iGEM 2023 — Team metropolis-shanghai

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![](https://static.igem.wiki/teams/4875/wiki/notebook.png)

July

2th

WET&DRY TEAM:

After a
quick break, Dr. Xie, the instructor of the wet team,
assisted to interpreted the whole project design and we determined the daily schedule. In the afternoon,
we had a brainstorm and  decided the team name,
Logo, VI, Mission, Slogan, team, team uniform, merch and created official account on social media. Then we
held the election for the preliminary leaders for team management.

3th

WET
TEAM:

l. Configuring LB Solid Medium

li. PCR preparation

lii. Culturing Escherichia Coli

liii. preparing LB culture medium

DRY
TEAM:

In the
morning, we did some training for the IHP (include questionnaire and interview), we confirmed the general
direction of the implementation and the questionnaire. After the lunch break, the team member finish the
writing of the implementation and the outline of the interview. We also got familiar with the first
education part we were going to start tomorrow.

4th

WET
TEAM:

liv. Centrifuging E.Coli in LB Broth

lv. Plasmid Enzyme cutting/digestion

lvi. Gel electrophoresis

lvii. Purification of target DNA

a. Purification of the target gene involves
isolating the specific DNA sequence of interest from a complex mixture.

lviii. Gel recycle

DRY
TEAM:

In the
morning, we decided the format and person in charge of the public tweets on Wechat social account
 and three online education activity. We completed the first draft of questionnaire 2. In the
afternoon, we conducted an interview with Dr. Qin Ruomeng (including material arrangement), completed the
first tweet, posted the first questionnaire to the public, and wrote the outline of the second
interview.

5th

DRY
TEAM

In the
morning, we finalized the visit plan for the afternoon of July 7 at Lujiazui Street, finished writing the
daily tweets for the public website, and finalized the outline of Interview 2 and the link to the meeting;
in the afternoon, we finished recording the video of Online Edu1, wrote the outline of Interview 3, and
completed the PPT and speech of the popularization of science for the elderly; because Professor Shen
suddenly had a very difficult task to handle, Interview 2, which was scheduled for tonight, was
rescheduled to tomorrow night.

WET
TEAM：

l. continuing gel electrophoresis and
recycling

li. T4 DNA ligase connects vectors and fragments

a. To perform heat shock transformation of E. coli
and facilitate the docking of the target DNA with the vector

lii. PCR product purification

a. To select the target DNA using a centrifuge and
purify it using a wash solution

6th

DRY
TEAM:

During
the day the team took business planning classes, and at night they conducted a second interview with
Professor Shen and prepared for tomorrow's interview materials.

WET
TEAM:

l. Colony PCR

a. Using pcr thermocycler to verify our colony

li. Plasmid Extraction

a. Using plasmid to seperate solution and the cell
debris

Gel electrophoresis

For checking and finding the
correct strand of target DNA

7th

DRY
TEAM:

In the
morning we made the final preparation and completed our business plan. We went to a senior center in the
afternoon and interviewed the staff members.

WET
TEAM:

l. Plasmid extraction

a. The plasmid extraction method is to remove RNA,
separate the plasmid from the bacterial genome DNA, and remove proteins and other impurities to obtain
relatively pure plasmids.

li. Culture of HEK293 cells

a. To culture HEK293 cells in an appropriate broth
at a temperature of 37°C and at a concentration of 5% CO₂ to obtain the raw material for cell
transfection.

lii. Cell transfection

a. Change the DMEM culture medium for HEK293 cell
for nutrients.

b. Mix DMEM, α2A-AR-Rluc8 plasmid, Venus K-Ras
plasmid, and PEI into 6 centrifuge tubes for cells to be fed to promote growth.

c. Put into an incubator for cells to conseve energy
and prolong their viability and cell recovery.

liii. LB culture media configuration

liv. Gel electrophoresis

8th

DRY
TEAM

We
conducted the EDU Workshop the entire day and completed the Edu block activity design. We also completed
the interview records at the senior center yesterday and published the third article.

WET
TEAM:

l. WIKI Documentation

li. RUSH-BRET and protein purification lecture

lii. enzyme digestion, connection

liii. Agarose gel purification

liv. adding IPTG

lv. BRET expirament

lvi. cell transfer

9th

DRY
TEAM:

In the
morning we completed the preparations for our pitch. In the afternoon we conducted our pitch and completed
our interview with the business expert. Completed the promotion video’s design, and discussed our
business plan book

 WET TEAM:

l. making SDS-PAGE gel

li. cell transferring

lii. Protein purification and gel produce

10th

DRY
TEAM: Day off

WET
TEAM:

l. BRET excitement - 1

li. Taking Gel purification and extraction
picture

lii. Continue writing wiki

11th

DRY
TEAM:

Finished
up the promotion video recording. Finished the PowerPoint slides show creation. We went to Tianlin Road
and take class for the children, introduce Alzheimer’s disease to them.

WET
TEAM: Day off.

12th

WET&DRY TEAM:

Today we
take group photos in the morning and after the lunch break we learn how to do a presentation. We learn how
to abstract key point. I'm in charge of the project goal part of the tomorrow's whole team's speech

13th

WET&DRY TEAM:

 Internal presentation session and review meeting for the next step
of the project.

