iGEM 2023 — Team ulc

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4959/wiki/contribution/notebook.png)

**21.7.2023**

**Events:**Visited the laboratory, have the basic concept of
the layout of the laboratory. Learned how to prepare LB solution to breed bacteria. Use the LB prepared
by students to feed and increase the number of E.coli for the experiment next day.

**22.7.2023**

**Events:**Extract plasmid base from some E.coli prepared
yesterday and detect the concentration, this procedure is failed as the concentration is very low. Put
the lasted E.coli to LB in the clean bench to ensure the bacteria in the solution only have E.coli,
these LB-bacteria solution is for next day’s experiment.

**23.7.2023**

**Events:**Extract plasmid base and target fragment from some
E.coli prepared yesterday and detect the concentration, this procedure is seccessful as the
concentration is at normal value. The digestion is then used to get the target fragment and plasmid base
and then use agarose gel electrophoresis to detect whether the digestion is seccessed .

**24.7.2023**

**Events:** Cut the agarose gel containing target fragment and
extract the fragments out, detect the concentration. The extraction of fragments in the agarose gel is
successful as the concentration is at normal value. Use enzymic linkage to construct a hole plasmid with
target fragment with plasmid base. Move the plasmid in to competent cell and feed the target-plasmid
E.coli in the LB solution with Kana.

**25.7.2023**

**Events:**The E.coil cannot grow in LB-Kana solution. The
transform was failed. Attract the plasmid and target fragment from the lasted E.coli and detect the
concentration. The extraction is successful as the concentration is at normal level. The digestion is
succeccful as the concentration is at normal level. The agarose gel electrophoresis is successful as the
graph under UV lamp shows two strips. The part of gel contain fragments are then recycled and detected
the concentration of the recycled fragments. The recycle is successes as the concentration is
considerable.

**26.7.2023**

**Ecvents:**The enzymIc linkage was down and the plasmid was
transformed in to the competent cell .

**27.7.2023**

**Events:**Extract and digest the plasmid, the graph of gel
under the UV lamp shows two strips which mean the target plasmid was constructed. The E.coli with
plasmid was them moved to large volume of LB and cultivated.

**28.7.2023**

**Events:**Prepared the solution IPTG to inducing the protein
expression.

**29.7.2023**

**Events:**Nickel column purification and nickel column
recovery to get the target protein.

**30.7.2023**

**Events:**Construct SDS-PAGE and do anion
chromatography.

**31.7.2023**

**Events:**EMSA detection of protein and DNA
binding.

**1.8.2023**

**Events:**Detect the cleavage ability of protein.

**12.9.2023-22.9.2023**

**Events:** Nickel column purification of protein sva/asp,
SDS-PAGE and EMSA

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4959/wiki/engineering/engineering.png)

## **Overview**

The COVID-19 pandemic in recent years markedly propelled the nucleic acid
detection industry in China. However, popular techniques, notably those based on CRISPR-Cas9, had
patents held internationally. This situation escalated the production costs and curtailed the
flexibility of using such technologies. Consequently, there's a pressing need to establish a nucleic
acid thermostatic detection technology grounded in foundational research and pioneering innovations,
ensuring independent intellectual property rights.

DNA phosphorothionylation, or sulfur modification, is a distinct backbone
alteration where non-bridging oxygen atoms on the DNA's phosphodiester bond are replaced by sulfur
atoms. Restriction enzymes that are specific to this sulfur modification can precisely target and
cleave this uniquely modified DNA. Given their strong affinity for nucleic acids, understanding the
binding and cleavage mechanisms of these enzymes can pave the way for their incorporation into
thermostable nucleic acid detection systems.

In our project, we aim to synthesize these potential
sulfur-modification-dependent restriction enzymes. By constructing them in an expression vector, we
intend to purify the resulting protein. Our subsequent focus will be on the exploration of the
protein's binding and cleavage capabilities using EMSA assays and cleavage assays.

## **Round1**

### **Design**

DNA phosphorothionylation modification (also known as
sulfur modification) is a backbone modification that replaces non-bridging oxygen atoms on the
phosphodiester bond of DNA with sulfur atoms, and sulfur modification-dependent restriction enzymes can
target cleavage of this type of modified DNA.
Through bioinformatics analysis, our project identified three potential
sulfur modification-dependent restriction enzymes: Sga, Sva, and Asp.

|  |  |
| --- | --- |
| **Name** | **Length** |
| Sga | 1314bp |
| Sva | 1230bp |
| Asp | 939bp |

Then, we chose pET28a as the vector to carry three
target DNA segments, respectively, since the T7 promoter and T7 RNA polymerase greatly benefit protein
induction [1]. The next step was enzyme digestion with Nde1 and Xho1, acting on template plasmids of
three enzymes and vectors, allowing the subsequent protein DNA insertion to vectors (ligation with T4
DNA ligase, T4 ligase binds preferentially to phosphorylated nicks and catalyzes the sealing reaction )
and transformation [2]. Double enzyme digestion was also necessary since the bands on the gel
represented by different DNA fragments could help us confirm if the preview construction was booming.

