iGEM 2022 — Team nanjing-hs

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{% block title %}Notebook{% endblock %}
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![](https://static.igem.wiki/teams/4289/wiki/banner.jpg)
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Notebook

Day 1

1. Configurate (solid / liquid) LB medium

| Component | Mass/volume |
| --- | --- |
| Tryptone | 10g |
| Yeast Extract | 5g |
| Sodium Chloride | 10g |
| Deionized Water | 1L |

Solid LB medium: Besides the component above, add 20g agarose (2%), mix well and
sterilize under high pressure for later use.

2. Inoculate glycerol bacteria pQE-30

The glycerol bacteria pQE-30 is inoculated into 15ml liquid LB culture medium containing
ampicillin/chloramphenicol, and cultured overnight in a constant temperature shaker (37℃, 220 rpm).

![](https://static.igem.wiki/teams/4289/wiki/t-nanjing-hs-notebook01.jpg)

Day 2

1. Amplify the hGK2 gene using PCR
2. Extract Plasmid pQE-30
3. Double-digestion of BamHI and SalI
4. Gel electrophoresis identification and Gel extraction
5. Use T4 Ligase to ligate
6. Transform into M15 E. coli competent cells & Coating plate

![](https://static.igem.wiki/teams/4289/wiki/t-nanjing-hs-notebook02.jpg)

Day 3

1. Positive transformant amplification
2. Extract plasmid from positive bacteria
3. Double enzyme digestion and Gel electrophoresis, send for Sanger sequencing

![](https://static.igem.wiki/teams/4289/wiki/t-nanjing-hs-notebook03.jpg)

Day 4

1. Inoculate the sequenced correct M15 transformants in 20 LB (ampicillin/chloramphenicol) and culture at 37℃ 220rpm
overnight.

![](https://static.igem.wiki/teams/4289/wiki/t-nanjing-hs-notebook04.jpg)

Day 5

1. Transfer all of the transformant cultured medium into 1L LB (ampicillin/chloramphenicol) culture medium and
incubate at 37℃ 220rpm until the OD is arranged 0.6-0.8.
2. Induce protein expression with IPTG after OD values of bacterial fluid is 0.6-0.8, and incubate at 25℃ 220rpm for
20h.
3. Ins-832 / 13 cell culture

1) Cryopreserved cells should be thawed and resuscitated immediately in a 37 ° C water bath after removal. Pipette
the cell fluid into a 15 ml centrifuge tube and add 3 ml complete medium.The whole medium was resuspended and moved
into a Petri dish. Centrifugate at 100 rpm for 5 min, and discard the supernatant. After resuspension with complete
medium, move it into a Petri dish.

2) Culture Ins-832 / 13 cells in RPMI 1640 medium containing 10% FBS, 10mM HEPES, 2mm L-glutamine, 50μM β-M
mercaptoethanol, 1 mM propylene, M sodium ketone, 100 U / mL penicillin with 100 μ G / ml streptomycin
(penicillin-streptomycin a cell culture incubator containing 5% CO2 at 37 ° C. When the cells grow to 80% - 90%, use
0.25% trypsin for digestion and generation. Generate it every 2 days approximately.

4. Configuration of GK enzyme activity detection reagent
Glucose: 42 mM (7.56 g)
G6PDH: 8.5 mM (7.6 g)
NAD：42U/mL
Buffer: 210 mM HEPES （50g），210 mM KCl （15.645g），17 mM MgCl2（1.62g），8.5 mM DTT （1.31g）
BSA:1.6%

![](https://static.igem.wiki/teams/4289/wiki/t-nanjing-hs-notebook05.jpg)

Day 6

1. Bacterial fluid collection

Centrifugation: 500 mL × 4, 4000 rpm for 15 min.

Collect the bacterial fluid.

2. Bacteria Lysate and protein purification
3. Protein identification by SDS-PAGE.
4. Protein dialysis and concentration.

![](https://static.igem.wiki/teams/4289/wiki/t-nanjing-hs-notebook06.jpg)

Day 7

1. Screen Glucokinase agonist

stablish a screening evaluation system for glucokinase agonists. The total volume of the
reaction system is
120μL.