Midterm Break

August

14th

WET
TEAM:

l. Culture of HEK293 cells

li. Cell transfection

15th

WET
TEAM:

l. Add IPTG

li. Prepare BRET samples

liii. making SDS-PAGE gel

16th

WET
TEAM:

l. Protein purification and concentration

li. Sent protein to Nanjing for IP-MS

lii. Sent cell samples to SubCat for BRET

17th

WET
TEAM:

l. Received BRET data for analysis

24th

WET
TEAM:

li. Received IP-MS result and analysis for wiki
documentation

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![](https://static.igem.wiki/teams/4875/wiki/engineering.png)

Overview

α2-Adrenergic receptors
(α2ARs) are present in the brain and are required for memory and learning, such receptors may be a
potential target for the treatment of AD.To investigate whether our target protein function is consistent
with our expectations, we will explore Str KDEL\_ SBP-α2A-AR-Rluc8 detached from
Biotin Streptavidin Combining with Venus K-Ras, through vector construction, protein purification, cell
transfection, and RUSH-BRET detection.

![](https://static.igem.wiki/teams/4875/wiki/engineering/0.png)

**Experimental Procedure
Flowchart**

**E****ngineering** **Cycles****:**

§ Enginering Cycle#1 Plasmid construction (DBTL)

§ Enginering Cycle#2 Protein purification
(DBTL)

§ Enginering Cycle#3 RUSH-BRET (DBTL)

**Explain the
cycle****s**

Ø **Enginering
Cycle#1** **Plasmid****construction**

**Design:**

![](https://static.igem.wiki/teams/4875/wiki/engineering/1.png)

**Figure****1.****Plasmid****M****ap of****G****ST****-α2A-AR-****C****T**

![](https://static.igem.wiki/teams/4875/wiki/engineering/2.png)

**Figure****2.****Plamid****M****ap of****Str-KDEL\_SBP-α2A-AR-Rluc8**

Studies have shown that the 2A
adrenergic receptor (2A-AR) in the G-protein-coupled receptor (GPCR) family is associated with Alzheimer's
disease and is a potential target for the treatment of neurodegenerative diseases. In order to obtain the
protein, firstly we will construct the GST-α2A-AR-CT plasmid and Str-KDEL\_SBP-α2A-AR-Rluc8
plasmid as shown above.

**Build:**

1) GST-α2A-AR-CT plasmid
construction

We
used primers to amplify α2A-AR fragment from
α2A-AR-GFP and inserted into the vectors pGEX-4T-1 to obtain GST-α2A-AR-CT plasmid.

2) Str-KDEL\_SBP-α2A-AR-Rluc8
plasmid construction

First,
using the human α2A-AR-Rluc8 plasmid as a template, point mutation at Sda1 to eliminate Sda1 enzyme
restriction site in order to get mutant α2A-AR-Rluc8 plasmid.

Second,
mutant α2A-AR-Rluc8 plasmid as a template to amplify target sequence by using primers. Gel DNA
Extraction purified 2106 bp product.

Third,
The plasmid Str-KDEL\_SBP-EGFP-Ecadherin (purchased from Addgene) was double digested with Sda1 and Xba1
and purified the product at 5234 bp to obtain the Str-KDEL\_SBP vector. we inserted the α2A-AR-Rluc8
fragment to Str-KDEL\_SBP vector to obtain Str-KDEL\_SBP-α2A-AR-Rluc8 plasmid.

**Test:**

①　Colony
PCR identification and cloning

We
found that only three results NO.7, 10, and 11 are in line with our expectations (700
bp around). In
this way, we would say these three ones are our target
strips.

![](https://static.igem.wiki/teams/4875/wiki/engineering/3.jpg)

**Figure****3.****Gel electrophoresis
results of colony PCR****: Marker-15K; 1,19,20-Negative control; 2-18-Clony PCR samples
and 7,10,11 are confirmed positive**

②　Sending
to the company for Sanger sequencing

The following alignment and base pairs which
confirmed that we have obtained the expected plasmid.

![](https://static.igem.wiki/teams/4875/wiki/engineering/4-1.png)
![](https://static.igem.wiki/teams/4875/wiki/engineering/4-2.png)

**Figure****4.****Global sequence
alignment of sequencing**

**Learn:**

Above all, we could confirm that we demonstrated the successful
construction of the plasmid GST-α2A-AR-CT and Str-KDEL\_SBP-α2A-AR-Rluc8, which will serve as
the basis for the following experiments.

Ø **Enginering
Cycle#2 Protein purification**

**Design:**

Protein
purification is vital for the specification of the protein function, structure, and interactions of the
protein of interest.(Protein\_purification-Wikipedia, 2023) The
purification process may separate the protein and non-protein parts of the mixture, and finally separate
the desired protein from all other proteins. In order to
obtain the pure protein, we designed a GST tag attached to α2A-AR-CT so that our protein could be
purified by Ni-NTA Agarose
Resin.

![](https://static.igem.wiki/teams/4875/wiki/engineering/5.png)

**Figure****5.****Schematic diagram of
GST-α-AR-CT protein purificatio****n****. Image by Xu
Xing.**

**Build：**

①　Collect
bacteria and divide the sample containing BL21(DE3) and GST-α2A-AR-CT into two 50mL centrifuges
tubes

②　Centrifuge
three times with 4000rpm, 5 min, and 25℃

③　Fix
the mold: fix two thin glass plates to the base

④　Configure
the lower layer glue:2.7ml 2x lower layer buffer,55μl coagulant

⑤　Injection
into the mold

⑥　Configure
the upper layer glue:0.75μl upper layer gel solution,0.75μl colored upper layer gel buffer,15μl
coagulant

⑦　Injection
into the mold

**Test:**

At the end of the experiment
we got the target protein by purification, and we compared it with the sample from each process during
purification by using protein gel electrophoresis. The electrophoresis result showed that our target
protein now became pure and we finally identified that we got the right protein (25
kDa).

