iGEM 2023 — Team rainsaca-china

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{% block title %}Notebook{% endblock %}
{% block cover\_img %}https://static.igem.wiki/teams/4996/wiki/cover/notebook.jpg {% endblock %}
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[Notebook](#a1)

Notebook

**Period 1: 2023.1.10-2023.4.30:**

Because we come from different areas in China, we are planning to learn some knowledge online together based on our teachers and development of project plan. We have learnt as below:

1) the iGEM Project manual

2) Books on Scientific Research and synthetic Biology

3) Concept, technical vocabulary, text and video materials

4) The charm of synthetic biology

5) In-depth Analysis of iGEM Award-winning cases

6) Thinking and skills of scientific research

7) Introduction to iGEM Competition

8) Introduction to Synthetic Biology

9) Introduction to Molecular Biology

10) Introduction to Cell Biology

11) Skill Training: Literature Retrieval Methods and Skills

12) Skill Training: Human Practices Method

13) Experimental Methods and Skills Learning in Synthetic Biology

14) Skill Training: Modeling and Data Processing

15) iGEM Awards Criteria Analysis (Entrepreneurship/Human Practices）

16) iGEM Awards Criteria Analysis (Life science)

**Period 2: 2023.7.25-2023.8.15:**

We begin to do the tasks separately. For wet laboratory activities, we had prepared experimental research & divide tasks、conduct experimental research & record data with mentors team、conduct literature research & compare experimental methods、conduct experiment records & develop professional lab habits、analyze experiment result & process、work with dry team member to conduct promotion & education work etc.

**Some details are below:**

**Stage1:**

1) Familiar with laboratory equipment and drugs and learn about laboratory safety matters；2）Introduction to laboratory instruments and drugs；3）Laboratory safety precautions；4）Configure required reagents；5）Organize supplies；6）Prepare culture medium.

**Stage2:**

1）Competent cell preparation and recombination and Principles of plasmid transformation；2）HIV-1 RRE plasmid transformation；3）Receptive cell transformation；4）Coating plate culture.

**Stage3:**

1）Obtaining the HIV-1 RRE gene sequence；2）RNA gene analysis processes and methods；3）Explain the acquisition of HIV-1 RRE gene sequence；4）Explain the RNA gene analysis process and methods.

**Stage4:**

1）Single bead selection culture；2）Shake the bacteria and take samples regularly to measure the growth curve (OD value)；3）Regular sampling to measure growth curve (OD value)；4）Seamless cloning technology explained；5）Purify plasmid and measure concentration.

**Stage5:**

1）Primer design principles and precautions；2）Nucleic acid electrophoresis and photography；3）Analyze electrophoresis results；4）HIV-1 Rev protein plasmid preparation)；5）Competent intracellular transformation；6）Plate overnight culture.

**Stage6:**

1） Observe the growth of strains on the plate; 2）Pick a single strain and shake it for culture；3）Take samples regularly to measure OD；4）Preparation of DNA template for PCR amplification reaction；5）Nucleic acid electrophoresis verification template, photos

；6）Preparation of HIV-1 Rev Glycerol Bacteria Preservation；7）Pick a single colony again and culture it overnight to prepare for plasmid extraction the next day.

**Stage7:**

1）T7 RNA polymerase in vitro transcription principle；2）Transcription reagent preparation and configuration；3）HIV-1 RNA in vitro transcription；4）Purification of in vitro transcribed RNA；5）RNA electrophoresis, photography；6）HIV-1 Rev shaken, overnight.

**Stage8:**

1） HIV-1 Rev protein expression；2）Regular OD measurement；3）Regular OD measurement again; 4）Measure OD regularly to 0.6-0.7, IPTG induces expression; 5）Bacteria collection.

**Stage9:**

1）Break bacteria, extract Rev protein, take samples, and retain samples to verify Rev protein expression；2）Configure protein purification gradient buffer；3）Protein column purification；4）Collect the purified fractions of the target protein Rev and verify by SDS-PAGE protein electrophoresis.

**Stage10:**

1）Experiments on RRE and Rev protein interactions at different ratios；2）EMSA conducts interaction verification; 3）Electrophoresis results and photos.

**Stage11:**

1）Explain the acquisition of protein gene sequences；2） Compare the analysis processes, functions and methods of protein and nucleic acid RNA；3）Selection and research significance of RNA and protein action sites.

**Period 3: 2023.8.10-2023.9.20:**

For dry laboratory activities,we had conducted social research& field research、conduct expert Interview、establish team brand promotion channel & promotion、fundraising & business entrprenuership、outreach & collaboration/communication、film team promotion videos & edit etc.

