iGEM 2021 — Team Shanghai_United

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# Team:Shanghai United/Notebook

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Notebook

![c6e6151cedecf75dbdbc9b3e079800c](https://static.igem.org/mediawiki/2021/0/07/T--Shanghai_United--Notebook01.jpg)

Jul.
28, 2021

1. Learnt and clarified research goals and
   methods as a team. Learnt safety precautions necessary for entering the laboratory.
2. Learnt and practiced the use of
   micropipettes.
3. Performed PCR of amilGFP gene; performed gel
   electrophoresis of said PCR products and tested for results.
4. Researched and learnt the reaction
   mechanisms of PCR and the preparation protocol of LB cultivation medium.
5. Used LB cultivation medium with KANA
   antibiotics to cultivate three E. coli cell lines that are KANA resistant and with
   ArsA, ArsD, and ArsR genes inserted, respectively.

Jul.
29, 2021

1. Performed plasmid extraction of three
   E.
   coli cell lines, cultivated
   since Jul. 28. Said bacteria have KANA resistance
   and ArsA, ArsD, or ArsR depending on the cell line. Extracted plasmids are tested for and yielded
   desirable concentrations.
2. Cleaned up amilGFP PCR products,
   obtained in Jul. 28. Cleaned up PCR products are tested
   for and yielded desirable concentrations.
3. Using restriction enzyme BsaI, digested
   ArsA, ArsD, and ArsR plasmids. Reaction mix is left overnight to react.
4. Using restriction enzymes BsaI and DpnI,
   digested cleaned up amilGFP PCR products. Reaction mix is left overnight to react.

Jul.
30, 2021

1. Harvested restriction enzyme digestion
   products of ArsA, ArsD, and ArsR plasmids as well as amilGFP PCR products; digestion was
   conducted in Jul. 29.
2. Performed gel electrophoresis on restriction
   enzyme digestion products of ArsA, ArsD, ArsR, and amilGFP genes.
3. Performed agarose gel DNA extraction of gel
   containing restriction enzyme digestion products. Extracted ArsA, ArsD, ArsR, and amilGFP genes are
   tested for and yielded suboptimal concentrations.
4. Performed T4 DNA ligase ligation of Ars
   genes and amilGFP genes.
5. Using ligation products of Ars genes and
   amilGFP genes, transformed DH5-α E. coli by heat shock method.
6. Prepared LB cultivation medium agar plates
   with 0.1% KANA antibiotics.
7. Spread Ars/amilGFP E. coli onto KANA antibiotics agar plates.

Jul.
31, 2021

![](https://static.igem.org/mediawiki/2021/0/08/T--Shanghai_United--Notebook02.jpg)

Gel
Electrophoresis Results of amilGFP PCR

![](https://static.igem.org/mediawiki/2021/7/76/T--Shanghai_United--Notebook03.jpg)

Gel
Electrophoresis Results of Colony PCR

1. Examined growths of Ars/amilGFP E. coli, cultivated since Jul. 30. Results were suboptimal: only 4
   colonies were found in a total of 6 plates.
2. Performed colony PCR and gel electrophoresis
   to test for Ars/amilGFP genes in observed E. coli colonies. Only one colony resulted to be
   ArsD/amilGFP positive, indicating failure of experimental procedure.
3. Performed amilGFP restriction enzyme
   digestion on cleaned up amilGFP PCR products, obtained in Jul. 29, to prepare for first retry of
   experimental procedure.
4. Performed PCR of amilGFP gene to prepare for
   second retry of experimental procedure. Performed electrophoresis to test for amilGFP PCR products.
5. Performed gel electrophoresis to test for
   amilGFP restriction enzyme digestion products. Performed agarose gel DNA extraction for digested
   amilGFP genes.
6. Cleaned up amilGFP PCR products.
7. Performed T4 DNA ligase ligation of digested
   amilGFP genes and digested Ars genes, obtained in Jul. 30.
8. Cultivated three cell lines of E. coli with KANA resistance and ArsA, ArsD, or ArsR
   genes inserted, depending on the cell line, to prepare for second retry of experimental procedure.
9. Using ligation products of Ars genes and
   amilGFP genes, transformed DH5-α E. coli by heat shock method.
10. Spread Ars/amilGFP E. coli onto KANA antibiotics agar plates, prepared
    in Jul. 30.