![](https://static.igem.wiki/teams/4875/wiki/engineering/6.png)

**Figure****6.****SDS-PAGE analysis of
GST-α-AR-CT**

**Learn:**

This picture is the result of
SDS-PAGE which is used to test proteins. P represents precipitate; S represents cell lysate; T represents
flow through; W represents wash and E represents elution. GST-α2A-AR-CT protein is found on column
E, marker 25KDa, this shows GST-α2A-AR-CT protein is purified from BL21(DE3) bacteria.

The electrophoresis result
showed our target protein was purified and we finally
identified that we got the right protein. After that, we could further use this sample for our IP-MS and
RUSH-BRET test.

Based on the IP-MS results, we
could further find our candidate proteins that have the potential ability to affect the α-AR-CT
transportation to the membrane.

Ø **Enginering
Cycle#****3****RUSH-BRET**

**Design:**

We need a way
to measure the level of α2A-AR within a cell. The
bioluminescence resonance energy transfer (BRET) method is based on resonance energy transfer between a
light-emitting enzyme and a fluorescent acceptor.

![](https://static.igem.wiki/teams/4875/wiki/engineering/7.png)

**Figure****7.****Schematic diagram of
RUSH-BRET assay****. Image by Xu
Xing.**

The Renilla
luciferase Rluc8 was fused to the C-terminus of the α2AR
(α2AR-Rluc8) to
serve as a BRET donor. The fluorescent protein venus was fused to the N-terminus of a C-terminal fragment
of KRas (V-kras) to serve as a plasma membrane-associated BRET acceptor. BRET donor and BRET acceptor
interaction produced a substantial BRET signal. KDEL is connected to Streptavidin
(Str), this complex was
fused to the SBP-α2AR -Rluc8, KEDEL can help
Str- SBP-α2AR to localize
to endoplasmic reticulum (ER). Biotin can bind Str to disturb KDEL and
SBP-α2AR -Rluc8 interaction,
SBP-α2AR -Rluc8 will be released
and transform from ER to cell membrane.
On the cell membrane, Energy is transferred when Rluc8 of
SBP-α2AR -Rluc8 replace the RAS
of the Venus-K-RAS on the cell
membrane.

**Build:**

In order to stimulate the transportation of α2A-AR-CT
protein to the cell membrane, we used HEK293 cell lines as the host.
After the cultivation, we processed cell transfection by PEI to transfer the plasmids,
Str
KDEL\_ SBP-α2A-AR-Rluc8 and Venus-K-Ras
plasmid into HEK 293 cell.

**Test:**

Procedure

①　Add
40µL of Biotin to each well and
incubate
for 0, 15, 30, 1, and 3 hours. The group without biotin for 0 minutes was used as the control group

②　Discard
the culture medium, wash the cells twice with DPBS, add 1ml of DPBS to resuspend cells.

③　Remove
a 200 μL sample of suspension to three wells in a black opaque 96-well plate and wait for 15min

④　Add
2µL Coelenterazine H to these 3 wells

⑤　Put
the plate into the ELISA Microplate Reader

⑥　Record
the value

⑦　BRET was processed by SubCat using (Spark® Cyto PLATE READER
WITH LIVE CELL IMAGING AND REAL-TIME CYTOMETRY) and
the measurement wavelength is 535nm/485nm.

BRET result

Based on the data in table 1 where the value is Venus/Rluc8, we
could draw the curve as shown in Figure 8.

![](https://static.igem.wiki/teams/4875/wiki/engineering/8.png)

**Table****1****. Fluorescence intensity
of RUSH-BRET in different time**

![](https://static.igem.wiki/teams/4875/wiki/engineering/table-1.png)

**Figure****8.** **The surface expression of
α2A-AR was measured by RUSH-based BRET assays**

**Learn:**

These increasing trends mean that the Str-KDEL\_SBP-α2A-AR-Rluc8
structure has been disturbed by the biotin and α2A-AR-Rluc8 move
from the endoplasmic reticulum to cell membrane.  Rluc8 of SBP-α2AR -Rluc8 replaced
the RAS of the Venus-K-RAS on the cell membrane and produced BRET signal that represents the amount of
the protein α2A-AR on the membrane.

To conclude, our RUSH-BRET system was working to measure the
amount of the protein α2A-AR on the membrane and it could be more
reliable for our future research on the drug
screening. Next move we will design more inquiry experiments to evaluate our RUSH-BRET system and
evaluate the candidate proteins we targeted from IP-MS results.    Based on our
discussion with our instructors, we will test those candidate proteins by building gene knock-out cells
to check if they possess the ability to affect the transportation of α2A-AR-CT to the
membrane.    In the future, we will try to develop more effective drugs targeted for
Alzheimer's disease by targeting the alpha2A-adrenergic receptor in collaboration with pharmaceutical
companies.

Reference

Protein purification (2023) Wikipedia. Available at:

https://en.wikipedia.org/wiki/Protein\_purification (Accessed: 14 September 2023).