Component of the reaction system

| Component | Volume (μL) | Final Concentration |
| --- | --- | --- |
| Ultrapure Water | 51.6 |  |
| Glucose | 12 | 5mM |
| G6PDH | 12 | 1mM |
| NAD | 12 | 5U/mL |
| 10×Buffer | 12 | 25mM HEPES, 25mM KCl,  2mM MgCl2,1mM DTT |
| BSA | 6 | 0.10% |
| LGK2 | 1.2 | 18.7μg/mL |
| Compound | 1.2 |  |

Finally, a flexstation 3 multi-purpose microplate reader workstation was used to add 12
μL ATP to initiate the reaction and immediately detect the change in absorbance at 340 nm. The Maximum reaction rate
is reflected by the slope of the curve.

2. Cell plating

After digesting the cells, inoculate them into 24 hole plate at the density of 2×105
each well, or into 96 hole
plate at the density of 5×105 each well.

![](https://static.igem.wiki/teams/4289/wiki/t-nanjing-hs-notebook07.jpg)

Day 8-11

1. Detect the effect of active compounds on insulin secretion induced by glucose

1) Incubate 2×105 of Ins-832/13 cells in a 24-well cell culture plate with 500 μL medium which is
placed in an
incubator with 5% CO2, 37°C overnight.

2) The next day, aspirate the medium. Add 500μLof KRB buffer (115 mM NaCl, 5 mM KCl, 24 mM NaHCO3, 10 mM HEPES, 2.5
mM CaCl2, 1 mM MgCl2, 0.1% BSA, pH = 7.3) with 2.8mM glucose. Starve the cells for 2h. And then replace the material
with KRB buffer containing 5.5 mM glucose and 20 μM compound 13926. Incubate the cells for 2h.

3) Supernatant of the absorbing medium was transferred to a 1.5 mL centrifuge tube at 600 rpm and centrifuged at 4℃
for 5 min. Take out the supernatant, and dilute it until the volume grew to 10 times the original. Test it with an
insulin assay kit. After the cells were lysed at 100μL RIPA, the protein concentration was determined as an internal
reference using the BCA protein concentration determination kit.

2. Detect the protective effect of active compounds on pancreatic islet cell

1) Ins-832 / 13 cells were incubated with a density of 5 × 105 for each well. Incubate the cells
in 96-well plates. Culture them overnight in a 5% CO2, 37°C cell incubator.

2) On the second day, add STZ with a final concentration of 0.4 mM and 13926 with a final
concentration of 20μM. Incubate them for 24 h.

3) On the third day, suck out the medium, add the medium containing 0.5 mg / mL MTT, and continue
to incubate for 4 h. After that, aspirate the medium. Add 100 μL DMSO to each hole, and shake the plate at 400 rpm
for 10 min. Detect the absorbance at 490 nm with a spectrum max M5 multi-functional micro-plate reader.

![](https://static.igem.wiki/teams/4289/wiki/t-nanjing-hs-notebook08.jpg)

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{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
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![](https://static.igem.wiki/teams/4289/wiki/banner.jpg)
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Engineering

Introduction

Type 2 diabetes accounts for 90% of all kinds of diabetes caused by a declining response
to insulin, the victims suffer from huge side effects. And the number of cases is increasing rapidly. Moreover, it
is gradually spreading among younger crowds. Therefore, we find it helpful to many people to design a drug used to
prevent symptoms of type 2 diabetes.
Glucokinase is a monomeric enzyme and serves as a “glucose-sensor” or “glucose receptor” in pancreatic cells and the
liver, eliciting glucose-stimulated insulin secretion, and as glucose “gate-keeper” in hepatocytes, promoting
hepatic glucose uptake and glycogen synthesis and storage. Due to its essential role in glucose homeostasis, it is
important to develop a screening platform for GK activators. In this project, we chose pQE30 as a protein expression
vector and purified the hGK2 protein in E. coli M15, and then the protein could be used in the screening platform.

How we design our plasmid

We design the plasmid: The DNA fragment of the hGK2 was inserted into the BamHI and SalI
sites of the pQE-30
vector (Figure 1).