Aug.
1, 2021

1. Examined growths of Ars/amilGFP E. coli, cultivated since Jul. 3[1](h). Results were optima[l:
   colonies were observed on all six plates](h).
2. Performed colony PCR and gel electrophoresis
   to test for Ars/amilGFP genes in observed E. coli colonies. All selected colonies yielded
   positive results of Ars genes.
3. Extracted ArsA, ArsD, and ArsR plasmids from
   Ars E.
   coli, cultivated Jul. 31. Because
   the first retry of experimental procedure yielded positive results, the second retry had been
   terminated.
4. Cultivated Ars/amilGFP positive E. coli.

Aug.
2, 2021

1. Preserved Ars/amilGFP positive E. coli for future studies.
2. Conducted function test of Ars/amilGFP
   positive E.
   coli with different
   concentrations of NaH2PO4 solutions.
3. Mailed Ars/amilGFP positive E. coli samples to sequencing companies to perform
   Sanger sequencing of Ars/amilGFP genes.
4. Started construction of team Wikipedia
   reports.

Aug.
3, 2021

1. Examined function test results, conducted
   in Aug. 2. No green fluorescence was found in all
   tubes.
2. Conducted second function test of
   Ars/amilGFP positive E. coli with higher concentrations of NaH2PO4.
3. Continued construction of team Wikipedia.
4. Created product design information and user
   manual sheet for dry team for reference.

Aug.
4, 2021

![d3ee87f2c64666dd414a0897fc4172d](https://static.igem.org/mediawiki/2021/f/fe/T--Shanghai_United--Notebook04.jpg)

1. Examined second function test results,
   conducted in Aug. 3. No green fluorescence was found in
   all tubes, indicating the inability of Ars/amilGFP E. coli to detect phosphate.
2. Analyzed results of Ars/amilGFP gene
   sequencing results, sequenced in
   Aug. 2. Constructed Ars/amilGFP genes
   are correct.
3. Conducted third function test of Ars/amilGFP
   positive E.
   coli with different
   concentrations of C2H6AsNaO2.

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# Team:Shanghai United/Engineering

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Engineering

Introduction

Arsenic is widely distributed in the natural environment.
Trace amounts of arsenic can be detected in soil, water, minerals, and plants. Among many environmental
pollutants, arsenic ranks first on the blacklist of polluting toxic elements and is also a powerful carcinogen.
In many countries, arsenic causes water, soil and crop pollution. We design an arsenic-induced response system
in the cell or on the surface of the engineered probiotic bacteria to detect the content of arsenite in the
environment, provide warnings to people, and reduce the health damage it causes to the human body.

Design

Three proteins ArsR, ArsD, and ArsA belong to the operon
family. The cysteine thiolates on their amino acid sequences can form a three-coordinate complex with arsenic
(iii), which is self-sensing to arsenic.

Under the induction of arsenite, ArsD gene is transcript
and enxpressed in E.
coli cells. At the same time, a reporter gene
GFP is connected to the ArsD gene. Then, when the concentration of arsenite in the environment, the expression
of ArsD-GFP can be detected by observing the intensity of the fluorescent signal.

Build

Three recombinant plasmids pUC57-ArsD-amilGFP (Figure 1),
pUC57-ArsR-amilGFP, and pUC57-ArsA-amilGFP were constructed in order to select the most sensitive sensor to
arsenic. All the constructs are confirmed by DNA sequencing.

![](https://static.igem.org/mediawiki/2021/4/4e/T--Shanghai_United--Engineering01.jpg)

Figure 1. Schematic map of plasmids.

Test

Fluorescence intensity generated by an ARSD/amilGFP
transformed E. coli reacting for 1 hour in C2H6AsNaO5 solutions. As seen in the figure, fluorescence was
detected for all C2H6AsNaO5 concentrations that are above 0, which confirms that the
designed plasmid worked as intended (Figure 2).