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![](https://static.igem.wiki/teams/4875/wiki/experiments.png)

CONTENT

Ø Culture
Mediums preparation

Ø PCR

Ø Plasmid
Extraction

Ø& E.
coli Transformation

ØAgarose
gel Electrophoresis

Ø Enzyme
digestion

Ø Enzyme
ligation

Ø Protein
Purification and Concentration

Ø SDS-PAGE

Ø Function
Test: BRET

*\* Samples were sent to SubCat for IP-MS test and analysis thus no
IP-MS procedures will be listed below.*

2）Apparatus

**Preparatory
Work**

**Configuring LB
Solid Medium**

|  |  |
| --- | --- |
|  | Name |
| 1 | 250mL Erlenmeyer Flask |
| 2 | Drug Spoon |
| 3 | Weighing Paper |
| 4 | Electronic Weight |
| 5 | Petri Dish |
| 6 | Sealing Film |
| 7 | 500mL Measuring Cylinder |

**Culturing****Escherichia
Coli**

|  |  |
| --- | --- |
| 1 | Single Channel Pipette |
| 2 | Pipette Tips |
| 3 | Centrifugal Tube |
| 4 | Incubator Shaker Series |

**Centrifuging E.coli in LB Broth**

|  |  |
| --- | --- |
| **1** | Micro Centrifuge |
| **2** | Micro centrifuge tubes |
| **3** | Incubator shaker series |
| **4** | Purification column |
| **5** | Outer thimble |
| **6** | Pipette tips |
| **7** | Single Channel pipette |

 **Plasmid
Construction**

**Plasmid Enzyme
digestion**

|  |  |
| --- | --- |
| 1 | Micro centrifuge tube |
| 2 | Single channel Pipette |
| 3 | Pipette tips |
| 4 | Ice tube |
| 5 | Thermostat Water Bath Cauldron |

**Agarose gel
electrophoresis and recycling**

|  |  |
| --- | --- |
| 1 | Centrifuge tube |
| 2 | Erlenmeyer flask |
| 3 | 20/200μL Single channel Pipette |
| 4 | Pipette tips |
| 5 | Microwave Oven |
| 6 | Electronic Balance |
| 7 | Electrophoresis tank |
| 8 | Comb |
| 9 | High channel level electrophoresis apparatus |
| 10 | Imagine System |
| 11 | Eppendorf Micro Test Tubes |
| 12 | UV Imaging System |
| 13 | Scalpel |

 **action**

|  |  |
| --- | --- |
| 1 | Micro Centrifuge Tube |
| 2 | DNA Purification Column |
| 3 | 14mL Polypropylene Round-Bottom Tube |
| 4 | Centrifuge |
| 5 | 200µL/1mL Single Channel Pipette |
| 6 | Pipette Tips |
| 7 | Vortex Oscillator |
| 8 | NANODROP 2000 |

**Enzymatic
digestion**

|  |  |
| --- | --- |
| 1 | Centrifuge |
| 2 | Pipette tips |
| 3 | 1mL/200μL/2μL Single Channel Pipette |
| 4 | NANODROP 2000 |
| 5 | Thermostat Water Bath Cauldron |
| 6 | Electrophoresis System |
| 7 | Ice Tube |
| 8 | Eppendorf Micro Test Tubes |
| 9 | Scalpel |
| 10 | UV Transilluminator |

**Colony
PCR**

|  |  |
| --- | --- |
| 1 | Thermal Cycler |
| 2 | Benchtop |
| 3 | Single channel pipette |
| 4 | Pipette tips |
| 5 | Micro centrifuge tube |
| 6 | Ice Tube |
| 7 | Benchtop High Speed Refrigerated Centrifuge |
| 8 | Culture Dish |

**BRET**

|  |  |
| --- | --- |
| 1 | Single Channel Pipette |
| 2 | Pipette Tips |
| 3 | Micro Centrifuge Tubes |
| 4 | 6 Well Cell Culture Plates |
| 5 | Microplate Reader |
| 6 | ELISA Microwell Strip Plate, 96-Well, black |

**Product
acquisition**

**Cell
transfection**

|  |  |
| --- | --- |
| 1 | Fluorescent Inverted Microscope |
| 2 | Electrothermal Thermostatic Bath |
| 3 | Air-jacketed CO2 Incubator Panasonic |
| 4 | Cell Counters |
| 5 | Benchtop |

**P****rotein
purification**

|  |  |
| --- | --- |
| 1 | Single Channel Pipette |
| 2 | Pipette Tips |
| 3 | Centrifuge Tubes |
| 4 | Small Vertical Electrophoresis Tank |
| 5 | Comb |
| 6 | Glass Pane |
| 7 | Glue Mold |

3）Procedure

**Preparatory
Work**

**Preparation of LB agar
Plate**

1. Weigh
2.5g LB broth and 1.5g agar powder add to a 250mL Flask.

2. Measure
out approximately 100 mL of deionized double distilled water(ddH2O) and add to Flask.

3. Use
a sealing film to seal the Erlenmeyer flask

4. Mix
until powder is dissolved.

5. Label
the date, sample name, and group number on the flask and then Autoclave sterilization at 121℃.

6. Add
antibiotic to the culture medium and mix well in the aseptic laboratory benchtop

7. Pour
the culture medium into the Petri dish in the aseptic laboratory benchtop.

**Culturing
Escherichia Coli (E. coli)**

1. Prepare
LB liquid culture medium.

2. add
Venus-K-Ras plasmid (Kan+),
 GST-α2A-AR-CT plasmid(Amp+),
 α2A-AR-Rluc8 plasmid(Amp+）to
different tubes with LB liquid medium