**Stage1:**

1）Familiar with laboratory equipment and drugs；2）Learn about laboratory safety matters；3）Team name, logo design；4）And peripheral and product promotion product design；5）Explanation of experimental principles；6）Laboratory tour；7）Laboratory safety precautions；8）Improve team culture product content

**Stage2:**

1）Create team IP；2）Complete peripheral order purchases (financial statement records), etc. 3）Conduct social research on the topic, including questionnaire design and preparation for social interviews；4）Formulate team work plan (publicity, science popularization, education, charity； 5）Completed social research, collected feedback data, and completed the first content release on publicity channels.

**Stage3:**

1）Design and planning of education and publicity activities (which groups are targeted, educational activity methods, required materials); 2）design and planning of Inclusive Award activities；3）Dry and wet team communication; 4）Bio-bank visit; 5）Optimize education and publicity activity plans.

**Stage4:**

1）Project solution acceptance questionnaire design, release preparation, expert interview questions；2）The first expert interview (technical feasibility) and feedback recording; 3） the completion of the second publicity channel content release.

**Stage5:**

1）(Collaboration) Contact other iGEM teams to confirm joint activity plans；2）Complete the design and preparation of promotional posters, leaflets and other materials；3）Communication and discussion on the progress of wet and dry teams；4）Team promotional video idea.

**Stage6:**

1）Outbound implementation of education and publicity activities；2）Activity summary；3）Writing the wiki: Communication section；4）Write a team video script；5）Completed the third promotion channel content release.

**Stage7:**

1）Promotional video material shooting；2）iGEM team cooperation and communication；3）Business planning explanation；4）Project business concept and product design.

**Stage8:**

1）Plan fundraising events；2）Prepare questions for second expert interview (product application)；3）Second expert interview and feedback record；4）Optimize commercial product design；5）Completed the fourth promotion channel content release.

**Stage9:**

1）Write a business plan；2）Wet and dry team communication；3）Team Booth Poster Design；4）Wiki web interface concept design.

**Stage10:**

1）Team promotional video shooting and editing；2）Conduct fund-raising planning activities and sustainable development creative activities；3）Completed the fifth promotion channel content release；4）Write a business plan；5）Impact part of the content.

**Stage11:**

1）Check for omissions and fill them in, and prepare relevant materials；2）Clarify tasks and plans for subsequent online activities.

**Period 4: 2023.9.10-2023.10.10:**

Building the Team Wiki. Our team members begin to record the team wiki based on our performances we have done before.

**The experiments we have used as below:**

1）Molecular experiments

2）Explanation of seamless cloning technology (including primer design, etc.)

3）DNA extraction and testing

4）PCR amplification of target genes

5）PCR product recovery-vector ligation-competent cell transformation

6）DNA/RNA electrophoresis

7）RNA in vitro transcription

8）Obtaining gene sequences and analyzing processes and methods of gene functions

9）Screening and significance of RNA-protein interaction sites

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block cover\_img %}https://static.igem.wiki/teams/4996/wiki/cover/engineering.jpg {% endblock %}
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[RCA Engineering Success](#a1)
[Overview](#b1)
[1) Design: In this project, we selected the HIV-1 isolate ARV-2/SF2
strain and designed the HIV-1 RRE RNA full-length gene and HIV-1 Rev full-length gene, respectively.](#c1)
[2) Build: (1)Our team constructed the HIV-1 RRE full-length gene
sequence, thought to prepare RRE RNA, and synthesized the plasmid and corresponding forward and
reverse primers from Prime Biologics, where the length of the HIV-1 RRE gene is 2484bp;（2）We also
constructed the gene sequence of HIV-1 Rev protein at the same time and ordered the over-expression
vector, HIV-1 Rev of 5,662 bp, through DNA Technologies as mentioned above, in order to prepare the
HIV-1 Rev protein.](#c2)
[3) Test: (1) We performed plasmid extraction and preparation, in vitro
transcription, and purification of the HIV-1 RRE designed and constructed；（2）We sequenced,
over-expressed, and purified the designed and constructed HIV-1 Rev；（3）Interaction of HIV-1 RRE RNA
with HIV-1 Rev protein was verified by gel migration electrophoresis (Electrophoretic Mobility Shift
Assay, EMSA).](#c3)
[4) Learn: Throughout the project testing, our team found that:
high-quality transcription of HIV-1 RRE RNA, stable over-expression and efficient isolation and
purification of HIV-1 Rev protein, as well as a certain proportion of mutual binding effect of the
two can be achieved. All of these will support the development of HIV-1 RNA vaccines and targeted
drugs in the future.](#c4)
[Design:](#c5)
[Build:](#c6)
[a）Successful cloning of HIV-1 RRE](#c7)
[b）Successful cloning of HIV-1 Rev](#c8)
[Test:](#c9)
[a) HIV-1 RRE template preparation](#c10)
[b)HIV-1 RRE RNA was obtained by in vitro transcription and HIV-1 RRE
RNA was isolated and purified.](#c11)
[c)HIV-1 Rev protein overexpression, isolation and purification](#c12)
[d)Interaction between HIV-1 RRE RNA and HIV-1 Rev protein](#c13)
[Learn:](#b2)