![](https://static.igem.org/mediawiki/2021/7/74/T--Shanghai_United--Engineering02.jpg)

Figure 2: Fluorescence Intensity of Transformed E. coli In
Different Concentrations of C2H6AsNaO5 Solutions

Learn

C2H6AsNaO5 was used for this function test. The observed
fluorescence intensity suggested that the engineered bacteria are functional. However, the fluorescence was
rather minor, so we speculate that E. coli responds poorly to organic arsenic compounds. Further
experiments would be conducted to test for fluorescence intensities of E. coli in inorganic arsenic solutions.

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# Team:Shanghai United/Experiments

Document

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Experiments

Synthetic Biology Protocols

General Protocols

Function Test

# PCR of the target Gene

A.      Materials

|  |  |
| --- | --- |
| ddH2O | 32 μL |
| dNTP | 5 μL |
| MgSO4 | 4 μL |
| Forward primer | 1 μL |
| Reverse primer | 1 μL |
| DNA template | 1 μL |
| KOD+ buffer | 5 μL |
| KOD+ | 1 μL |

B.      Protocol

1.       Prepare and mark PCR tubes and align tubes in order on tube rack.

2.       Under normal circumstances,
ddH2O should be added into tubes
first, and
enzymes should be
added last. All other materials
uncluding, but are not limited to dNTP, primers, buffers, and templates should be added in between.

3.       PCR tubes should then be centrifuged briefly to
ensure all liquids collect at the bottom of the tube.

4.       Move PCR tubes to PCR machine to initiate the process.

C.      Reacting
conditions

|  |  |  |
| --- | --- | --- |
| 98 °C | 10 minutes | 1 cycle |
| 98 °C | 15 seconds | 26 cycles |
| 55 °C | 15 seconds |
| 68 °C | 1 minute |
| 68 °C | 10 minutes | 1 cycle |
| 4 °C | Infinite | N/A |

# Gel Electrophoresis of the Gene PCR Product A. Materials

|  |  |
| --- | --- |
| Loading buffer & DNA dye | 1 μL |
| PCR product | 5 μL |
| TAE buffer | 200 mL |
| Agarose | 2 g |

B.      Protocol

1.       Mix TAE buffer and agarose
powder in a ratio of 100 mL TAE buffer : 1 g agarose powder.

2.       Heat the mixed solution in a microwave until
agarose powder completely dissolves into TAE buffer.

3.       Pour the liquid into gel trays of suitable sizes. Insert a comb onto the tray to

4.       Mix 6x loading buffer premixed with DNA dye with PCR product in a ratio of 1 unit loading buffer : 5 units PCR product.

5.       Load proper ladder into first well of gel.

6.       Load samples into wells.

7.       Perform electrophoresis at 185 V for 10 minutes.

8.       Harvest gel, take image of gel under untraviolet light, and analyze results.

# PCR Product Clean Up

A.      Protocol
(MN
NucleoSpin
Gel
and
PCR
Clean-up
Kit)

1.       Combine obtained PCR products into one centrifuge tube.

2.       Add buffer NTI to the product in a ratio of 2 μL buffer NTI : 1 μL PCR product to allow DNA to bind to the membrane.

3.       Transfer the solution to a column placed within a collection tube. Centrifuge

at 12000 rpm for 30 seconds.

4.       Transfer the liquid in the collection tube to the column. Centrifuge at 12000 rpm for 30 seconds.

5.       Dispose of the liquid in the collection tube. Add 700 μL buffer NT3 to wash the membrane. Centrifuge at 12000 rpm for 30 seconds.

6.       Repeat step 5.

7.       Add
30
μL
ddH2O
preheated
to
65
˚C
to
the
column.
Centrifuge
at
12000
rpm
for 30 seconds.

B.      Buffers

Buffer NTI: Adjust DNA binding conditions and allow DNA to bind to membrane.

Buffer NT3: Wash membrane.

# Plasmid Extraction

A.      Protocol
(TIAN
Prep
Mini
Plasmid
Kit)

1.       Harvest and transfer cultivated bacteria into marked 2 mL centrifuge tubes.

2.       Centrifuge tubes at 12000 rpm for 30 seconds to
deposit bacteria to the bottom of the tube. Dispose of the clear liquid on top.

