ concentration at 24h after IFN-γ addition
Orange →NC, Blue →IFN-γ 0 nM, Red → IFN-γ 10 nM

Learn:

We showed that the MESA system operates as a chimeric receptor of its own design, supporting the SWIFT concept. Compared to the MESA system in the previous paper mentioned above, the MESA system worked equally well, suggesting that the system can work even if the receptor parts are recombined in a modular fashion.

In addition, as described in detail in the engineering cycle, we found that a GS linker between the TM and the intracellular part in the MESA system had a lot of leakage and mistakenly operated without a ligand. Therefore, we decided not to use a GS linker in the MESA system.

In this case, we used a CAG promoter to express each part, and we found that if the number of copies incorporated into the genome was too large, the expression would be too high, crowding the system at the cell membrane and causing a lot of leakage. Also, if the copy number incorporated into the genome was low, there were fewer receptors that functioned as a MESA system in response to rapamycin and there was less tendency. In this study, they changed the concentration at which transfection was performed and succeeded in finding the appropriate concentration.

It was also found that the concentration of plasmid during transfection changes the amount of recombinant gene in the cell line, and that it is necessary to select the appropriate concentration.

When the SWIFT system is actually used, it is assumed that cell lines that are responding well will be extracted by cell sorter and used. Although we did not have time to do so this time, we would like to conduct experiments in the future by using cell sorter to extract cell lines that respond to the desired reaction.

Design:

The mechanism of secretion unit forms most of the second half of SWIFT system.This unit works after the protease signal emitting from the MESA system is amplified; Amplified protease cleaves the ER retention signal that contains a protein that inhibits the trans-signaling and then releases the protein that inhibits the trans-signaling out of the cell.

We have admitted that the better way for PoC to do co-staining with ER/Golgi markers to clarify ER retention signaling works. However, our advisor declared that we lack the skills to do that. Also, in the first plan, we planned to utilize SEAP to quantify the secreted amount of protease. However, the cloning of SEAP has not been successful.

AAYL, which is not an ER retention signal, was used as a control against the ER retention signal KKYL.

Plasmid 6 encoding CD4signal-eGFP-FCS-CD28TMD-PPVcs-KKYL, and the ER retention signal is cleaved in the presence of PPV protease. Plasmid 7 used as control encoding CD4signal-eGFP-FCS-CD28TMD-PPVcs-AAYL and is also not retained in the ER.

Build:

Figure 14: Completed Plasmids of Plasmid 6

Figure 15: Completed Plasmids of Plasmid 7

DNA fragments and vectors were transformed into E. coli by InFusion, followed by colony PCR, and only those with bands at the correct positions were cultured for Miniprep. The extracted plasmids were determined for sequencing by the Sanger sequencing (ordered to Eurofins Genomics) and compared with the sequence of the plasmid of interest. Only E. coli that yielded the correct plasmids were further cultured for Midiprep to extract a high concentration of the plasmid. The resulting plasmids were transfected into human cell lines together with transposase.

Test:

The cell lines were observed after transfection and eGFP was observed to be withheld in the ER. Figure 16: Photographs taken at 40x after about 72h in plasmid 6

Figure 17: Photograph at 40x about 72h after transfection in plasmid 7

Learn:

In Figure 14, with KKYL, the protein localization is more distinct, rather than as homogenous as plasmid 7 that showed in Figure 15.

However, we ourselves do not believe that what is here is a clear PoC. We also tried to measure and quantify the secreted amount using SEAP. However, we could not succeed in cloning SEAP despite repeated trial and error, including redesigning the Forward primer, adjusting the Tm, and changing the type of E. coli. Therefore, in Dry Lab, we did modeling of secreted Amount of POI (Protein Of Interest) and got the data that was advantageous to us.

For more details, see Modeling Page.

Improvement:

We attempted to measure and quantify secretion using SEAP. However, despite repeated trial and error, including redesigning the forward primer, adjusting the Tm, and changing the type of E. coli, we were unable to successfully perform PCR of SEAP. In the future, we will increase the number of trials and succeed in PCR, then quantify using SEAP and observe using confocal microscopy.