![](https://static.igem.wiki/teams/4289/wiki/t-nanjing-hs-engineering01.jpg)

Figure 1. The plasmid map in this project

How we build our plasmid

In order to obtain the target fragments, we selected the appropriate endonuclease and
digested both the DNA fragments and plasmid carrier simultaneously. We digested the DNA fragment hGK2 and plasmid
pQE-30 with BamHI and SalI. Then we obtained the target DNA fragments (Figure 2) and ligated the fragments with T4
DNA ligase. Afterward, we transformed the recombinant plasmid into E. coli M15 competent cells and coated on the LB
culture medium plate.

![](https://static.igem.wiki/teams/4289/wiki/t-nanjing-hs-engineering02.jpg)

Figure 2. Gel electrophoresis results of target gene fragments. A. double
enzymes digested hGK2 DNA fragments, B. double enzymes digested pQE-30 plasmid.

We inoculated 3 single colonies and extracted the plasmids. To verify the plasmids, we
digested these plasmids with BamHI and SalI (Figure 3A). We send the constructed recombinant plasmid to a sequencing
company for sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF
region (Figure 3B), and the plasmid was successfully constructed. So far, we have successfully constructed the
pQE30-hGK2 vector.

![](https://static.igem.wiki/teams/4289/wiki/t-nanjing-hs-engineering03.jpg)

Figure 3 Agarose gel electrophoresis diagram of the clone. (A) Verify the
colony in lanes 1-3
(B) Sequenced results mapped to the plasmid

In Figure 3A, we can see that there are obvious bands of hGK2 and pQE30, proving that
our recombinant cloning products were constructed successfully. In Figure 3B, we can see that there is no difference
in the result of the template and construction, which represents the success of construction. This meant that we can
carry out subsequent cell transfection and characterization qualitative detection.

Protein expression and verification

In order to purify the protein, we cultured the M13 transformants in LB
(Ampicillin/Chloramphenicol) and add IPTG to induce the protein expression when the OD600 reached 0.6-0.8. After
overnight induction and culture, we collected the cells and ultrasonic fragmentation of cells to release the
intracellular proteins. Next, we used Ni-NTA column purification to purify the hGK2 protein. As shown in Figure 4,
there are several clear bands which means the hGK2 protein was successfully expressed in the strain.

![](https://static.igem.wiki/teams/4289/wiki/t-nanjing-hs-engineering04.jpg)

Figure 4. SDS-PAGE detection of hGK2.

How we learn from our project

We have already collected the data and figures from our experiments. GK plays a role in
transferring Glucose into Glucose 6-phosphate(G6P). hGK2, as a glucose receptor in pancreatic islets β cells, is
mainly responsible for promoting insulin release and biosynthesis under glucose stimulation, as well as regulating
pancreatic islets β cell survival and proliferation.
After transforming the hGK2 gene containing plasmid in the E. coli M13 strain, we detected the gene expression
through SDS-PAGE. Because of the activity of the hGK2, we believe that if we can develop a glucokinase agonist
compounds screen platform with hGK2 protein, it may be applied to clinical disease treatment, and improve the
quality of life of patients.

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{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
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![](https://static.igem.wiki/teams/4515/wiki/banner.jpg)
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Experiments

1. Construction of pQE-30-hGK2 recombinant plasmid:

(1) Material: human glucokinase sequence with BamHI and SalI
restriction enzyme cutting site and pQE-30 vector
(2) Digested the hGK2 sequence and pQE-30 vector with BamHI and SalI. Subject the digested products to agarose gel
electrophoresis, cut the gel and extract the DNA fragments.
(3) Use T4 ligase for ligation at 22℃ for 1h, and take 2 μL of ligation product. The ligation product was
transformed into the competent cells of E. coli M15 by the heat shock method. Add 1mL of antibiotic-free LB medium
and culture at 37 ℃ for 1h. After centrifugation at 6000rpm for 2min, remove the supernatant, and the coat
precipitate on LB solid medium containing Ampicillin and Chloramphenicol for overnight culture.
(4) The next day, pick up three colonies on LB medium plate and incubate each of them into 3mL LB medium
containing ampicillin for overnight culture. Preserve the bacterial solution with glycerol, and extract the
plasmids contained in the transformants with the Plasmid Extraction Kit.
(5) Use BamHI and SalI double enzyme digestion to identify, and sequence the positive plasmids.