3.       Add 250 μL buffer P1 into the 2 mL centrifuge tubes to levitete the bacteria persent in the tubes.

4.       Add 250 μL buffer P2 to the tubes to lyse bacterial cell wall. Mix the solution gently; do not vortex.

5.       Add 350 μL buffer P3 to the tubes to neutralize buffer P2 and cause proteins in the solution to percipitate. Mix the solution gently; do not vortex.

6.       Centrifuge the solution at 12000 rpm for 10
minutes to collect percitipation to the bottom of the tube.

7.       Add 500 μL buffer BL to a column placed within a collection tube to wash the membrane. Centrifuge at 12000 rpm for 30 seconds.

8.       Transfer the clear liquid obtained in step 6 to the column washed in step 7 and centrifuge the column at 12000 rpm for 30 seconds.

9.       Dispose the clear liquid in the collection tube. Add 500 μL buffer PD to the column and centrifuge at 12000 rpm for 30 seconds.

10.   Dispose of the liquid in the collection tube. Add 600 μL buffer PW to the column. Centrifuge at 12000 rpm for 30 seconds.

11.   Repeat step 10.

12.   Dispose of the clear liquid in the collection
tube. Centrifuge at 12000 rpm for 1 minute.

13.   Transfer the column to a new 2 mL centrifiuge tube. Dispose of the collection tube.

14.   Add
40
μL
ddH2O
preheated
to
65
°C
to
the
column.
Centrifuge
at
12000
rpm
for 30 seconds.

B.      Buffers

Buffer P1: Levitate bacteria and lyse
RNA.
Buffer P2: Lyse bacteria cell wall.

Buffer P3: Neutralize P2 and cause proteins to precipitate. Buffer BL: Wash memnbrane pre-use.

Buffer PD: Wash proteins off the membrane. Buffer PW: Wash salts off the membrane.

# Restriction Enzyme Digestion

A.      Materials

|  |  |
| --- | --- |
| ARSA/ARSR/ARSD | |
| 10x NEB buffer | 5 μL |
| ARSA/D/R Plasmid (300 ng/μL) | 10 μL |
| BsaI restriction enzyme | 1 μL |
| ddH2O | 34 μL |
| amilGFP | |
| 10x NEB buffer | 5 μL |
| amilGFP (400 ng/μL) | 10 μL |
| BsaI | 1 μL |
|  |  |
| DpnI | 1 μL |
| ddH2O | 33 μL |

B.      Protocol

1.       Mix the materials in a 1.5 mL centrifuge tube.

2.       Place the centrifuge tube in 37 °C metal bath for at least 15 minutes.

# Gel Electrophoresis of Restriction Enzyme Digestion Product

A.      Materials

|  |  |
| --- | --- |
| Loading buffer with DNA dye | 10 μL |
| Digestion product | 50 μL |

B.      Protocol

1.       Mix 6x loading buffer premixed with DNA dye with PCR product in a ratio of 1 unit loading buffer : 5 units PCR product.

2.       Load proper ladder into first well of gel.

3.       Load samples into wells.

4.       Perform electrophoresis at 185 V for 10 minutes.

5.       Harvest gel, take image of gel under untraviolet light, and analyze results.

# Agarose Gel DNA Extraction

A.      Protocol
(MN
NucleoSpin
Gel
and
PCR
Clean-up
Kit)

1.       Collect gel with desired
DNA fragments into 2 mL centrifuge
tubes. Weigh
the mass of gels on an electronic balance.

2.       Add buffer NTI to the tube in a ratio of 2 μL buffer NTI : 1 mg agarose gel.

3.       Heat the mixture at 50 ˚C in a metal bath until the gel dissolves completely.

4.       Transfer the solution to a new column placed within a collection tube. Centrifuge at 12000 rpm for 30 seconds.

5.       Transfer the liquid in the collection tube to the column. Centrifuge at 12000 rpm for 30 seconds.

6.       Dispose of the clear liquid in the collection tube. Add 700 μL buffer NT3 to

the column. Centrifuge at 12000 rpm for 30 seconds.