![](https://static.igem.wiki/teams/4959/wiki/engineering/engineering-1.png)

Figure 1 Plasmids construction overview

After that, we induced our proteins with IPTG,
a molecular mimic
of [allolactose](https://en.wikipedia.org/wiki/Allolactose "Allolactose"), a [lactose](https://en.wikipedia.org/wiki/Lactose "Lactose") [metabolite](https://en.wikipedia.org/wiki/Metabolite "Metabolite") that
triggers [transcription](https://en.wikipedia.org/wiki/Transcription_%28genetics%29 "Transcription (genetics)") of the [*lac operon*](https://en.wikipedia.org/wiki/Lac_operon "Lac operon"), and it is therefore used to
induce protein expression where the gene is under the control of the [lac
operator](https://en.wikipedia.org/wiki/Lac_operator "Lac operator"). [4] Since the proteins remained
in the E. coli after induction, we first changed the pH of the protein
system. We carried out
ultrasonication
 to
break
 the cell wall of bacteria,
managing to get target proteins. As another centrifuge was done, proteins that we needed were suspended
in the supernat of the solution. The extracted proteins were not pure and couldn’t be used directly for
function analysis, so we decided to do Ni-chelating affinity chromatography, Q column
affinity
 chromatography, an anion
exchange chromatography to remove
impure
 proteins, DNA attachment on
proteins and imidazole in solution, which may disturb subsequent experiments respectively. Then, we sent
the pure proteins to concentrate with a centrifuge. During the process, two SDS-PAGE[5] were done to assess protein purity.
One was done after Ni-chelating affinity chromatography,
and the other was after
concentration.

Finally, three pur
ified
 extracted target proteins were tested for their
binding ability with phosphorothioated DNA (EMSA) and the cleavage ability (nucleic cleavage test) of
cutting specific DNA sequences to see if they could be applied to nucleic acid testing.

![](https://static.igem.wiki/teams/4959/wiki/engineering/engineering-2.png)

Figure 2
Diagrammatic drawing
 of target enzymes’ cleavage ability to PT
DNA

### **Build**

We first transformed the template plasmids of Sga,
Sva, and Asp into E. coli competent cells for conservation and thawed the cells containing pET28a
plasmid. After that, we prepared a system to lyse these cells and extracted plasmids. To create the
sticky ends on cutting sites, we used two endonucleases, Nde1 and Xho1, to digest four plasmids with PCR
amplifier. Then, AGE and gel extraction were performed to separate DNA segments in each plasmid. After
purification and concentration, we used T4 DNA ligase to ligate the sticky ends of each enzyme DNA
segment and vector pET28a; the recombinant plasmids were finally constructed.

![](https://static.igem.wiki/teams/4959/wiki/engineering/engineering-3.png)

Figure 3 Plasmids profiles of pET28a-Asp, pET28a-Sva,
pET28a-Sga

To completely ligate enzyme DNA and vector and
temporarily conserve the recombinant plasmids, the transformation into DH5ɑ competent cell through
heatshock was needed. After double enzyme digestion confirming we got the ideal plasmids, we could
transform three plasmids into BL21 (an E.coli used for high-level protein expression [3] to do protein
induction.)

### **Test**

1.
**Enzymatic validation of recombinant plasmids**

We use nanodrop to measure the concentration and
purity of plasmids to ensure we extracted enough of them to carry out digestion. After gel extraction, a
concentration test of each DNA segment (Sga, Sva, Asp, and pET28a) was conducted to see if they reached
the minimum amount to move to the next step --- ligation.

To verify that we successfully constructed the
recombinant plasmids, we performed the enzymatic digestion again. In the expected result, the gel should
appear to have two large and two small bands for each lane under UV light.

![](https://static.igem.wiki/teams/4959/wiki/engineering/engineering-4.png)

Figure 4 Gel electrophoresis results of vectors and target gene fragments

**2. Protein extraction and purification**

IPTG was used to induce protein in E. coli cells.
Ultrasonication was performed to extract them, and target proteins were suspended in the supernatant of
the solution. The next step was purification.

First, we did Ni-chelating affinity
chromatography purification to remove impure proteins. SDS-PAGE
of three samples of each protein (supernat after ultrasonic
ation
, effluent of Ni-chelating affinity
chromatography, purified proteins) was
followed
 to assess purification. The results showed no
bands in all three
lanes
 of Sva, so it hadn’t been collected and purified
successfully. Then we did Q column affinity chromatography, anion exchange column to remove DNA
attachment, and imidazole sending. After one night's concentration of purified proteins, we did another
SDS-PAGE; the results showed a loss of Asp during purification, and some
impure
 proteins still existed in it. The band of Sga
was clear, indicating a successful purification. Meanwhile, the ELISA test (Measuring the OD of BSA with
known
 concentrations to draw a best-fit line showing
the
math
 relationship between protein concentration and
OD, using this relationship to
calculate
 the concentration of unknown proteins) was
carried out to measure the exact engagement of Sga and Asp, the concentration of Asp was relatively low
(about 0.16ng/µl). Therefore, only Sga could used to do function tests.

![](https://static.igem.wiki/teams/4959/wiki/engineering/engineering-5.png)

Figure 5 SDS-PAGE

（
A
）
R
esults of target protein after purification
（
B
）
R
esults of target protein after concentration

**3****. Function Test 1****：****Electrophoretic mobility shift assays (EMSA)**

After extracting the target proteins, purification
(nickel affinity chromatography, Q column chromatography, gravity column) and concentration were done,
preparing for two function analyses: EMSA and nucleic acid cleavage test.

AGE conducted EMSA, dyed using SYBR Gold. It could
test the binding ability of Sga and Pt-DNA since when Sga binds to Pt-DNA, the molecular weight of DNA
increases, and DNA travels at a lower velocity in the gel than regular DNA strands. To further explore
the influence of DNA-protein ratio on binding capacity, we also set 6 different balances of DNA and
protein (DNA:protein=1:0; 1:1; 1:2; 1:4; 1:8) in each group.

![](https://static.igem.wiki/teams/4959/wiki/engineering/engineering-6.png)

Figure 6 EMSA results
of
 Sga

（
A
）
R
esult of BL21 EMSA
（
B
）
Result of B7A EMSA

**4****. Function Test 2****：****Nucleic acid cleavage test**

A nucleic acid cleavage test was also done by AGE. It
is used to examine the cutting ability of Sga to related sites on Pt-DNA. We set several comparison
groups: different protein concentrations (0; 0.077; 0.15; 0.31; 0.625; 1.25; 2.5; 5; 10; 20; 40; 80;
160ng/µl),

different pH (6.0; 6.5; 7.0), if DNA contain sulfur
modification. The severed DNA fragments would travel faster than complete DNA in gel, and the bands
would show a luminance trend as the concentration of proteins changes.