2. Expression and purification of 6×his-hGK2 protein:

(1) Inoculate the positive strains to 20 mL of Ampicillin (100 μg/mL) and Chloramphenicol (50 μg/mL) in LB medium
overnight in a shaker at 37°C, 210 rpm. The next day, all the bacterial cultured medium was transferred into 1L of
LB medium containing Ampicillin (100 μg/mL) and Chloramphenicol (50 μg/mL) at 37°C 210 rpm until OD600 is around
0.6-0.8. Add IPTG until the final concentration is 0.3 mM, and the culture was continued to incubate at 25 ℃, 210
r/min for 20 h.
(2) Collect the bacteria cells at 4 ℃, 12000 rpm, centrifuge for 2 min, remove the supernatant and collect the
precipitate.
(3) Add 30mL of binding buffer (20 mM Tris, 0.5 M NaCl, 10 mM Imidazole, pH = 8.0) and mix well. Then use an
ultrasonic crusher to break the bacteria (4 ℃, 300W, sonicate for 10 s, stop for 5 s, a total of 30 min). collect
the supernatant after centrifuging at 4℃, 12000 rpm for 40 min. During centrifugation, balance the Ni-NTA resin
column with binding buffer, and then add the supernatant to the resin column and bound at 4 ℃ for 2 h.
(4) During this period, mix the protein several times to fully combine with the resin. Make the supernatant slowly
pass through the resin column, and then remove the non-specific binding protein with 7.5mL binding buffer, 15mL
elution buffer1 (20 mM Tris, 0.5 M NaCl, 20 mM Imidazole, pH = 8.0), and elute it with 7.5mL elution buffer2 (20
mM Tris, 0.5 M NaCl, 150 mM Imidazole, pH = 8.0).
(5) SDS-PAGE verification and stain it with Coomassie blue staining solution. Select the flow-through containing
high-purity protein for dialysis.
(6) Load the purified protein into a 35 KDa dialysis bag and dialyze three times at 4°C for 5 h each time using
dialysis buffer (25 mM HEPES, 25 mM KCl, 2 mM MgCl2, 1 mM DTT, pH = 7.1). Concentrate the protein to 2.24 mg / mL
at 3000 g, 4°C using a 30 KDa concentration tube. After sub-packaging, store it at -80 ℃ for subsequent
experiments.

3. Screening of glucokinase agonists:

(1) The principle of this experiment is that GK catalyzes the phosphorylation reaction of ATP and glucose to
generate glucose-6-phosphate. Glucose-6-phosphate reacts with excess NAD+ to produce NADH under the action of
glucose-6-phosphate dehydrogenase. Therefore, the activity of GK can be reflected by measuring the absorbance
change of NADH at 340 nm.

(2) Based on this principle, we established a screening and
evaluation system for glucokinase agonists. The volume
of the reaction system is 120 μL. First, each component except the compound is dissolved into solution and then
added to a 96-well plate, then DMSO and the compound to be tested are added in the sequence. The content of each
component is as the following chart shows,

| Component | Volume (μL) | Final Concentration |
| --- | --- | --- |
| Ultrapure Water | 51.6 |  |
| Glucose | 12 | 5mM |
| G6PDH | 12 | 1mM |
| NAD | 12 | 5U/mL |
| 10×Buffer | 12 | 25mM HEPES, 25mM KCl,  2mM MgCl2,1mM DTT |
| BSA | 6 | 0.10% |
| LGK2 | 1.2 | 18.7μg/mL |
| Compound | 1.2 |  |

(3) The reaction is initiated by adding 12 μL ATP using the Flexstation 3 Multifunction Microplate reader
workstation and immediately detecting the absorbance changes at 340 nM, reflecting the maximum reaction rate of GK
by the slope size.
(4) We validated the platform with the positive compound PF-04937319, and the EC50 of the positive compound is
0.93 μM.
(5) We used this platform to screen the compounds and found a small molecule 13926 with glucokinase activation
activity. The EC50 was 87 nM and the maximum activation rate was 1.21.