7.       Repeat step 6.

8.       Dispose of the liquid in the collection tube.
Centrifuge at 12000 rpm for 1 minute.

9.       Add 30 μL ddH2O preheated to 65 ˚C to the column. Centrifuge at 12000 rpm for 30 seconds.

# T4 DNA Ligase Ligation

A.      Materials

|  |  |
| --- | --- |
| ARSA/R/D plasmid (30 ng/μL) | 1.5 μL |
| GFP (100 ng/μL) | 4.5 μL |
| T4 DNA ligase | 1 μL |
| T4 DNA ligase buffer | 1 μL |
| ddH2O | 2 μL |

B.      Protocol

1.       Add ddH2O, T4 DNA ligase buffer, and T4 DNA ligase into a PCR tube with GFP gene and ARS plasmid
in a ratio of 1 units ARS plasmid : 10
unit GFP
gene.

2.       Transfer the solution to PCR machine at 16˚C.

# Transformation of DH5-α E. coli Using Heat Shock

A.      Protocol

1.       Mix the tube introduced DNA material with DH5-α
competent cells. The volumn of DNA material must not exceed 10% of that of the competent cells.

2.       Gently fick the tube 4-5 times to mix competent cells and DNA material. Do not vortex.

3.       Place the tube on ice for 30 minutes.

4.       Place the tube at exactly 42 ˚C for 90 seconds to perform heat shock.

5.       Immediately place the tube back on ice after heat
shock. Keep the tube on ice for 2-3 minutes.

6.       Add 900 μL LB culture medium without KANA antibiotics to the tube. Incubate the transformed bacteria at 37 ˚C for 30 minutes.

7.       Centrifuge the tube at 12000 rpm for 30 seconds.

8.       Dispose half of the clear liquid in the centrifuge
tube. Mix and levitate the bacteria on the bottom of the tube with a micropipette.

9.        In a sterile environment, transfer the transformed bacteria to prepared agar plates.

# Colony PCR Identification

A.       Materials

|  |  |
| --- | --- |
| 2x PCR mix | 10 μL |
| Forward primer | 0.5 μL |
| Reverse primer | 0.5 μL |
| Template | Desired E. Coli |
| ddH2O | 9 μL |

B.      Protocol

1.       Refer to PCR
of
amilGFP
Gene, Jul.
28,
2021.

2.       Notes to this reaction:

a.       Primers were delivered in the form of dry powder. Centrifuge primers at 12000 rpm for 5 minutes to deposit powder at the bottom of its tube.

b.       Dilute primers to a concentration of 10 nmol/ml.

c.       Mix the 2x PCR mix, R.primer, Templalate, ddH2O as master mix.

C.      Reacting
conditions

|  |  |  |
| --- | --- | --- |
| 98 °C | 10 minutes | 1 cycle |
| 98 °C | 15 seconds | 26 cycles |
| 55 °C | 15 seconds |
| 72 °C | 30 seconds |
| 72 °C | 5 minutes | 1 cycle |
| 4 °C | Infinite | N/A |

# LB Culture Medium Preperation

1. Formula

|  |  |
| --- | --- |
| Yeast extract | 5 g/L |
| Tryptone | 10 g/L |
| NaCl | 10 g/L |
| Agar\* | 1.5% of total mass |
| ddH2O | Desired volume |

\*Agar
is
only
necessary
when LB
culture
medium
is
prepared
as
solid
agar
gel.

2. Sterilization

Sterilize
at
121
°C
for
30 minutes.

3. KANA
   antibiotics

Concentration
of
KANA
antibiotics
in
solution
is
50
mg/μL.
Functional
concentration
of
KANA
antibiotics
is
50
μg/μL.

E. coli Cultivation

A.      Protocol

1.       Clean workstation with 75% ethanol
and work near a lit alcohol lamp in steril
conditions.

2.       Prepare
LB
culture
medium
without
KANA
antibiotics.

3.       Prepare
three
lines of E. coli with KANA
resistance and
ARSA,
ARSD,
or
ARSR
plasmids depending on
the
cell line.