![](https://static.igem.wiki/teams/4959/wiki/engineering/engineering-7.png)

Figure 7 Nucleic acid cleavage result of
Sga

### **Learn****:**

In effect, we failed enzymatic digestion verification
several times. The AGE test either showed the vague bands or the bands with non-ideal base pairs. We
concluded that the reasons for this phenomenon include too low a concentration of plasmids and the
contamination of restriction enzymes (Nde1 and Xho1).

About the failure in purify
ing
 Sva and Asp, we deduced that something went
wrong when performing protein induction with IPTG; not all E. coli cell bodies
precipitate
 successfully after centrifuge. Therefore, the
concentration of extracted proteins would be too low, and it would lose even more during the subsequent
purification. Unfortunately, the function analysis couldn’t be conducted due to the low protein
amount.

When we first did EMSA, someone accidentally dropped a
marker into the sample lane in the non-pt-DNA group, causing a sample loading error. Besides, bands of
all pt-DNA groups were at the bottom of the gel and showed no luminance change. It told us that SBD
didn’t successfully bind to Pt-DNA. We thought it was due to the too-short DNA probe bought from the
company. So the next time, we used the longer one (BL21 and B7A) as a probe and made the
success.

The first-try results showed no differences between
the groups of BL21 and B7A. This might be due to improper temperature, improper incubation time,
problems during gel making, etc. A sampling loading error also appeared; someone added the opposite
protein concentration in one pH group.

##

## **Round****2**

### **Design**

Since we didn’t get Asp and Sva in round one, we carried out round two to manage to purify
these Asp and Sva. The general process was the same as
the
previews one.
First
ly, digesting
Asp and Sva template plasmids
 and pET28a with two restriction enzymes
,
 Nee1 and Xho1. Then
,
 the T4 DNA ligase was used to ligate sticky ends on vector and two template plasmids
to form recombinant plasmids. After double enzyme digestion
verification, we
induced
 Asp and Sva in E. coli using IPTG. When protein
was expressed in the cell body, we used ultrasonication to extract Asp and Sva, purifying them with
Ni-chelating affinity chromatography, Q column affinity chromatography, and anion exchange
chromatography for the next concentration. SDS-PAGE was done to measure the purities of proteins in each
stage; the EMSA and nucleic acid cleavage were done for highly purified proteins.

**Build**

As in the first round, we constructed Sva-pET28a and
Asp-pET28a plasmids. To obtain Sva-pET28a and Asp-pET28a plasmids, we used Nde1 and Xho1 to digest
vector and template plasmids. After purification and recovery of the vector and target fragments, we
ligated them using T4 DNA ligase. To conserve, stabilize, and completely ligate enzyme DNA,
transformation into DH5ɑ competent cell through a heatshock was needed. When double enzyme digestion
confirmed we got the ideal plasmids, we transformed two plasmids into BL21 (an E.coli used for
high-level protein expression to do protein induction.)

### **Test**

In the second round of our experiment, the basic
methodology of protein extraction and purification, EMSA, and nucleic acid cleavage tests are all kept
the same as usual to test our newly synthesized enzymes.

**1. Protein extraction and purification**

IPTG was used to induce protein in E. coli cells.
Ultrasonication was performed to extract them, and target proteins were suspended in the supernatant of
the solution. The next step was purification. Our protein purification has two steps. The first is
nickel column purification and the second is anion chromatography.

![](https://static.igem.wiki/teams/4959/wiki/engineering/engineering-8.png)

Figure 8 SDS PAGE
r
esults of the target protein

(A) SDS PAGE of Asp ;(B) SDS PAGE of Sva

“S”: Protein from the supernate after ultrasonication

“P”: Protein from the precipitate after ultrasonication

“Ni”: Protein obtained after nickel column purification

“Q”: Protein obtained after ion-exchange chromatography

“M”: Marker

Protein extraction, purification and concentration are
all in preparation for subsequent functional testing. We similarly performed the following two
functional tests on Sva and Asp - EMSA and nucleic acid cleavage.

**2. Function Test 1****：****Electrophoretic mobility shift assays (EMSA)**

We performed EMSA using vertical DNA gels, using SYBR
Gold for dyeing. It could test the binding ability of Sga and Pt-DNA since when Sga binds to Pt-DNA, the
molecular weight of DNA increases, and DNA travels at a lower velocity in the gel than regular DNA
strands. To further explore the influence of DNA-protein ratio on binding capacity, we also set 6
different balances of DNA and protein (DNA:protein=1:0; 1:1; 1:2; 1:4; 1:8) in each group.

![](https://static.igem.wiki/teams/4959/wiki/engineering/engineering-9.png)

Figure 9 EMSA result
s
 of BL21 and B7A

(A) EMSA result of Asp ;(B) EMSA result of Sva

**3. Function Test 2****：****Nucleic acid cleavage test**

A nucleic acid cleavage test was also done by AGE. It
is used to examine the cutting ability of Sga to related sites on Pt-DNA. We set several comparison
groups: different protein concentrations (0; 0.077; 0.15; 0.31; 0.625; 1.25; 2.5; 5; 10; 20; 40; 80;
160ng/µl), different pH (6.0; 6.5; 7.0), if DNA contain sulfur
modification. The severed DNA fragments would travel faster than complete DNA in gel, and the bands
would show a luminance trend as the concentration of proteins changes.