3. Mark
the name of different tubes

4. Put
these tubes into in incubator shaker series overnight

**Plasmid
Construction**

**PCR****preparation**

(1)
PCR system

|  |  |
| --- | --- |
| PCR systems | |
| Reagent | Volume（μL） |
| 2× PCR Mix | 25 |
| Primer F (10 μM) | 2.5 |
| Primer R (10 μM) | 2.5 |
| DNA template | 2 |
| ddH2O | 18 |
| Total | 50 |

(2) PCR Program :

1.
95 ℃ 3 min;

2.
95 ℃ 30 s

3.
56 ℃ 30 s

4.
72 ℃ 2 min; 2-4 for 30 cycles

5.
72 ℃ 5 min

**Plasmid Enzyme
digestion**

1. Transfer
the plasmid solution into new centrifuge tubes, 30 μL each.

2. Add
10 μl water into each new centrifuge tube.

3. Add
5 μl green buffer into each new centrifuge tube.

4. Add
2.5 μl fast digest Xba1 enzyme into each new centrifuge tube.

5. Add
2.5 μl fast digest Sba1enzyme into each centrifuge tube.

Mix
these samples and put the tube with the mixture into the 37°C water bath for 1.5h.

**Purification
target DNA**

1. Weigh
1% Agarose, 0.5g on a scale.

2. Add
the Agarose into a flask.

3. Weigh
50mL of TAE buffer using a graduated cylinder

4. Add
weighted TAE buffer into the flask.

5. Fully
mix the TAE Agarose solution.

6. Place
the flask into a microwave and heat the solution until Agarose fully dissolves.

7. Place
separating comb on the gel tray.

8. Pour
the mixture into a gel tray.

9. Add
5 μL of YeaRed nucleic acid gel stain using a pipette.

10. Wait
for the gel mixture to solidify.

11. Carefully
remove the gel from gel tray and place it into an electrophoresis apparatus.

12. Add
TAE buffer until the solution fully covers the gel.

13.  Load
previous PCR sample using a pipette into the sample holes created by the separating comb.

14. Electrify
solution with a negative charge on the left and a positive charge on the right to make the solution flow
from left to right. 140V 40min

**Agarose gel
electrophoresis and Gel Extraction**

1. Put
the Dual Sided Comb on the gel board

2. Weigh
and add 0.25g of agarose into the flask

3. Add
25mL of 1×TAE into the flask

4. Heat
the mixture with a microwave oven to let the agarose completely dissolved in the TAE

5. Add
2.5μL of YeaRed into the flask

6. Pour
the dyed solution onto the gel tray and cool it

7. Take
the dual sided comb out of the gel tray

8. Take
the casting tray out of the gel tray

9. Put
the tray on the side that is near to the cathode of the gel box

10. Add
all of the 3 samples into each corresponding groove of the gel

11. Add
5-10μL of marker into one of the grooves of the gel

12. Close
the gel box and start the electrophoresis with 160V for 30 minutes

13. Take
the gel out and put it under the imaging system

14. Identify
each DNA strand after the gel electrophoresis

15. Excise
the DNA fragment from the agarose gel with a clean, sharp scalpel

16. Put
each gel slice into the centrifuge tube and add 500μL Buffer B2 to each tube

17. Incubate
at 50°C for 10 min

18. Transfer
the binding mix into the miniprep column

19. Centrifuge
the miniprep column at 8000rpm for 30 seconds and then discard flow-through

20. Add
500μL of wash buffer to the miniprep column and centrifuge at 9000rpm for 30 seconds and discard
flow-through

21. Repeat
step 20

22. Place
the miniprep column back into the same tube and centrifuge for 1 minute at 9000rpm, then place miniprep
column into a clean 1.5 ml microcentrifuge tube

23. Add
15-40μL of EB to the center of column membrane and let the column stand for 1 min in room temperature,
and then centrifuge for 1 min.

**Gel DNA
Purification Kit and enzyme digestion**

1. Weigh
the target gel

2. Add
3-6× buffer
B2

3. Water
bath the solution at 50℃ until the gel is completely dissolved

4. Combine
miniprep column and collection tube and transfer the binding mix to the column

5. Place
the tubes into a centrifuge (12000rpm 28℃, 30sec)

6. Pour
out the discard solution and add 500μL wash solution

7. Place
the tubes into a centrifuge (12000rpm 28℃, 30sec)

8. Repeat
steps 6-7

9. Pour
out the discard solution and put it in the centrifuge (1200rpm, 28℃, 2mins) without adding anything

10. Change
a collection tube and add in 30μL ddH2O

11. put
it in the centrifuge (1200rpm, 28 degrees, 2mins)

12. Collect
the solution in the collection tube and add it back to the column

13. Repeat
step 11

14. Label
the solution in the tube

15. Measure
the concentration of the DNA solution

16. Use
an ice tub to contain the enzymes

Add
enzyme, buffer solution\*2, H2O into the PCR solution

**T4 ligase link target gene
and vector and junction product transformation**

1. Add
1 µL T4 DNA Ligase Buffer, 3µL
XbaI-SdaI α2A-AR-Rluc8, 5µL XbaI-SdaI α2A-AR-Rluc8-vector, 1µL T4 DNA Lisase to
the PCR tube and mix well.