4.       Add
KANA
antibiotics
to
LB
culture
medium
in
a
ratio
of 1
μL
KANA
antibiotics
: 1 mL
LB
culture
medium.

5.       Prepare
three
marked 15
mL
centrifure
tubes.
Distribute
4
mL
LB
culture
medium
to
each tube.

6.       Transfer
10
μL
of
bacterial
solution
to
its
corresponding
tube.

7.       Cultivate
in a shaker
in
37 °C
at 220
rpm.

# Preparation of Agar Plates

A.      Protocol

1.       Heat the pre-prepared solid LB
culture medium in a microwave until the solid
completely
melts.

2.       Add
KANA
antibiotics
in
a
ratio of
1
μL
KANA
antibiotics
:
1000
μL
culture
medium.
Mix
the
solutions
throughly.

3.       In a
sterile
environment,
carefully pour
approximately
20-30 mL
culture
medium
to
each empty plate.

4.       Tilt
the
plate
genly to
cover
the
plate
completely
with
culture
medium.
Set
the
plates
still
and
wait for
culture medium
to
cool
and
solidify.

# Preservation of Transformed E. coli

A.       Materials

# LB Culture Medium with Phosphate Preparation

A.      Materials
(LB
culture
medium)

1. Refer
   to
   LB
   Culture
   Medium
   Preparation

B.      Materials
(phosphate
solution)

|  |  |
| --- | --- |
| Na2HPO4.12H2O | 25 mg |
| NaH2PO4.2H2O | 13 mg |
| ddH2O | 200 mL |

C.      Protocol

1. Weigh
   25
   mg
   Na2HPO4
   and
   dissolve
   in
   100
   mL
   ddH2O.
2. Weigh
   13
   mg
   NaH2PO4
   and
   dissolve
   in
   100
   mL
   ddH2O.
3. Sterilize
   solutions
   at
   121˚C
   for
   30
   minutes
   in
   a
   sterilization
   pot.

# Function Test Attempt 1

A.      Protocol

1.       Sterilize
workstation
and
work
near
a
lit
alcohol
burner.

2.       Prepare
marked
15
mL
test
tubes
for
testing.

3.       Distribute
4
mL
LB
culture
medium
without
KANA
antibiotics
to
each
test
tube.

4.       Add
40
µL
E.coli
to
its
corresponding
tube.

5.       Cultivate
at
37˚C
for
2
hours
on
a
shake
bed.

6.       Dilute
NaH2PO4
to
10
mg/L.

7.       Add
NaH2PO4
to
each
test
tube
depending
on
the
testing
concentration.

B.      NaH2PO4
concentrations

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
|  | 0 µg/L | 10 µg/L | 20 µg/L | 50 µg/L | 100 µg/L | 150 µg/L |
| NaH2PO4  (10 mg/L) | 0 µL | 4 µL | 8 µL |  |  |  |
| NaH2PO4 |  |  |  | 2 µL | 4 µL | 6 µL |
| (100 mg/L) |  |  |  |  |  |  |

# Function Test Attempt 2

A.      Protocol

1.       Harvest
3
mL
E.coli
bacterial
solution.
Centrifuge
at
12000
rpm
for
30
seconds to
deposit bacterial at the button of the tube. Dispose of the
supernatant
in
the
tube
.

2.       Prepare
new 15 mL test tubes. Add LB
culture medium depending on the
target
concentration
of
phosphate.

3.       Transfer
a
moderate
amount
ofLB culture medium
from the test tube to
the
centrifuge
tube.

4.       Levitate
the
bacteria
in
the
centrifuge
tube.
Transfer
the
bacterial
solution
back
to
test
tube.

5.       Add
phosphate
solution
to
the
test
tube,
depending
on
the
concentration.

6.       Cultivate
at
37˚C
and
220
rpm
for 2
hours.

B.      Solution
amounts

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
|  | 0 mg/L | 1 mg/L | 10 mg/L | 20 mg/L | 50 mg/L | 100 mg/L |
| NaH2PO4  (100 mg/L) | 0 µL | 40 µL | 400 µL | 800 µL | 2000 µL | 4000 µL |
| LB culture medium | 4000 µL | 3960 µL | 3600 µL | 3200 µL | 2000 µL | 0 µL |

# Function Test Attempt 3

A.      Protocol

Refer
to
Function
Test
Attempt
2.