Design:

Though we did not make it to do the concrete assay, we designed 2 plasmids for the Amplification unit, based on the result of protein structure prediction; a disordered one and a stable one.

TVMV^Thr-AI in both plasmids,plasmid 8 and plasmid 9, is composed of TVMV Protease, THR-CS, TVMV-AI, and HisTag linked by a linker in this order.

The difference of each plasmid is the stability of its linker. Plasmid 8 is more stable and less leaky, whereas plasmid 9 is more reactive and more leaky.

Figure 19: Completed Plasmids of Plasmid 8

Figure 20: Completed Plasmids of Plasmid 9

Build:

We constructed plasmids for Amplification unit like this. Due to lack of time, we couldn’t utilize it to make plasmids.

We would like to do experiments in the future.

Design:

All promoters of the reporter cell line created in this study are mini promoter, and minP-ZsGreen was created as a positive control. The reporter of all reporter cell lines was ZsGreen.

The reporter cell line used STAT3 and AP-1 as cis-elements, which are factors that induce the expression of downstream genes in response to IL-6 and IL-1β, respectively.

minP-ZsGreen (Control) STAT3 reporter-ZsGreen (IL-6) AP-1 reporter-ZsGreen (IL-1β)

Once the reporter cell lines were constructed as described above, they had many leaks and were useless. Thus we added a puromycin resistance gene to the plasmid to select the cell lines that contained the plasmid after transfection using cell sorter. The cell lines with fewer leaks were sorted, and those that functioned properly were cultured and used as reporter cell lines.

plasmid 10: minP-ZsGreen, EF1α core promoter-PuroR (Control) plasmid 11: STAT3 reporter-ZsGreen, EF1α core promoter-PuroR (IL-6 reporter cell lines) plasmid 12: AP-1 reporter-ZsGreen, EF1α core promoter-PuroR (IL-1β reporter cell lines)

Build:

Figure 21: Completed Plasmids of Plasmid 10

Figure 22: Completed Plasmids of Plasmid 11

Figure 23: Completed Plasmids of Plasmid 12

DNA fragments and vectors were transformed into E. coli by InFusion, followed by colony PCR, and only those with bands at the correct positions were cultured for Miniprep. The extracted plasmids were determined for sequencing by the Sanger sequencing (ordered to Eurofins Genomics) and compared with the sequence of the plasmid of interest. Only E. coli that yielded the correct plasmids were further cultured for Midiprep to extract a high concentration of the plasmid. The resulting plasmids were transfected into human cell lines together with transposase.

Then, puromycin was used to kill cell lines that did not contain the plasmid.

Test:

Each ligand was added at 10000 pg/ml, 100 pg/ml, 50 pg/ml, 10 pg/ml, and 0 pg/ml.

Fluorescence was analyzed by flow cytometry (BD FACS Melody) 24h after the ligands were added.

10000 pg/ml is the reference concentration of IL-1β, IL-6 when CRS is occurring.

Cell lines that did not show much leakage were sorted by Cell sorter and cultured.

FACS results before sorting are shown below.

Figure 24: Mode of fluorescence intensity for each concentration of IL-6 in plasmid 10 (reporter cell line for IL-6) at 24 h after IL-6 addition.
Error bar indicates standard error.

Figure 25: Graph of fluorescence intensity per concentration of IL-6 in plasmid 10 (reporter cell line for IL-6) at 24 h after addition of IL-6. error bar indicates standard error.
Red → 0 pg/ml Blue → 100 pg/ml

Learn:

In our experimental environment, IL-6 is difficult to easily quantify in terms of cost and equipment, but in future experiments, IL-6 can now be easily quantified by reporter cell line. The classification by cell sorter completed making reporter cell lines. Therefore, a single clone of reporter cell lines can now be used to compare increases or decreases in IL-1 and IL-6 concentrations in experiments throughout Secretion and SWIFT.