2. Put
the sample into the PCR instruments and set up in 25℃ for
1 hour

3. Add
10µL mixture of T4 carrier and purpose gene to DH5α competent cell and put on ice for 25min

4. Add
1µL GST- α2A-AR-CT plasmid to BL21(DE3) chemical competent cell and put on ice for 25min

5. Heat
shock 90 seconds at 42 °C in H2O
bath.

6. Put
these samples on ice for 2min

7. Add
700µL LB without resistance into the sample

8. Put
these sample into water-soluble pot as 37°C for 30min

**Transformation of E. coli by
heat shock and spread plate cultivation**

1.
Take
50μl each of BL21(DE3) and DH5α and place each of them into two test tubes.

2.
Add
1μl of GST- α2A-AR-CT Plasmid to BL21(DE3) competent cells and 10μl of ligation product to
DH5α.

**Colony PCR**

1. Divide
the solution into 10 equal parts and replace them in 10 separate tubes.

2. Using
a pippete tip, move parts of the bacteria colony into centrifugal tubes （in
a benchtop）

3. Put
the tubes into the thermal cycler for 95℃ for
3 minutes. 95℃ for
30 seconds,55℃ for
30 seconds, and 72℃ for
2 minutes. This prodecdure should be repeated for 32 times.

4. Place
the tubes at 72℃ for
5 minutes and store them at 12℃.

**Plasmid
Extraction**

1. Add
500µl of Buffer S into each of the 4 collection tubes with Miniprep columns

2. Centrifuge
these tubes for 30 seconds at 12000×g

3. Add
250µl of buffer SP1 into the tubes

4. Make
the bacteria in the tubes floating using a shaker

5. Move
the bacteria into the collection tubes.

6. Add
250µl of buffer SP2 into the collection tubes

7. Add
350µl of buffer SP3 into the collection tubes and
invert immediately but gently 4–6 times.

8. Centrifuge
these tubes for 10minutes at 8000×g

9. Transfer
the cleared lysates to the new collection tubes

10. Centrifuge
these tubes for 30 seconds at 8000×g

11. Discard
flow-through

12. Add
500µl Buffer DW1 into each of the 4 collection tubes

13. Centrifuge
these tubes for 30 seconds at 9000×g

14. Add
500µl wash solutions into each of the 4 collection tubes

15. Centrifuge
these tubes for 30 seconds at 9000×g

16. Discard
flow-through

17. Add
500µl wash solutions into each of the 4 collection tubes

18. Centrifuge
these tubes for 60seconds at 9000×g

19. Discard
flow-through

20. Move
the column into a centrifuge tube

21. Add
35-50µl of Elution buffer into the tube, centrifuge for 1 min.

**Protein
Purification**

**Induction of
bacteria and Protein induced expression**

1.  Transform
the α2A-AR-CT plasmid into E. coli BL21(DE3)

2. Inoculate
the strains into 5mL LB medium, incubate overnight

3. Transfer
0.5 mL of bacterial solution into fresh LB medium (100 mL)

4. incubate
until OD600=0.5

5. Add
IPTG (1 mM) and incubate 37 ℃, 220 rpm overnight

**Protein
purification**

1. Centrifuge
and collect the bacteria at 4,000 rpm for 5 minutes, discard the supernatant.

2. Mix
the strain with 4ml lysis buffer and then Ultrasonically lysed bacteria on ice (500W, 3s/3s, 10
min).

3. Centrifuge
at 10,000 rpm at 4℃ for
20 min, and collect the supernatant.

4. Add
1 mL mixed well GST-tag purification resin, centrifuge at 1000g for 1min, discard the supernatant. Mix the
resin with 0.5 mL lysis buffer and transfer the mixture to the column.

5. Add
the supernatant to the column and flow through, repeat for 5 times.

6. Add
1 mL lysis buffer to the column and collect the fluid. Repeat for 4-5 times.

7. Collect
the sample for SDS-PAGE.

**SDS-PAGE**

1. Mix
the protein solution with 6× loading buffer

2. Place
it in the 100℃-water bath for 5 minutes

3. Extract
20 μL of the product for SDS-PAGE electrophoresis

4. Stain
the gel using Coomassie Blue Staining Solution, then destain it using Destaining Solution
overnight.

5. Blots
were imaged using an LI-COR Biosciences Odyssey imager.

**Product
acquisition**

**HEK293****Cell culture and
transfection**

1.Culture
the HEK293 cell in DMEM culture medium at 37℃ 5% CO2 overnight.

2. Change
the DMEM culture medium used to cultivate HEK293 cell into each of the six platforms culture plate

3. add
150µL of DMEM, Str
KDEL\_ SBP-α2A-AR-Rluc8 plasmid 250ng, Venus-K-Ras plasmid 750ng ,
and 3µL of PEI into each of the 6 centrifuge tubes

4. pipette
up and down to mix solution

5. Put
the 6 centrifuge tubes into an incubator for10 minutes

6. Transfer
the solution in each centrifuge tube to its corresponding platform culture plate

**Cell passage**

1. Aspirate
cell media from culture dish.

2. Wash
dish with 5mL PBS

3. Aspirate
PBS

4. Add
2mL of Trypsin-EDTA to digest the cells

5. Add
6mL of DMEM in order to neutralize typsin-EDTA activity

6. Count
cell number, then compute the volume of the DMEM we need to add.

7. Add
13.3mL DMEM into the petri dish to dilute the cell liquid

8. Add
3mL of the diluted liquid into each cell in the six-well cell culture plate

9. Place
the six-well cell culture plates in the incubator at least 16 hours

**α2A-AR RUSH-BRET**

1. Add
40µL of Biotin to each well and incubate for 0, 15, 30, 1, and 3 hours. The group without biotin for
0 minutes was used as the control group.