B.      Notes:

C2H12AsNaO2
is
used
instead
of
NaH2PO4.

C.      C2H12AsNaO2
amounts

Same
as
the
amounts
of
NaH2PO4
in
Function
Test
Attempt
2.

To
be
continued....

CONTACT INFO

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# Team:Shanghai United/Results

Document

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Results

First Attempt to
Construct the Plasmids

AmilGFP
PCR

![](https://static.igem.org/mediawiki/2021/9/95/T--Shanghai_United--result01.jpg)

Figure 1: Gel
Electrophoresis Results of PCR of amilGFP Genes

Figure 1 shows gel
electrophoresis results of amilGFP PCR. Column M is a 2K marker ladder. Columns 1-6 are PCR
products of amilGFP genes

All 1-6 columns displayed
successful results at 700bp which could be used for later
experiments.

ARS
Plasmids
Extraction

|  |  |  |
| --- | --- | --- |
|  | Group 1 | Group 2 |
| ARSA | 425.9 ng/μL | 307.8 ng/μL |
| ARSD | 293.4 ng/μL | 300.8 ng/μL |
| ARSR | 214.4 ng/μL | 154.0 ng/μL |

Table 1:
Concentrations of ARS Plasmids Extraction Results

Table 1 shows that all
concentrations except the concentration of ARSR
from Group 2 are within
the desired range. While Group 2 was collecting ARSR, half of the sample was
accidentally mixed with ARSA
samples and must be
discarded -
hence the
deviation.
The concentrations were
used in to calculate the amount of ARS genes to be added in restriction
enzyme digestion.

AmilGFP
PCR
Products Clean-up

|  |  |  |
| --- | --- | --- |
|  | Group 1 | Group 2 |
| amilGFP | 382.6 ng/μL | 350 ng/μL |

Table 2:
Concentrations of amilGFP PCR Products
Clean-up Results

Table 2 shows the concentrations
of products of amilGFP PCR clean-up. The concentrations of
the samples are optimal and could be used to calculate the
amount of samples added when performing
restriction enzyme digestion.

ARS/amilGFP
Digestion

![](https://static.igem.org/mediawiki/2021/3/3a/T--Shanghai_United--result04.jpg)

Figure 2: Gel
Electrophoresis Results of Restriction Enzyme Digestion of ARS Plasmids and amilGFP Genes

Figure 2 shows gel
electrophoresis results of gel electrophoresis on restriction enzyme digestion products of ARSA, ARSD,
ARSR, and amilGFP genes. All products showed
successful results that could be used for later
experiments and these
target genetic
materials were extracted from the desired bands.

ARS/amilGFP
Gel
Extraction

|  |  |  |
| --- | --- | --- |
|  | Group 1 | Group 2 |
| ARSA | 31.0 ng/μL | 28.1 ng/μL |
| ARSD | 48.3 ng/μL | 23.3 ng/μL |
| ARSR | 5.2 ng/μL | 20.5 ng/μL |
| amilGFP | 106 ng/μL | |
|  |  |  |

Table 3: the
Concentrations of 7.30
ARS/amilGFP Gel
Extraction from Two Groups

We recycled GFP and the three plasmids ARSA, ARSD
and ARSR after electrophoresis. We used Nanodrop, a common lab
spectrophotometer, to test the concentration of the four products. According to Table 3, all data
except the concentration of ARSR
from Group 1 are
within the acceptable range. A possible reason for the anomaly
is that when the sample containing ARSA
was loaded into the gel box, the pipette pierced into the gel, causing a small amount of
leakage of the sample. It is noted that the concentration
of amilGFP is also abnormally high. The reason
is that since the two samples of amilGFP used by the two groups are the
same, we mixed them together and then tested theri concentration.

After we found out the
concentration of amilGFP, ARSA, ARSD and ARSR, it will be much easier to
calculate the volume of amilGFP needed to mix with each plasmid to
construct T4 DNA ligase ligation system according to the ratio between
amilGFP and plasmid is 1:10.

