The iGEMs involved in the wet experiment each prepared 100µl of PCR system:

1. The synthesized primers are dissolved in ddH2O after a short period of discovery in a laboratory and stored at -20°C in a 100 µM storage solution. Dilute the primers to 10 µM of working solution before use.

2. Configure the following PCR system in the PCR center and mix well (Note: Reagents are added in the order shown in Table 1).

3. Place it in the PCR machine and run according to the following procedures: 95℃ 3 min; 95℃ 15 sec, 63℃ 30 sec,

72℃ 30s, 30 cycles; 72℃ 5 min; 4℃ insulation.

4. The PCR products are removed and analyzed by agarose electrophoresis.

After testing and stabilizing HIV-1 RRE DNA template preparation, HIV-1 RRE RNA was obtained by in vitro transcription, and high quality and stable HIV-1 RRE RNA was obtained by isolation and purification, thus realizing a stable in vitro transcription RNA process.

1. Reagent thawing

Optimized T7 RNA Polymerase freezing tubes were briefly centrifuged on ice; thawed 10× Transcription Buffer and 4 ribonucleotides (ATP, CTP, GTP, UTP) were mixed and centrifuged to the bottom of the tubes and set aside on ice; 10× Transcription Buffer was placed at room temperature.

2. Configuration of transcription reaction system at room temperature

Prepare the reaction system according to the following system (Reaction volume will be 100μL so add 62μL of water ).

3. Incubate at 37℃ for 1.5h, and transcribe to obtain HIV-1 RRE RNA.

The above reaction solution was mixed well, briefly centrifuged to the bottom of the tube, and incubated at 37°C for 1.5 h. The length of the transcript was 358 nt, and the length of the reaction was critical to the quality of the transcription.

Note: It is recommended to use RNase-free tips and EP tubes, wear disposable latex gloves and masks, and prepare all reagents with RNase free H2O to prevent RNase contamination during the experimental procedure.

4. Molecular sieve purification of HIV-1 RRE RNA.

The obtained transcription products were first precipitated by centrifugation at low temperature and high speed, the purpose of which was to remove unreacted template DNA and possible white precipitate of the reaction products (this is the formation of magnesium pyrophosphate from free pyrophosphate and magnesium ions in the reaction solution during the reaction process); and then purified to remove free pyrophosphate and so on by Supedx200 molecular sieves, and then replaced into the desired preservation buffer HEPES buffer containing 6 mM Mg2+. The target RNA fractions were collected and subjected to gel electrophoresis to verify the quality of nucleic acids, and Nanodrop to measure the concentration and then sorted for use.

Overexpression of HIV-1 Rev protein and optimization of the isolation and purification process as a means to obtain stable monomeric Rev proteins. and to provide stable samples for interaction with HIV-1 RRE RNA.

A. The successfully cloned HIV-1 Rev plasmid was transformed into E. coli DH5α cells for single colony culture.

1. Remove the receptor cell DH5α from the -80℃ ultra-low temperature refrigerator and place it on ice for 5 min, and thaw it.

2. Add target HIV-1 Rev DNA (10ng and volume <10µl) to the sensory cell suspension, gently spin the tube to mix the contents and ice bath for 30min.

3.The centrifuge tube was placed in a 42°C water bath for 90 s. The tube was quickly transferred to ice for 3 min.

4.Add 900µl of sterile LB medium (without antibiotics) to the centrifuge tube, mix well and incubate at 37℃ with shaking for 1h (220rpm/min).

5.The transformed sensory cells were centrifuged at low speed (5000rpm, 4min) and part of the supernatant was discarded, 100µl of medium was reserved, and the suspension was gently blown with a pipette, and then all of it was added to LB solid agar medium containing the corresponding antibiotics, and the cells were spread evenly with a sterile applicator stick.

6.Place the plate at room temperature until the liquid is absorbed, invert the plate and incubate at 37°C for 12-16h, after the appearance of single colonies, store at four degrees for spare parts（see the picture A below).

A) HIV-1 Rev monoclonal colony

B. HIV-1 Rev protein overexpression, isolation and purification.

1. Select single colonies in A to 100 ml of sterile LB medium corresponding to resistance, and incubate overnight at 220 rpm/min on a shaker at 37°C for 16h.

2. After measuring the OD600 of the overnight cultured bacterial solution at 0.6, take 15ml of bacterial solution to 1L and add it into the corresponding resistant sterile LB medium, incubate it at 37℃ on shaker at 220rpm/min until the OD600 is at 0.6-0.8, and start to induce the expression of the target proteins by using 1mM IPTG, and then continue to incubate it at 16℃ on shaker at 220rpm/min for 12-16h.

3. Cell fragmentation: centrifuge to collect the organisms, weigh the wet organisms, add the lysis solution and protease inhibitor, and perform ultrasonic fragmentation to release the proteins.

4. Collect protein solution: collect protein solution by centrifugation at high speed to remove cell debris.

5. Purification: Crude purification of target proteins using Ni column and gradient elution of heterogeneous proteins using different imidazole concentrations, and finally elution of target proteins with high concentration of imidazole.

6. Dialysis: The target protein eluted with high concentration of imidazole buffer is dialyzed overnight to remove imidazole.

7. Molecular sieve purification: The dialyzed proteins are further purified by Superdex75 columns, displacing their buffer to be consistent with the buffer in which HIV-1 RRE RNA is preserved, in order to prevent RNA degradation or protein aggregation/degradation due to buffer inconsistency.

8. Collect the protein fraction, measure its concentration, and freeze it.

The HIV-1 RRE RNA, which was successfully prepared for testing, was subjected to gel migration electrophoresis experiments with HIV-1 Rev proteins through a series of RNA-to-protein ratios as a means of verifying the interaction between the two.

1. 20µl of HIV-1 RRE RNA and HIV-1 Rev protein were configured with n(RNA/Rev) of 1:12.5; 1:25; 1:50; 1:75, respectively, and after mixing them well, they were placed on ice for 40min for reaction, and then 8% TBM gel was used for the gel migration experiment.

2. The results of gel migration experiments were analyzed.