4. Detect the effect of active compounds on insulin secretion induced by glucose:

(1) Incubate 2×105 of Ins-832/13 cells in a 24-well cell culture plate with 500 μL medium which is placed in an
incubator with 5% CO2, 37°C overnight.
(2) The next day, aspirate the medium. Add 500μLof KRB buffer (115 mM NaCl, 5 mM KCl, 24 mM NaHCO3, 10 mM Hepes,
2.5 mM CaCl2, 1 mM MgCl2, 0.1% BSA, pH = 7.3) with 2.8mM glucose. Starve the cells for 2h. And then replace the
material with KRB buffer containing 5.5 mM glucose and 20 μM compound 13926. Incubate the cells for 2h.
(3) Supernatant of the absorbing medium was transferred to a 1.5 mL centrifuge tube at 600 rpm and centrifuged at
4℃ for 5 min. Take out the supernatant, and dilute it until the volume grew to 10 times the original. Test it with
an insulin assay kit. After the cells were lysed at 100 μL RIPA, the protein concentration was determined as an
internal reference using the BCA protein concentration determination kit.

5. Detect the protective effect of active compounds on pancreatic islet cells:

(1) Ins-832 / 13 cells were incubated with a density of 5 × 105 / well. Incubate the cells in 96-well plates.
Culture them overnight in a 5% CO2, 37°C cell incubator.
(2) On the second day, add STZ with a final concentration of 0.4 mM and 13926 with a final concentration of 20 μM.
Incubate them for 24 h.
(3) On the third day, suck out the medium, add the medium containing 0.5 mg / mL MTT, and continue to incubate for
4 h. After that, aspirate the medium. Add 100 μL DMSO to each hole, and shake the plate at 400 rpm for 10 min.
Measure the absorbance at 490 nm with a spectrum max M5 multi-functional microplate reader.

6. Expected Results:

1. The recombinant plasmids pQE-30-hGK2 and pGEX-4t-1-hGK2
were successfully constructed.
2. Successfully purified 6×His-hGK2 protein and GST-hGK2 protein.
3. Establish an hGK2 agonist screening platform, evaluate the activities of the two proteins, and use the platform
to screen the active compounds.
4. It is proved that the compound can enhance insulin secretion and protect pancreatic islet cells.

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{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block header\_content %}
![](https://static.igem.wiki/teams/4289/wiki/banner.jpg)
{% endblock %}
{% block page\_content %}

Results

1. Construct the hGK2 expression plasmid

We design the plasmid: The DNA fragment of the hGK2 was inserted into the BamHI and SalI
sites of the pQE-30 vector (Figure 1).

![](https://static.igem.wiki/teams/4289/wiki/t-nanjing-hs-results01.png)

Figure 1. The plasmid map in this project

In order to obtain the target fragments, we selected the appropriate endonuclease and
digested both the DNA fragments and plasmid carrier simultaneously. We digested the DNA fragment hGK2 and plasmid
pQE-30 with BamHI and SalI. Then we obtained the target DNA fragments (Figure 2) and ligated the fragments with T4
DNA ligase. Afterward, we transformed the recombinant plasmid into E. coli M15 competent cells and coated on the LB
culture medium plate.

![](https://static.igem.wiki/teams/4289/wiki/t-nanjing-hs-results02.png)

Figure 2. Gel electrophoresis results of target gene fragments. A. double
enzymes digested hGK2 DNA fragments, B. double enzymes digested pQE-30 plasmid

2. Identification of plasmid construction

We inoculated 3 single colonies and extracted the plasmids. To verify the plasmids, we
digested these plasmids with BamHI and SalI (Figure 3A). We send the constructed recombinant plasmid to a sequencing
company for sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF
region (Figure 3B), and the plasmid was successfully constructed. So far, we have successfully constructed the
pQE30-hGK2 vector.

![](https://static.igem.wiki/teams/4289/wiki/t-nanjing-hs-results03.png)

Figure 3 Agarose gel electrophoresis diagram of the clone. (A) Verify the
colony in lanes 1-3
(B) Sequenced results mapped to the plasmid

In Figure 3A, we can see that there are obvious bands of hGK2 and pQE30, proving that
our recombinant cloning
products were constructed successfully. In Figure 3B, we can see that there is no difference in the result of the
template and construction, which represents the success of construction. This meant that we can carry out subsequent
cell transfection and characterization qualitative detection.