Colony
PCR

![](https://static.igem.org/mediawiki/2021/0/08/T--Shanghai_United--result06.jpg)

Figure 3: Gel
Electrophoresis Results of Colony PCR of First Experiment Attempt

We examined growths of
ARS/amilGFP E.
coli,
cultivated since Jul. 30. Results were suboptimal:
only 4 colonies were found in a total of 6 plates. We then performed colony PCR and gel electrophoresis
to test for ARS/amilGFP genes in observed E.
coli colonies. Figure 3 indicates that
only one colony resulted to be ARSD/amilGFP positive, indicating failure of
the experimental procedure.

Second Attempt to
Construct the Plasmids

AmilGFP
Digestion

![](https://static.igem.org/mediawiki/2021/7/7e/T--Shanghai_United--result07.jpg)

Figure 4: Gel
Electrophoresis Results of amilGFP
PCR in
Attempt 2

We performed PCR of the amilGFP gene to prepare for the second retry of the experimental procedure, then we
performed electrophoresis to test for amilGFP PCR products. Figure 4 indicates that the second
sample of group 1 resulted in failure, but the other samples are successful which could be retrieved for the
later experiments use.

AmilGFP
Gel
Extraction

|  |  |
| --- | --- |
| amilGFP | 92.3 ng/μL |

Table 4:
Concentrations of Gel Extraction of amilGFP
Enzyme
Digestion Products

Table 4 showed the concentration
of all products, extracted from gel from amilGFP Digestion, combined. The result is
optimal and could be used to calculate the amount of
sample added when performing T4 DNA Ligase Ligation.

Colony
PCR

![](https://static.igem.org/mediawiki/2021/3/3d/T--Shanghai_United--Proof_of_Concept04.jpg)

Figure 5: Gel
Electrophoresis Results 1 of Colony PCR of Second Experimental Attempt

![](https://static.igem.org/mediawiki/2021/5/58/T--Shanghai_United--Proof_of_Concept05.jpg)

Figure 6: Gel
Electrophoresis Results 2 of Colony PCR of Second Experimental Attempt

Figures 5 and 6 show the result
for colony PCR identification on the E.coli with ARS/amilGFP inserted that were cultivated
previously. The purpose is to examine whether the E.coli contains expected gene segment of ARSA/D/R and amilGFP.

As showing
above, all samples are
determined to have bands of corresponding molecular sizes, which shows that all selected colonies
yielded positive results of ARS
and
amilGFP
genes.

Function
Tests

Function
Test With
NaH2PO4,
Attempt 1

Such function tests were
performed under the hypothesis that
NaH2PO4 is structurally similar to arsenic compounds that
were meant to be detected. NaH2PO4 is used to substitute for arsenic
compounds due to experimental safety. No fluorescence was detected in all samples, indicating that the
concentrations of NaH2PO4 may be too low or that genetically
engineered E.
coli will not react to
NaH2PO4.

Function
Test With
NaH2PO4,
Attempt 2

In order to verify our assumption
in attempt 1, NaH2PO4 was used again in this testing,
with greater concentration
than the last test. No fluorescence was detected in all samples, indicating the inability of the
genetically engineered E.
coli to react with
NaH2PO4.

Function
Test With
C2H6AsNaO2

![](https://static.igem.org/mediawiki/2021/8/8e/T--Shanghai_United--Proof_of_Concept06.jpg)

Figure
7: Fluorescence
Intensity of Transformed E. coli In Different Concentrations of C2H12AsNaO5 Solutions

Given that NaH2PO4 cannot be used to stimulate
the expression of amilGFP in genetically engineered
E.
coli, C2H6AsNaO5 was used for this function test.
Extremely minor fluorescence was detected by a microplate reader after 19 hours of reaction time. This
result confirms the design of E.
coli to be correct and
functional.

Figure 7 displays the fluorescence
intensity generated by an ARSD/amilGFP transformed E.
coli reacting for 1
hour in C2H6AsNaO5 solutions. As seen in the figure,
fluorescence was detected for all C2H6AsNaO5 concentrations that are above 0,
which confirms that the designed plasmid worked as intended. However the fluorescence was rather minor, so we speculate that E.
coli responds poorly
to organic arsenic compounds. Further experiments would be conducted to test for fluorescence intensities of E.
coli in inorganic arsenic solutions.

To
be continued ...

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