![](https://static.igem.wiki/teams/4959/wiki/engineering/engineering-10.png)

Figure 10 Nucleic acid cleavage result
s
 of BL21 & B7A

(A)Result of Asp ;(B) Result of Sva

### **Learn**

We generally considered our second round of
experiments more successful than in round one. In the protein extraction and purification test, it is
clear enough to identify that all the irrelevant proteins disappeared gradually as the experiment
continued, eventually revealing a clear, bright, and uniform band at 45 and 35 for SVA and ASP,
respectively; this result corresponded with our previous estimation of the mass of the two proteins. In
the EMSA binding experiment, it is also straightforward to observe that the binding of both proteins -
SVA and ASP – turned out to be successful inside the B7A bacteria, where phosphorothioated-DNA strands
occur naturally, as the concentration of the prokaryotic DNA increases. Finally, in the nucleic acid
cleavage test, although it is clearer to identify the cleavage results of ASP on the B7A gDNA than those
results of SVA on the B7A gDNA, both results proved an increase in cleavage level and success rate with
the increase of gDNA concentration. In conclusion, our second round of protein expression and
verifications turned out to be a great success that consolidated the
practicality
 and functionality of our three enzymes as
potential raw materials for nucleic acid testing and POCT technologies.

Reference
:

[1]
*Addgene: PET28-MHL*. (n.d.). <https://www.addgene.org/26096/>

[2]
Doherty, A. J., & Dafforn, T. R. (2000). Nick recognition by DNA ligases one 1Edited by K.
Nagai. *Journal of Molecular Biology*, *296*(1), 43–56. <https://doi.org/10.1006/jmbi.1999.3423>

[3]
*BL21(DE3) competent cells*. (n.d.). <https://www.thermofisher.cn/order/catalog/product/EC0114>

[4]
Wikipedia contributors. (2023). Isopropyl
Β-D-1-thiogalactopyranoside. *Wikipedia*. <https://en.wikipedia.org/wiki/Isopropyl_%CE%B2-D-1-thiogalactopyranoside>

[5]
*Protein electrophoresis using SDS-PAGE: A detailed overview |
GoldBio*. (n.d.). <https://goldbio.com/articles/article/Protein-Electrophoresis-SDS-PAGE-Overview>

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4959/wiki/experiments.png)

## **Preparation of LB medium**

|  |  |
| --- | --- |
| LB (Lysogeny Broth) Culture | 1L |
| Tryptone | 10g |
| Yeast Extract | 5g |
| NaCl | 10g |
| Agar | 15g (1.5%) |

Pour 10 plates, need to add Kana before pouring, 100ml one 100ul Kana

250ml for solid, 250ml for liquid

Simultaneous sterilization: water, two boxes of small, medium and large gun tips, 1.5 ml
tubes

## **Diamond Plasmid Mini Prep Kit**

1.
Preparation

•
Add the RNase A to Buffer SP1.

•
Add the volume of 100% ethanol indicated on the bottle label to the Wash Solution.

•
Check Buffer SP2 and SP3 for precipitation.

1.
Equilibrate column: Place a Miniprep column into an collection tube. Add 500 ul Buffer S to
column, centrifuge at 12,000 X g for 1 min. Discard the filtrate in collection tube. Return the column
to the collection tube.

2.
Collect 1.5-5ml of overnight LB bacteria culture. Centrifuge at 8,000 X g for 2 min to pellet the
bacteria. Discard as much of the supernatant.

3.
Resuspend the bacterial pellet in 250plof Buffer SP1 by vortexing. Make the pellet completely
resuspended.

4.
Add 250 pl of Buffer SP2, and mix by gently inverting the tube for 5-10 times. And incubate at
room temperature (15-25 C) for 2-4 min.

5.
Add 350pl of Buffer SP3, and mix immediately by gently inverting the tube for 5-10
times.

6.
Centrifuge at 12,000 X g for 5-10 min. Transfer the supernatant to column, centrifuge at 8,000 X
g for 30 sec, discard the filtrate in the collection tube.

7.
(Optional) Add 500pl Buffer DW1, Centrifuge at 9,000 X g for 30 sec, discard the filtrate in the
collection tube. Return the column to the collection tube.

8.
Add 500pl Wash Solution, centrifuge at 9,000 X g for 30 sec, discard the filtrate in the
collection tube. Ret turn the column to the collection tube.

9.
Repeat step 9.

10.
Place the column back into the collection tube. Centrifuge at 9,000 X g for 1 min.

11.
Transfer the column into a clean 1.5 ml Eppendorf tube. To elute plasmid, add 50~100pl of Eluent
buffer to the centre of the membrane. Incubate for 1 min at room temperature. Centrifuge at 9,000 X g
for 1 min. Keep the DNA solution.

## **Enzymatic digestion**

Enzymatic cleavage of target fragments and vector:

1.
Ndel/Xhol double digested target fragments: ①Asp, ②Sga, ③Sva, 900~1000bp

2.
Ndel/Xhol double digestion pET-28a(+) plasmid, about 6000bp.

Purpose fragment 3 samples, make two copies of each, total 6 samples. Plasmid 3 samples. Total 9
samples.

Reaction conditions: 37℃/30min

## **Gel Electrophoresis**

|  |  |
| --- | --- |
| Reagents | Volume |
| Agarose | 1.5% - 1.5g |
| TBE | 100ml |
| Loading Buffer per sample | 2µl |
| DNA Marker | 2ul |

## **Gel Extraction - Diamond DNA Gel Extraction Kit**

1.
Preparation: before use the kit:

–
Check Wash Solution for addition of 100% ethanol.

–
Check Buffer B2 for precipitation.

–
Adjust water bath to 50'C.

2.
Excise the agarose gel slice containing the DNA fragment of interest and weight.

3.
Add a 3-6 times of sample volume of Buffer B2, heat at 50'C in water both for 5-10 min until the
gel is completely dissolved.