RCA Engineering Success

Overview

1) Design: In this project, we selected the HIV-1 isolate
ARV-2/SF2 strain and designed the HIV-1 RRE RNA full-length gene and HIV-1 Rev full-length gene,
respectively.

2) Build: (1)Our team constructed the HIV-1 RRE full-length
gene sequence, thought to prepare RRE RNA, and synthesized the plasmid and corresponding forward and
reverse primers from Prime Biologics, where the length of the HIV-1 RRE gene is 2484bp;（2）We also
constructed the gene sequence of HIV-1 Rev protein at the same time and ordered the over-expression
vector, HIV-1 Rev of 5,662 bp, through DNA Technologies as mentioned above, in order to prepare the
HIV-1 Rev protein.

3) Test: (1) We performed plasmid extraction and preparation,
in vitro transcription, and purification of the HIV-1 RRE designed and constructed；（2）We sequenced,
over-expressed, and purified the designed and constructed HIV-1 Rev；（3）Interaction of HIV-1 RRE RNA with
HIV-1 Rev protein was verified by gel migration electrophoresis (Electrophoretic Mobility Shift Assay,
EMSA).

4) Learn: Throughout the project testing, our team found
that: high-quality transcription of HIV-1 RRE RNA, stable over-expression and efficient isolation and
purification of HIV-1 Rev protein, as well as a certain proportion of mutual binding effect of the two
can be achieved. All of these will support the development of HIV-1 RNA vaccines and targeted drugs in
the future.

Design:

Our iGEM team designed and constructed the full HIV-1 RRE gene, the HIV-1 Rev full-length amino acid
sequence, and designed its vector, which included PMV and RRE reverse primer sequences. To ensure that
the HIV-1 RRE target gene is ready for amplification, we designed and determined primers optimized to
the best.

![](https://static.igem.wiki/teams/4996/wiki/engi/img1.png)

c) PMV Forward Primer: 5’GTAACCCACTCGTGCACCCAACTGATCTTC3’

RRE Reverse Primer: 5'GGTAGCATTCCAAGGCACAGCAGTGGTG3’

Figure 1 a). HIV-1 RRE vector map; b). HIV-1 Rev vector map; c). RRE
forward and reverse primers.

Build:

a）Successful cloning of HIV-1 RRE

The PCR process was used to react the specifically designed primers, enzymes and constructed plasmid
vectors in order to obtain the target gene fragments of HIV-1 RRE. Sequencing results showed that HIV-1
RRE was successfully cloned, in which the gene length was 708bp.

![](https://static.igem.wiki/teams/4996/wiki/engi/img2.png)

![](https://static.igem.wiki/teams/4996/wiki/engi/img3.png)

Fig. 2. a) Agarose electrophoresis of the target gene of HIV-1 RRE; b) PCR reaction process of the target
gene of HIV-1 RRE; c) The target DNA gene of HIV-1 RRE

b）Successful cloning of HIV-1 Rev

DNA transformation, plate coating, and sequencing of the designed and constructed HIV-1 Rev vector
indicated that the HIV-1 Rev vector was successfully cloned.

![](https://static.igem.wiki/teams/4996/wiki/engi/img4.png)

![](https://static.igem.wiki/teams/4996/wiki/engi/img5.png)

Figure 3. a) HIV-1 Rev monoclonal colony; b) HIV-1 Rev target gene
sequencing analysis results (dark grey represents HIV-1 Rev target gene sequences); c) Target gene
sequence comparison results are identical.

Test:

a) HIV-1 RRE template preparation

Successful acquisition of HIV-1 RRE RNA for in vitro transcription is firstly to obtain high quality
HIV-1 RRE template after optimized PCR process and primers by constructed and prepared plasmid. The
construction has been shown in Figure 2 that the HIV-1 RRE target gene after PCR is accurate and has
clear bands, which can be the condition for HIV-1 RRE template preparation. Therefore, we will carry out
the PCR process through the same conditions to prepare enough HIV-1 RRE template as the key substrate to
realize in vitro transcription to obtain HIV-1 RRE RNA.