3. Construction of transcriptional activation platform

In order to purify the protein, we cultured the M13 transformants in LB
(Ampicillin/Chloramphenicol) and add IPTG to induce the protein expression when the OD600 reached 0.6-0.8. After
overnight induction and culture, we collected the cells and ultrasonic fragmentation of cells to release the
intracellular proteins. Next, we used Ni-NTA column purification to purify the hGK2 protein. As shown in Figure 4,
there are several clear bands which means the hGK2 protein was successfully expressed in the strain.

![](https://static.igem.wiki/teams/4289/wiki/t-nanjing-hs-results04.png)

Figure 4. SDS-PAGE detection of hGK2.

4. hGK2 protein activity detection

Overview

GK takes part in the first step of glycolysis, catalyzing glucose into Glucose
6-phosphate(G6P). We purified the hGK2 protein and developed an activity detection system using INS-832/13 cells to
verify if our hGK2 works well.

a) Establish the screen system for glucokinase agonists

We established a screening system for glucokinase agonists. The total volume of the
reaction system is 120μL.

Component of the reaction system:

| Component | Volume (μL) | Final Concentration |
| --- | --- | --- |
| Ultrapure Water | 51.6 |  |
| Glucose | 12 | 5mM |
| G6PDH | 12 | 1mM |
| NAD | 12 | 5U/mL |
| 10×Buffer | 12 | 25mM HEPES, 25mM KCl,  2mM MgCl2,1mM DTT |
| BSA | 6 | 0.10% |
| LGK2 | 1.2 | 18.7μg/mL |
| Compound | 1.2 |  |

Finally, we used Flexstation 3 Multifunction microplate reader workstation to add 12 μL
ATP to initiate the reaction and immediately detect the change in absorbance at 340 nm. The Maximum reaction rate is
reflected by the slope of the curve.

![](https://static.igem.wiki/teams/4289/wiki/t-nanjing-hs-results05.png)

Figure 5. Results of glucokinase agonists detection

As we can learn from the result (Figure 5), compound 13926 was finally discovered (maximum agitation rate, 1.21;
EC50, 87 nM), which with higher activation activity than PF-04937319 (EC50, 930 nM).

b) Effects of 13926 on insulin secretion

Incubate 2×105 of INS-832/13 cells in a 24-well cell culture plate with 500μL culture
medium overnight. The next day, replace the medium with 500μL of KRB buffer (115 mM NaCl, 5 mM KCl, 24 mM NaHCO3, 10
mM HEPES, 2.5 mM CaCl2, 1 mM MgCl2, 0.1% BSA, pH = 7.3) with 2.8mM glucose and cultured for 2h. Then replace the
medium with KRB buffer containing 5.5mM glucose and 20μM compound 13926. Incubate the cells for 2h. The supernatant
of the medium was transferred to a 1.5 mL centrifuge tube and centrifuged at 600 rpm, 4℃ for 5 min. Dilute the
supernatant and test it with an insulin assay kit. After the cells were lysed at 100μL RIPA, the protein
concentration was determined as an internal reference using the BCA protein concentration determination kit. hGK2
enzyme activity detection platform was established and a positive compound PF-04937319 was used to prove the
activity of hGK2 (Figure 6A).

![](https://static.igem.wiki/teams/4289/wiki/t-nanjing-hs-results06.png)

Figure 6. Effects of 13926 on insulin secretion and cell viability

c) Effects of 13926 on protecting effects of pancreatic islet cells

Ins-832/13 cells were incubated with a density of 5 × 105 for each well. Incubate the
cells in a 96-well plate. Culture them overnight in a 5% CO2, 37°C cell incubator. On the second day, add STZ with a
final concentration of 0.4 mM and 13926 with a final concentration of 20 μM. Incubate them for 24 h. On the third
day, discarded the medium, add the medium containing 0.5 mg / mL MTT, and continue to incubate for 4 h. After that,
aspirate the medium. Add 100 μL DMSO to each hole, and shake the plate at 400 rpm for 10 min. Detected the
absorbance at 490 nm with a spectrum max M5 multi-functional microplate reader (Figure 6B).
The results indicated that 13926 effectively promoted insulin secretion and protected it from STZ damage in
Ins-832/13 cells.

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