4.
(Optional) If the DNA fragments is less than 500 bp, add further with 113 volume of
isopropanol.

5.
Transfer the binding mix to the column and centrifuge at 8000 X g for 30 sec. Discard the
filtrate in the collection tube. Return the column to the collection tube.

6.
Add 500 pl Wash Solution to column and centrifuge at at 9,000 X g for 30 sec, Discard the
filtrate in the collection tube. Return the column to the collection tube be.

7.
Repeat step 6.

8.
Place the column back in the collecting tube and centrifuge at 9,000X g for 1 min.

9.
Place the column back in a new 1.5 ml Eppendorf tube, add 15-40 pl Elution Buffer to the center
of the column membrane, after sitting at room temperature for 1 min, centrifuge for 1 min. Keep the DNA
elution.

## **Ligation Protocol with T4 DNA Ligase (M0202) NEB**

1.
Setup the following reaction in a microcentrifuge tube on ice

(T4DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of
1:3 vector to insert for the indicated DNA sizes.) Use NEBioCalculator to calculate molar ratios.

|  |  |
| --- | --- |
| COMPONENT | 20ul REACTION |
| T4 DNA Ligase Buffer (10x)\* | 2 ul |
| Vector DNA (4 kb) | 50 ng (0.020 pmol) |
| Insert DNA (1 kb) | 37.5 ng (0.060 pmol) |
| Nuclease-free Water | Up to 20 ul |
| T4 DNA Ligase | 1 ul |

\*The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.

1.
Gently mix the reaction by pipetting up and down and microfuge briefly.

2.
For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10
minutes.

3.
For blunt ends or single base overhangs, incubate at 16 overnight or room temperature for 2hours
(alternatively, high concentration T4 DNALigase can be used in a 10minute ligation).

4.
Heat inactivate at 65C for 10 minutes.

5.
Chill on ice and transfom 1-5μl of the reaction into 50μl competent cells.

## **Culturing Bacteria**

Total 3 samples ①Asp-pET28a, ②Sga-pET28a, ③Sva-pET28a

Reaction conditions: 16°C/2h (overnight is best), 37°C/30min

Inactivation by heating at 65°C for 10 min

## **Conversion of DH5α by enzyme-linked products**

### **Experimental design**

1.
3 samples were transformed: ①Asp-pET28a, ②Sga-pET28a, ③Sva-pET28a.

2.
Coat on 3 Kana/LB plates (3 plates are required).

3.
37℃ overnight incubation culture

### **Transformation steps:**

1.
Remove 100ul load of E. coli receptor cells from -80℃ refrigerator and thaw on ice:

2.
Add 5ul of recombinant product, shake gently and leave on ice for 10min;

3.
42℃ water bath for 90s, rapidly transfer to ice for 2min;

4.
In the ultra-clean bench: add 700ul LB liquid medium to the EP tube, mix well with the bacterial
solution, and resuscitate at 37℃ for 1h;

5.
the bacterial solution coated with antibiotic-containing LB plate (prepared in advance or on-site
inverted plate), until the bacterial solution air-dried inverted culture, 37 ℃ overnight (about
16h)

## **Preparation of IPTG**

1.
IPTG: isopropyl-β-D-thiogalactopyranoside, molecular weight 238.3, a kind of strong inducing
agent, not metabolized by bacteria and very stable.

Preparation of storage solution: Dissolve 1g of IPTG in 4ml of distilled water, then dilute to
5ml with distilled water, sterilize with a disposable needle filter, and then divide into 1ml small
portions for storage at -20℃. (Concentration is 0.8mol/l)

## **Pick BL21 plate monoclonal shaking bacteria**

I. Pick DH5α plate monoclonal shaking bacteria

Induction of protein expression

1.
Transfer the bacterial solution to LB medium at 1% and incubate at 37℃ for 4h. 2.

2.
When the OD600 of the bacterial solution reaches 0.6, remove and cool the shake flask, then add
0.8MIPTG in the proportion of 0.2% to induce protein expression, and then incubate at 16℃ for 20
hours.

## **Purification reaction application proteins:**

1.
ultrasonic breaking of bacteria

1.
Sterilization: pre-cooled to 4 degrees using a cell breaker

2.
lubricate two times, the first time with water and the second time with Buffer before
feeding.

3.
Inject the sample, circulate for a while and then pressurize it to about 700-800 bar, and
calculate the running time.

4.
After the end of the run, rinse five times, except for the third time with 20% anhydrous ethanol,
the rest of the time with water.

2.
Centrifugation

1.
pre-cool to 4°C

2.
centrifuge leveling +/- 0.06g

3.
run at 9500 rpm for one hour

3.
Nickel column purification

1.
Equilibrate the column: add 20 column volumes or more of Buffer A to the column.

2.
Ultrasonically crush the supernatant for two minutes to break up the residual DNA.

3.
Take 0.45 µL of sample, syringe through the membrane and onto the nickel column.

4.
Collect the filtrate and repeat step 3.

5.
Buffer A washes out the non-specific binding proteins (about 20 column volumes).

6.
Buffer B elutes specifically bound proteins (use gradient elution when doing this for the first
time)

7.
Post-treatment

4.
Anion-exchange column chromatography to elute DNA-binding proteins.

1.
Decontamination: wash the adsorbent with 1M NaCl (column volume 5ml). 2.

2.
Wash the adsorbent with three times the column volume of water. 3.

3.
Equilibration: Wash the adsorbent with three times the column volume of Buffer.

4.
Sampling: take a sample before and after the sample passes through the column (if there is only
DNA on the adsorbent, leave out the liquid as the sample; if there is still protein on the adsorbent,
use FPLC to elute the protein).

5.
Post-treatment: Wash the column with 1M NaCl solution at 2-3 times the volume of the column and
water at 4 times the volume of the column.