**Protocol**

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).

![](https://static.igem.wiki/teams/4996/wiki/engi/img6.png)

Table 1 HIV-1 RRE template preparation PCR reaction substrate system

3. Place in a PCR instrument and run as follows: 95°C for 3 min; 95°C for 15 sec, 63°C for 30 sec, 72°C
for 30 s, and 72°C for 5 min.

The PCR products were then run in the PCR instrument for 3 min at 95 °C, 15 sec at 95 °C, 30 sec at 63
°C, 30 s at 72 °C, 30 cycles at 72 °C, 5 min at 72 °C, and held at 4 °C.

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

b)HIV-1 RRE RNA was obtained by in vitro transcription and
HIV-1 RRE RNA was isolated and purified.

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.

**Protocol**

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; and 10× Transcription Buffer was placed at room temperature.

2. Configure the transcription reaction system at room temperature

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

![](https://static.igem.wiki/teams/4996/wiki/engi/img7.png)

Table 2. Composition of substrate systems for configuring transcription
reactions

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

The above reaction solutions were mixed well, briefly centrifuged to the bottom of the tube, and
incubated at 37°C for 1.5 h. The transcript length 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.

c)HIV-1 Rev protein overexpression, isolation and
purification

Over-expression 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.

**Protocol**

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

1. Remove the DH5α sensory cells from the -80℃ ultra-low temperature refrigerator and place on ice for
5min, thaw. 2.

2. Add the target HIV-1 Rev DNA (10ng, <10µl) to the suspension of receptor cells, gently rotate the tube
to mix the contents, and place the tube in an ice bath for 30min.

3. Place the tube in a 42°C water bath for 90s, then quickly transfer the tube to ice for 3min.

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

5. Centrifuge the transformed sensory cells at low speed (5000rpm, 4min), discard part of the
supernatant, keep 100µl of medium, gently blow the suspension with a pipette, and then add all
of them to the LB solid agar medium containing the corresponding antibiotics, and spread the
cells uniformly with a sterile applicator.

6. Place the plate at room temperature until the liquid is absorbed, invert the plate and
incubate at 37℃ for 12-16h, after the appearance of single colonies, store at four degrees for
spare use (see Figure 3a).

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

**Protocol**

1. Select the single colony in A to 100ml of the corresponding resistant sterile LB medium, 37℃
shaker 220rpm/min overnight culture 16h.

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

3. Cell fragmentation: collect the bacterium by centrifugation, weigh the wet bacterium, add the
lysis solution and protease inhibitor, and perform ultrasonic fragmentation to release the
protein.

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

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

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

7. Molecular sieve purification: The dialyzed proteins are further purified by Superdex75
columns, replacing its buffer with the same buffer used for HIV-1 RRE RNA preservation to
prevent RNA degradation or protein aggregation/degradation due to buffer inconsistency.

8. Collect the protein fractions, measure their concentration, and freeze them.

d)Interaction between HIV-1 RRE RNA and HIV-1 Rev protein

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.

**Protocol：**

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.

Learn:

In our iGEM project, our team investigated the preparation of HIV-1 RRE DNA templates, in vitro
transcription, isolation and purification, and HIV-1 Rev protein overexpression, isolation and
purification, as well as the interactions between the two, and the results showed that not only did each
member of our wet-lab experiment obtain a uniform, high-quality, and stable HIV-1 RRE DNA template, but
also obtained a single, tight, and high-purity RNA band in the bulk RNA transcription and purification,
and obtained the effective target protein we expected in the HIV-1 Rev overexpression and isolation and
purification. The results showed that each member of our wet experiment not only obtained a uniform,
high quality and stable HIV-1 RRE DNA template, but also obtained a single, tight and high-purity RNA in
batch transcription and purification, and then obtained the effective target proteins in HIV-1 Rev
overexpression, isolation and purification, and successfully realized the interaction between the HIV-1
RRE RNA and the HIV-1 Rev protein. These successful experimental results and experiences will provide
valuable experience for the design of other viral RNA and protein studies. Meanwhile, these will not
only provide a reliable reference for RNA vaccine development and nucleic acid integrase inhibitors, but
also provide an effective molecular mechanism for drug development that alters viral RNA load and
interaction sites.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block cover\_img %}https://static.igem.wiki/teams/4996/wiki/cover/experiments.png {% endblock %}
{% block page\_content %}

{{self.title()}}

[Experiment](#a1)
[a) Preparation of HIV-1 RRE DNA templates](#b1)
[b) HIV-1 RRE RNA obtained by in vitro transcription](#b2)
[c)HIV-1 Rev protein overexpression, isolation and purification](#b3)
[d) Interaction between HIV-1 RRE RNA and HIV-1 Rev protein](#b4)

Experiment

a) Preparation of HIV-1 RRE DNA templates

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).