2. Discard
the culture medium, wash the cells twice with DPBS, add 1ml of DPBS to resuspend cells.

3. Remove
a 200 μL sample of suspension to three wells in a black opaque 96-well plate and wait for 15min.

4. Add
2µL Coelenterazine H to these 3 wells.

5. Put
the plate into the ELISA Microplate Reader and record the value.

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![](https://static.igem.wiki/teams/4875/wiki/results.png)

Overview

Results

Future plans

Reference

Overview

Evidence
has shown a distinct correlation between aging and Alzheimer’s Disease (AD); however, age is not the
only trigger of AD. Scientists have narrowed down and discovered a connection between Alzheimer’s
Disease and an adrenergic receptor named Alpha2a-AR (Sun
L,
et al, 2010). Our results mainly consist
of two parts.

The first part is to construct
GST-α2A-AR-CT plasmid and
Str-KDEL\_SBP-α2A-AR-Rluc8 plasmid, and express GST-α2A-AR-CT protein in
E.coli
BL21. The second part is to transfect the
Str-KDEL\_SBP-α2A-AR-Rluc8 plasmid into HEK293 cell line to express Alpha2a-AR and
construct RUSH-BRET
platform to monitor movement α2A-AR between plasma membrane and endoplasmic reticulum.

Results of your work

**Target1:****P****lasmid****s****construction**

· GST-α2A-AR-CT plasmid
construction

In
this part of the experiment, we used the following primers to amplify α2A-AR fragment
(TIFNHDFRRAFKKILCRGDRKRIV) from α2A-AR-GFP and inserted into the vectors pGEX-4T-1 to obtain
GST-α2A-AR-CT plasmid.

GST-α2A-AR-CT-Forward:
5’-GATCGGATCCACCATCTTCAACCACG-3’

GST-α2A-AR-CT-Reverse:
5’-GATCCTCGAGTCACACGATCCGCTTCC-3’

· Str-KDEL\_SBP-α2A-AR-Rluc8
plasmid construction

First,
using the human α2A-AR-Rluc8 plasmid as a template, point mutation at Sda1 to eliminate Sda1 enzyme
restriction site in order to get mutant α2A-AR-Rluc8 plasmid. The primer is as follows:

α2A-Sda1
mutant-F: 5’-CACCCCTTACTCCCTACAAGTGACGCTGACGCTG-3’

α2A-Sda1
mutant-R: 5’-CAGCGTCAGCGTCACTTGTAGGGAGTAAGGGGTG-3’

Second,
mutant α2A-AR-Rluc8 plasmid as a template to amplify target sequence by using the following primers.
Gel DNA Extraction purified 2106 bp product.

KDEL-SBP-2A-Rluc8-F:5'-GATCCCTGCAGGTATGGGCTCCCTGCAGCCG-3'

KDEL-SBP-2A-Rluc8-R:5'-GATCTAGATTACTGCTCGTTCTCAGCACGCG-3'

Third,
The plasmid Str-KDEL\_SBP-EGFP-Ecadherin (purchased from Addgene) was double digested with Sda1 and Xba1
and purified the product at 5234 bp to obtain the Str-KDEL\_SBP vector. we inserted the α2A-AR-Rluc8
fragment to Str-KDEL\_SBP vector to obtain Str-KDEL\_SBP-α2A-AR-Rluc8 plasmid.

To
confirm whether our experiment is successful. We ran the gel electrophoresis. We use this method to obtain
relatively pure bands of target genes. According to the gel below, we can see that the strip of
α2A-AR-Rluc8 is located at 2322 bp around and the strip of
Str-KDEL\_SBP-EGFP-Ecadherin is located at 5210 bp around. In that way, we could basically confirm that
we have obtained the expected plamids then we can recycle the strips for following
experiments.

![](https://static.igem.wiki/teams/4875/wiki/result/1.png)

**Figure1.****Gel
electrophoresis****results of colony
PCR****. 1,2: α2A-AR-Rluc8;
3,4: Str-KDEL\_SBP-α2A-AR-Rluc8; M:****Marker****-****15K**

· Colony
PCR

In
the past 12 days, we have inoculated 17 media regarding colony PCR. By using agarose gel electrophoresis
and contrasting with marker, yes, the first and last one with a letter M is our marker. So, after we test
the existence of the DH5alpha which is a kind of E. coli transfected by special plasmid, we found that
only three results NO.7, 10, and 11 are in line with our expectations (700
bp around). In
this way, we would say these three ones are our target
strips.