5.
Heparin adsorption (DNA removal)

1.
Equilibrate the column with four times the column volume of Buffer. 2.

2.
Dilute the sample using salt-free buffer

3.
Sample passes through heparin adsorption column (test for hang-ups, if no hang-ups then need to
continue diluting sample)

4.
FPLC

### **Nickel column chromatography**

Buffer A

|  |  |
| --- | --- |
| COMPONENT | PARAMETER |
| MES - pH6.7（Replaceable with Tris-Cl - pH 8.0） | 20 mM |
| NaCl | 300 mM |
| Glycerol | 5% |
| Imidazole | 50mM |

Buffer B

|  |  |
| --- | --- |
| COMPONENT | PARAMETER |
| MES - pH6.7（Replaceable with Tris-Cl - pH 8.0） | 20 mM |
| NaCl | 300 mM |
| Glycerol | 5% |
| Imidazole | 500mM |

### **Heparin adsorption column ( clear DNA )**

Buffer A

|  |  |
| --- | --- |
| COMPONENT | PARAMETER |
| Tris-Cl - pH 8.0 | 20 mM |
| NaCl | 50 mM |
| Glycerol | 5% |

Buffer B

|  |  |
| --- | --- |
| COMPONENT | PARAMETER |
| Tris-Cl - pH 8.0 | 20 mM |
| NaCl | 1 M |
| Glycerol | 5% |

Diluted Buffer

|  |  |
| --- | --- |
| COMPONENT | PARAMETER |
| Tris-Cl - pH 8.0 | 20 mM |
| Glycerol | 5% |

### **Desalting (similar to molecular sieve)**

1.
Equilibrate the column: Wash the column with four times the volume of Buffer.

2.
Add the remaining liquid from the upper portion of the ultrafiltration tube to the adsorption
column.

1.
if the volume of liquid is greater than 2.5 ml, aspirate 2.5 ml

2.
if the volume of liquid is less than 2.5 ml, aspirate liquid from the lower part and replenish to
2.5 ml

3.
3.5 ml of buffer is added to the column and the liquid is collected in a buffer-wetted
ultrafiltration tube.

4.
Post-treatment: the column is washed 4 times with water and stored.

### **Concentration (ultrafiltration, about 1 ml)**

1.
Ultrafiltration tubes are rinsed with buffer.

2.
centrifuge at 4500 rpm

3.
after concentration, transfer the liquid from the upper tube to a small blotting tube, freeze and
centrifuge at 12,000 rpm for 5-20 minutes, and transfer the sample to a new blotting tube.

4.
Post-processing: Use 0.1M NaOH for 15-30 minutes and soak in water and store in 20%
ethanol.

### **Protein Preservation**

•
Add 50% glycerol to half of the proteins and store at -40 degrees Celsius in 50 ul
tubes.

•
Add 5% glycerol to half of the proteins and store 50ul per tube at -80 degrees
Celsius.

## **Protein Quantification and Concentration Detection**

### **Plotting the standard curve**

1.
For 300 microliters of the system, 285 microliters of Bradford's solution is required, and for
the other fifteen microliters, 0-6 microliters of 1mg/ml BSA is added sequentially to the system and the
volume is replenished to 15 microliters by adding buffer.

2.
Mix for 5 to 10 minutes at room temperature.

3.
Absorbance at 595 nm was measured spectrophotometrically.

### **Protein samples**

1.
Take three dilutions, as dilute as possible, and measure more than one value.

2.
Place the plate in the enzyme counter.

## **Protein Function - Binding and Cleavage Test**

### **Detection of DNA composition using agarose gel electrophoresis**

1.
use DNA extracted from BL 21 and b7a strains, where b7a, the bacterium, carries natural
sulfur-modified DNA.

2.
Set the voltage to 120 volts and electrophoresis the gel for 40 minutes.

### **Configure 10 microliters of system sample**

|  |  |  |  |
| --- | --- | --- | --- |
| 5x Buffer | DNA (5pµ/µl) | Protein (15pµ/µl) | ddH2O |
| 2 | 1 | 0 | 7 |
| 2 | 1 | 0.3 | 6.7 |
| 2 | 1 | 0.6 | 6.4 |
| 2 | 1 | 1.2 | 5.8 |
| 2 | 1 | 2.4 |  |

5x Buffer: 1ml Tris-Cl and 0.5ml NaCl

1.
Add 0.5µl marker

2.
Add 5µl sample

3.
Use 15µA current and carry out electrophoresis for 40 minutes

### **Perform a gel blocking assay (mini-gel electrophoresis) to check if the protein is bound to the DNA.**

|  |  |
| --- | --- |
| Reagent | Volume/µl |
| Tris (pH 6, 6.5, 7) | 20 |
| NaCl | 2 |
| DTT | 2 |
| MnCl2 | 2 |
| ddH2O | 74 |

If the protein binds to the DNA, the total volume becomes larger and moves closer in the
gel

Configuration of cutting experiment buffer

### **Configure Cutting Experiment Sample (10µl)**

|  |  |
| --- | --- |
| Reagent | Volume/µl |
| 2x Buffer | 5 |
| DNA (150ng/µl) | 2 |
| Protein | 1 |
| ddH2O | 2 |

1.
set the metal bath at 37 degrees Celsius and wait for one hour for cutting.

2.
2 (control) x 3 (pH=6, 6.5, 7) x 6 (protein concentration) = 36 samples were
produced.

3.
1µl of proteinase k was added to degrade the protein so that the gel electrophoresis results
were not affected by the protein.

4.
set the metal bath at 50 degrees Celsius and wait for 30 minutes to degrade the
proteins.

5.
0.5µl was added and gel electrophoresis was carried out at 120V for 40 minutes.