![](https://static.igem.wiki/teams/4996/wiki/experi/img1.png)

Table 1 HIV-1 RRE template preparation PCR reaction substrate system

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.

b) HIV-1 RRE RNA obtained by in vitro transcription

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 ).

![](https://static.igem.wiki/teams/4996/wiki/experi/img2.png)

Table 2. Composition of substrate systems for configuring transcription
reactions

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.

c)HIV-1 Rev protein overexpression, isolation and
purification

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).

![](https://static.igem.wiki/teams/4996/wiki/experi/img3.png)

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.

d) Interaction between HIV-1 RRE RNA and HIV-1 Rev protein

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.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block cover\_img %}https://static.igem.wiki/teams/4996/wiki/cover/results.jpg {% endblock %}
{% block page\_content %}

{{self.title()}}

[Results](#a1)
[Reference:](#b1)

Results

a) By analyzing the electrophoresis results on the agarose gel Figure 4, we can see that each electrophoretic band is at the location of our target gene. Therefore our team came up with the result that each of our wet team members were able to complete the template experiment and get a stable HIV-1 RRE DNA template.

![](https://static.igem.wiki/teams/4996/wiki/result/img1.png)

Figure 4 HIV-1 RRE DNA template agarose electropherogram. The gel above is a 3% gel showing a DNA template near the target position of a gel .

b) The stable DNA template provided a high-quality template for our preparation of transcribed HIV-1 RRE RNA. After in vitro transcription, isolation and purification, the purified peak graph in Figure 5 shows a single symmetrical peak of the transcribed RNA, and the inlay gel electrophoresis indicates that the purified HIV-1 RRE RNA has a single, tight and high quality band, which is essential for EMSA experiments of interaction with viral proteins.

![](https://static.igem.wiki/teams/4996/wiki/result/img2.png)
![](https://static.igem.wiki/teams/4996/wiki/result/img3.png)

Fig. 5 a) Diagram of molecular sieves for HIV-1 RRE RNA isolation and purification, inset shows 8% TBM plot of gel electrophoresis of fractions collected from HIV-1 RRE RNA; b) HIV-1 RRE RNA sequence

c) HIV-1 Rev protein overexpression, isolation, purification and characterization. HIV-1 Rev protein was cultured at 37°C until the OD600 was at 0.6-0.8, induced with 1 mM IPTG, and overexpressed overnight at 16°C for 12-16 h. Bacteria were collected. Rev was purified on nickel resin by 5M urea denaturation and re-denaturation for nucleic acid removal and gradient imidazole elution of heteroproteins, followed by overnight dialysis and Superdex 75 molecular sieves for purification and target buffer replacement. As can be seen on the 10% SDS-PAGE electropherogram embedded in Figure 6, the purified Rev has a single band, which is basically sufficient for further study of the interaction of Rev with HIV-1 RRE RNA, as well as the basis for exploring the interaction site.

![](https://static.igem.wiki/teams/4996/wiki/result/img4.png)

Fig. 6 After dialysising at 4 degree for overnight in the (NH4)2SO4 buffer, HIV-1 Rev is further changed into the store buffer through Superdex 75 column. Inset shows the result of 10% SDS-PAGE gel of fractions.

d）The results of the experimental validation of HIV-1 RRE RNA and HIV-1Rev protein binding to each other are shown in Figure 7, which indicates that bands of HIV-1 RRE RNA complexed with HIV-1 Rev are clearly present and can form low, medium, and high levels of Rev-RRE multimers as the RNA-to-Rev molar ratio continues to increase until the monomeric RNAs are completely consumed to form a stable Rev -RRE complexes. The full-length HIV-1 RRE RNA and full-length HIV-1 Rev interaction results are consistent with the facts reported in the literature, which suggests that the full-length RNA and full-length Rev protein interactions are not affected[1],and this also provides a valid basis for further investigation of the HIV-1 RNA and viral protein sites of action.

![](https://static.igem.wiki/teams/4996/wiki/result/img5.png)

Fig. 7. Plot of EMSA electrophoresis results of HIV-1 RRE RNA binding to HIV-1 Rev each other.

Reference:

[1] Chringma Sherpa, et al. The HIV-1 Rev response element (RRE) adopts alternative conformations that promote different rates of virus replication. Nucleic Acids Research, 2015, 43(9) , 4676–4686.

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