![](https://static.igem.wiki/teams/4875/wiki/result/2.png)

**Figure2.****Gel electrophoresis
results of colony PCR****: Marker-15K; 1,19,20-Negative control; 2-18-Clony PCR samples
and 7,10,11 are confirmed positive**

· Detecting
base pairs of target plasmid

Finding
the base pairs of our target DNA is
shown
below:

DNA
ligation, transfer DH5a, colony PCR, choose numbered 71011. Inoculation of LB. plasmid extraction. Picked
1 of the three for Sanger sequencing. The three products were sent to a company for Sanger sequencing and
we got the following alignment and base pairs which
confirmed that we have obtained the expected plasmid.

![](https://static.igem.wiki/teams/4875/wiki/result/3-1.png)
![](https://static.igem.wiki/teams/4875/wiki/result-update01.png)

**Figure3.****Global sequence
alignment of sequencing**

**Target2: Expression and purification of GST-α2A-AR-CT protein**

· Expression

The
next part of our experiments is to transform the GST-α2A-AR-CT plasmid into E.coli BL21 which is
commonly used in plasmids transformation, and then to verify the protein expression in it.

· Purification

We
ran a PAGE gel of the precipitate, cell lysate, flow through, wash and elution to test proteins. Our
target proteins GST-α2A-AR-CT
molecular weight is in the range of 25 kDa. The Figure 4 showed that GST-α2A-AR-CT protein is found
on column E, marker 25, indicating GST-α2A-AR-CT is purified from E.coli BL21. At the end of the
experiment, we completely captured the pure GST-α2A-AR-CT protein by purification, and we compared
it with the sample from each process during purification by using protein gel electrophoresis. After the
electrophoresis process our target protein now is pure and we finally identified that we got the right protein.

![](https://static.igem.wiki/teams/4875/wiki/result/4.png)

**Figure4.****SDS-PAGE analysis of
GST-α-AR-CT**

**Target3:
IP-MS to screen for candidate****proteins**

Through
the GO data, we can know that our target gene is enriched at
the
biological process level, with the highest number of genes enriched in translation, and our differential
genes are most significantly enriched in this regulation of translation. Through the KEGG data, we can
know that the number of genes enriched on Ribosome is the highest, and our differential genes are most
significantly enriched in
Thermogenesis.

Based
on the IP-MS results
in figure 5 and 6,
we could
further find our candidate proteins that have the potential ability to affect the α-AR-CT
transportation to the membrane and the detailed result could be found in this [***EXCEL***](https://static.igem.wiki/teams/4875/wiki/result/proteingroups.pdf).

IP-MS was
completed by Professor Shen's
research group at Nanjing University of Traditional Chinese Medicine, providing IP-MS testing service
and
data analysis service.

![](https://static.igem.wiki/teams/4875/wiki/result/5.png)

**Figure5.****KEGG Pathways for
Significantly Expressed Proteins**

![](https://static.igem.wiki/teams/4875/wiki/result/6.png)

**Figure6.****GOBP for
Significantly Expressed Proteins**

**Target****4****:****RUSH-BRET
platform establishment in host cell lines**

We
established
the
RUSH-BRET platform in host cell lines. Since
the BRET instrument (Spark® Cyto PLATE READER WITH LIVE CELL IMAGING AND REAL-TIME CYTOMETRY) is only
allowed for trained users, our BRET was completed by SubCat Academy in ATLATL lab and the
measurement wavelength is 535nm/485nm.

The
raw data can be found in Table 1. We
select the
V/R data
from Table 1 and
 calculate
the average and difference values to create a fluorescence intensity of RUSH-BRET in different
time in
Table 2.

**Table1. Fluorescence
intensity of RUSH-BRET assays**

![](https://static.igem.wiki/teams/4875/wiki/result/7.png)

**Table2. Fluorescence
intensity of RUSH-BRET in different time**

![](https://static.igem.wiki/teams/4875/wiki/result/table1.png)

The
entire curve is smooth and the error bar is small, which is consistent with our ideal expectations. These
increasing trends mean that the Str-KDEL\_SBP-α2A-AR-Rluc8 structure has been disturbed by the biotin
and α2A-AR-Rluc8 move from the endoplasmic reticulum to cell membrane. Rluc8 of SBP-α2AR -Rluc8
replace the RAS of the Venus-K-RAS on the
cell membrane and produced
BRET signal that
represents the amount of the protein α2A-AR on the membrane.

![](https://static.igem.wiki/teams/4875/wiki/result/table2.png)

**Figure7.****The surface expression of
α2A-AR was measured by RUSH-based BRET assays**

Future plans/Further Discussion

There
are a wide variety of medications available for the treatment of Alzheimer's disease. Most of these drugs
only have a blocking effect on a certain part of AD and do not block the course of AD, which is why these
drugs can improve symptoms in early and middle stages of AD, but are less effective in late stages of AD.
Therefore, it is more important to find multi-target drugs that can block the progression of AD.

Since
we have made some progress in our RUSH-BRET system establishment and basic function test.  Next move
we will design more inquiry experiments to evaluate the system and evaluate the candidate proteins we
targeted from IP-MS results.  Based on our discussion with our instructors, we will test those
candidate proteins by building gene knock-out cells to check if they possess the ability to affect the
transportation of α2A-AR-CT to the membrane.  In the future, we will try to develop more
effective drugs targeted for Alzheimer's disease by targeting the alpha2A-adrenergic receptor in
collaboration with pharmaceutical companies.

Reference

 [1] Sun
Lei, Yang Ying, Dou Caiyan. Research progress in the treatment of Alzheimer's disease [J]. Medical
Information (Junten-Journal),2010,5(07):1969-1970.

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