## **Vazyme Biotech Co.,Ltd. Gram-Negative Bacteria DNA Extraction**

Experimental Procedure

Sample Processing for E.coli - Gram-negative bacteria

1.
Take 1-5 ml of bacterial culture (less than 1.0 x 10% bacteria), centrifuge at 10,000 rpm (11,500
x g) for 1 min, and pour off the culture.

–
The number of bacteria can be measured by spectrophotometer, 10D60 is about 1.5×10°
bacteria.

2.
Add 230 μl BufferGA and shake until the bacteria are thoroughly suspended.

3.
Add 20μulProteinaseK and shake to mix.

4.
Add 250ulBufferGB, shake and mix well, 70℃C water bath for 10min.

–
The addition of BufferGB may produce white precipitate, which will disappear at 70C and will not
affect the subsequent experiments. If the solution does not become clear, it means that the cell lysis
is not complete, which may result in a small amount of extracted DNA and impurity.

5.
(Optional) If the RNA residue has a big influence on the subsequent experiments, add 4μl
RNaseA to the digestion solution, shake for 15sec, and leave it at room temperature for
5-15min.

6.
Enter 08-2 for column purification.

## **Vazyme Biotech Co.,Ltd. DNA Purification**

1.
Add 180μl of anhydrous ethanol, shake and mix well, flocculent precipitate may appear,
centrifuge briefly to collect the liquid on the inner wall of the cap.

2.
Transfer the above mixture to a FastPuregDNAMini Columns Ill adsorbent column (adsorbent column
has been placed in the collection tube) and centrifuge at 12,000 rpm (13,400\*g) for 1 min. run the
filtrate.

3.
Add 500ul of BufferPB (please check that anhydrous ethanol has been added before use) to the
adsorbent column and centrifuge at 12,000 rpm (13,400\*g) for 1min, discard the filtrate.

4.
Add 600μl BufferPW (please check whether anhydrous ethanol has been added before use) to the
adsorption column, centrifuge at 12,000rpm (13.400×g) for 1min, discard the filtrate.

5.
Repeat step 4.

6.
Put the adsorption column back into the collection tube and centrifuge the empty tube at 12,000
rpm (13,400 × g) for 2 min.

–
After centrifugation of the empty column, it can be left uncapped for 2-5min to allow the
residual ethanol to evaporate completely.

7.
Transfer the adsorbent column to a new 1.5 ml centrifuge tube (supplied), add 50-100 u of Elution
Buffer to the center of the adsorbent column membrane, leave it at room temperature for 2-5 min, and
centrifuge at 12,000 rpm (13,400 × g) for 1 min.

–
Note: The following steps can all help to increase DNA yield.

–
Preheat the ElutionBuffer to 55 before eluting;

–
Repeat the elution step with new Elution Buffer (this procedure increases the yield but decreases
the concentration);

–
To increase the concentration of DNA, reintroduce the solution from the first elution to the
adsorption column for elution.

8.
Discard the column and store the DNA product at -30 to -15°C to prevent
degradation.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4959/wiki/results/results.png)

1.
**Construction of the** **recombinant****plasmids**

We prepared three DNA segments that can encode the
sulfur-modified dependent restriction enzyme we want. They are Asp, Sva, and Sga, respectively.
In
itially, we needed to extract pET-28a(+) from our E.
coli and test whether we got enough sample concentration. We used nanodrop and got an eligible
concentration.

To express it in the competent DH5 αwe have to cut and insert it into the carriers.
I
nitially, enzyme digestion is done with pET-28a(+) to
get our vector. The segments are inserted into the XhoI and
NdeI
 sites of the pET-28a(+) vector,
respectively, by ligase T4. We did another enzyme digestion and ran gel electrophoresis to see whether
we got the right recombinant plasmid ( Figure 1.1 ). Thankfully, we got the right bp for all three DNA
segments. Sga ran to the position between 1000 bp and 1500 bp, which matches its calculated number of
1314 bp. Sva ran to the position between 1000 bp and 1500 bp, a bit forward than Sva, which matches its
estimated number of 1230 bp. At the same time, Asp ran to the position around the 1000 bp line, which
matches its calculated number of 939 bp.

![](https://static.igem.wiki/teams/4959/wiki/results/result-1-1.png)

F
igure 1.1. Gel electrophoresis results of vectors and
target gene fragments

After determining that we had constructed the correct
recombinant plasmid, we retransformed it into BL21 receptor cells for subsequent protein expression. It
is worth mentioning that the solid medium used was supplemented with an antibiotic (Kanamycin), which
ensured that we screened to get the correct E. coli.

![](https://static.igem.wiki/teams/4959/wiki/results/result-1-2.png)

Figure 1.2. Growth of recombinant plasmids of Sga,Sva,
and Asp after transformation of Escherichia coli in plate culture

For further confirmation, we sent samples of the
bacterial fluid to the company for sequencing (Azenta). The sequencing results are shown below, which is
100% proof that we got the right plasmid. ( Figure 1.3 ).

![](https://static.igem.wiki/teams/4959/wiki/results/result-1-3.png)

F
igure 1.3. Test sequence of Sga-pET28a, Sva-pET28a and
Asp-pET28a

**2. Protein expression and purification**

After that, IPTG is added to induce protein
expression. To get a pure target protein, we used Nickel column purification, and an SDS PAGE ( Figure
2.1 ) was done to show whether we have our target protein and whether it is purified or not.

![](https://static.igem.wiki/teams/4959/wiki/results/result-2-1.png)

Figure 2.1. SDS PAGE results of target protein after
Nickel column purification

a: protein from solution with elution
buffer

b
: protein from solution with wash buffer

c: protein from supernate from
ultrasonication

Figure 2.1 shows that  B and C are supposed to
have little or no target protein, while A should have a single stripe. Sva shows no results in all three
samples, meaning we failed to extract the protein of Sva. But we still got target proteins in samples A
in Asp and Sga. Happily, the size of the protein obtained was in
accordance with the expected values.

