Experiments
For our wet work, we started the process by planning and ordering the parts (BBa_K2946010, BBa_K2946011, BBa_K2946013, BBa_K2946014, BBa_K2549001 and BBa_K321004). BBa_K321004 was ordered with a ScFv from our lab in order to improve a part (BBa_K2946003).
They were then ordered as gBlocks from IDT. As our backbone vector for the BBa_K2946010, BBa_K2946011, BBa_K2946013, BBa_K2946014 parts we used pLN75 & pLN193 (obtained from Nissim et al., Cell 2017).
Once the gBlocks arrived, they were resuspended, and the cloning process began.
Cloning
Our gBlocks were digested over night with relevant restriction enzymes.
BBa_K2946010 and BBa_K2946013 were digested with Asc1 and Xho1, while BBa_K2946011 and BBa_K2946014 were digested with Asc1 and BamHI
Each tube contains:
50 µL DNA, 1.5 µL from each enzyme, 5 µL cut-smart, 12 µL UPW – total 70 µL
After complete DNA digestion using the relevant restriction enzymes, the DNA segments were run and separated on 1% agarose gel with TAE buffer and crystal violet. The vectors were cut from the gel and cleaned using Wizard SV Gel and PCR Clean-up System from Promega according to their protocol. (protocol 1)
For extracted the gblocks from the restriction enzymes we used an extraction kit according to Gel/PCR Fragments Extraction Kit. (protocol 2)
Ligation
Ligation of DNA inserts into their vectors was done by using 1 unit of T4 DNA Ligase from NEB at room temperature for 2 hrs.
Each tube contains:
1µL buffer, 1µL T4 ligase, 1µL vector, 2µL insert and 5µL DDW.
| Plasmid | Gene | Part number |
|---|---|---|
| pLN193 | TS1 | BBa_K2946010 |
| pLN75 | TS2 | BBa_K2946011 |
| pLN193 | TS11 | BBa_K2946013 |
| pLN75 | TS12 | BBa_K2946014 |
| PGEM4Z | CD19 | BBa_K2549001 |
| PGEM4Z | kir3DL1+ ScFv | BBa_K2946003 |
Table 1: Ligations – Each part was ligated into a specific vector. TS1 and TS11 were each inserted to pLN193, TS2 and TS12 were inserted into pLN75. 1721 were insert into pGEM4Z 5'UT-eGFP-3'UT-A64 (PGEM4Z) and so as 1722.
Following ligation DNA was transformed into TOP10 competent cells according to our labs protocol. (protocol 3) Mini prep was done to 3-5 colonies for each part using Zyppy Plasmid Miniprep kit. (protocol 4) After determining positive colonies, one was used for maxi prep using Maxi prep endotoxin free- PureYield Plasmid Maxiprep System – Promega. (protocol 5)
DNA transfection using Hek293T
HEK293T cells were grown in DMEM complete medium (C.M.) containing 500 ml medium, 50 ml FCS, 5 ml sodium pyruvate and 2 ml Pen-strep.
Table 2 – DNA transfection plan
| Plasmid 1 (1µg) | Plasmid 2 (1µg) | |
|---|---|---|
| 1 | Positive control | - |
| 2 | GFP (pEGFP-N3) | - |
| 3 | Mock | - |
| 4 | TS1 | - |
| 5 | TS2 | - |
| 6 | TS11 | - |
| 7 | TS12 | - |
| 8 | TS1 | TS2 |
| 9 | TS11 | TS12 |
| 10 | Non-Transfected | - |
Transfections were done in 6 welled plates using transfection reagent FuGENE HD.
FuGENE HD transfection reagent (Promega, Madison, WI; Catalog #E2311) was mixed with OptiMEM medium (Life Technologies Catalog #31985) according to the materials found in Cell 2017 and methods. This was then added to the mixture of two plasmids (1 mg each).
During the 20-minute incubation of FuGENE HD with the DNA complexes at room temperature, HEK293T suspension cells were prepared and diluted to 1.2x106 cells/mL in culture medium. 0.5 mL of diluted cells were added to each tube, mixed well, and 5 minutes later plated into a 6-well plate containing 2 mL cell culture medium and incubation at 37C with 5% CO2.
24 hours post-transfection, the cells growth medium was replaced, and cells were collected 24 hours after that (48 hours post-transfection).
For parts containing mkate2 exons, cells were collected using 0.5ml trypsin + 1ml DMEM-C.M. These cells were then subjected to flow cytometry and measured by FACSCalibur by BD Biosciences or Attune NxT by Thermo Fisher scientific.
Improved part (BBa_K2946003) and characterization of part (BBa_K2549001)
As our backbone vector for CD19 (BBa_K2549001) and kir3DL1 (BBa_K2946003) parts we used PGEM4Z as our vector. The genes were cloned into the plasmid similarly to the described above.
RNA transfection using K562 (CML cell line)
In order to examine the expression of CD19 and kir3DL1 parts we used RNA transfection to insert the parts into K562 cells.
mRNA production was done according to T7 mScript Standard mRNA Production System - Cell Script. (protocol 6)
K562 cells were grown in RPMI complete medium (C.M.) containing 10% FCS, 1 mM sodium pyruvate, 2 mM L-glutamine, Pen-strep solution 1:200.
Transfection into K562 cells was done by Electroporation.
Electroporation was performed with Gene Pulser Xcell (Bio-Rad Laboratories, Hercules, CA) (protocol 7) according to the following conditions:
| Cell type | Amount of cells | Amount of mRNA | Pulse type | Volts | Ω | µF | Pulse duration | Cuvette width |
|---|---|---|---|---|---|---|---|---|
| K562 | 3x106 | 10µg | Exponential | 350V | - | 150µF | - | 4mm |
Table 3 - electroporation conditions
After the transfection we incubated the cells with antibodies for the FACS measurement.
10 µl Anti-Human c-Myc- APC (R&D SYSTEMS, Cat: IC3696A).
5 µl Anti Ha- Alexa Fluor 647 (R&D SYSTEMS, Cat: IC6875R).
Preparing for FACS
Cells were harvested, washed once with cold FACS buffer (PBS with 0.5M EDTA, 0.01% sodium azide and 1% BSA) and incubated for 45 minutes at 4֯C in the dark with the respective Ab-conjugate at concentration recommended by the manufacturer. Cells were washed again with 3 ml FACS buffer, resuspended in 0.3 ml PBS and subjected to flow cytometry using FACSCalibur (Becton Dickinson, San Jose, CA). Data were analyzed by FCSexpress (DeNovo Software, Los Angeles, CA).
Reference
1. Nissim, L. et al. Synthetic RNA-Based Immunomodulatory Gene Circuits for Cancer Immunotherapy. Cell 171, 1138-1150.e15 (2017).