![](https://static.igem.wiki/teams/4959/wiki/results/result-2-2.png)

Figure 2.2. SDS PAGE results of target protein after
concentration

Then, we use Bradford to test our samples'
concentration so we can do quantitative delusion. We first get a
standard

curve
 with a formula by adding 1-6mg/ul BSA, a protein
with known concentration as a contrast of concentration. The y-axis represents OD, and the x-axis
represents protein concentration. We tested the Asp and Sga samples, respectively, by subtracting the
background value from our data, which is the concentration of water, and putting the value of OD into
the formula to get protein concentration. We prepare three samples of the same kind and take the average
value from three repeating samples to get the most accurate concentration. The respective results are
0.853392145mg/ul for Sga and 0.164146188mg/ul for Asp. As the concentration for Asp was too low, we
decided to continue our experiment with Sga.

![](https://static.igem.wiki/teams/4959/wiki/results/result-2-3.png)

Figure 2.3. S
tandard

curve

of
 BSA

Since
we failed to get the Sva sample and didn’t have enough
concentration of Asp after nickel column purification,
w
e redid the protein purification of these two samples
and ran
SDS
 PAGE.

![](https://static.igem.wiki/teams/4959/wiki/results/result-2-4.png)

Figure 2.4 SDS PAGE results of the target
protein

(A) SDS PAGE of Asp ;(B) SDS PAGE of Sva

S: protein from supernate after
ultrasonication

p: protein from precipitate after ultrasonication

Ni: Protein obtained after nickel column
purification

Q: Protein obtained after ion-exchange
chromatography

F
rom Figure 2.4(A), we can see that there are all kinds
of proteins in supernate after ultrasonication precipitate after ultrasonication because the results shown on
them on SDS PAGE are scattered and with a large amount. However, after doing nickel column purification
and ion exchange chromatography, concentrated lines on each sample indicate that the purification is
successful, and we have only one kind of protein. We then checked the kDa of Asp, which is 36kDa, and
the result is successfully confirmed as the line is just about the same level of scale 35.

We then repeated the same procedure in the Sva sample.
F
rom Figure 2.4(B), we can also see that there are all
kinds of proteins in supernate after ultrasonication precipitate after ultrasonication because the results shown on
them on SDS PAGE are scattered and with a large amount. However, after doing nickel column purification
and ion exchange chromatography, concentrated lines on each sample indicate that the purification is
successful, and we have only one kind of protein. We then checked the kDa of Sva, which is 45kDa, and
the result is successfully confirmed as the line is just about the same level of scale 45.

# **3 Function Test**

After extracting the target proteins, purification
(nickel affinity chromatography, Q column chromatography, gravity column) and concentration were done,
preparing for two function analyses: EMSA and nucleic acid cleavage test.
T
his part presents the overview and experiment results
of the function test for the enzyme we obtained.

## **3.1 Electrophoretic mobility shift assays (****EMSA)**

The EMSA test aims to test the binding specificity
(phosphorothioate dependent in this case) of the Sga enzyme that is purified.
E
MSA 5x buffer is prepared with 100 mM Tris-Cl and 50
mM NaCl concentrations.
A
 10ul system is then used to achieve binding
between the target enzyme and the dsDNA – phosphorothioate B7A and non-phosphorothioate BL21. The system
consist
s
 of the following compositions. (Unit: ul)

Table 3.1 EMSA system (Unit: uL)

![](https://static.igem.wiki/teams/4959/wiki/results/results-table01.png)

T
he dsDNA is prepared from annealing of given ssDNA.
Enzyme binding is followed by SDS PAGE (Sodium dodecyl sulfate – polyacrylamide gel electrophoresis).
The product obtained is then
sta
ined using
SYBR

Gold
（
Invitrogen
）
 without light, thus observed using a
ge
l imager. We expect enzyme binding with only ptDNA,
thus no binding with non-ptDNA. The two result images are demonstrated in Figure 3.1
（
A
）
(non-pt DNA) and Figure 3.1
（
B
）
(mtDNA).

![](https://static.igem.wiki/teams/4959/wiki/results/result-3-1.png)

Figure 3.1 SDS-PAGE result

（
A
）
R
esult of BL21 EMSA
（
B
）
Result of B7A EMSA

A
s shown in Figure 3.1, non-pt DNA BL21 presents no
significant sign of successful binding with normal DNA chain length – around 25 bp. Non-pt DNA BL21
presents various DNA chain lengths under the SDS PAGE test, including a normal chain with a length of
around 25 bp and chains with a length of over 250 bp. The significantly longer chains observed on every
DNA protein level (not including the control level) represent successful binding between the target
enzyme and phosphorothioate B7A DNA.

I
n short, enzyme binding is observed in the ptDNA group
while not observed in the non-ptDNA control group.
T
herefore, phosphorothioate-dependent binding of the
target enzyme is confirmed.

Next shown are the results of our second round of
experiments. The methodology used is the same as in the first round, and in this round of experiments,
we obtained results for ASP and Sva.

![](https://static.igem.wiki/teams/4959/wiki/results/result-3-2.png)

Figure 3.2
R
esult of BL21 and B7A EMSA
for
 SVA

(A) EMSA result of Asp ;(B) EMSA result of Sva

As shown in Figure 3.2, non-pt DNA BL21 presents no
significant sign of successful binding with normal DNA chain length – around 25 bp. As with previous
analyses, the EMSA results for Asp and Sva also demonstrated binding of phosphorothioate to the target
enzyme

## **3.2 Nucleic acid cleavage test**

The nucleic acid cleavage test aims to test the
cleavage specificity (ptDNA dependent in this case) of the enzyme we obtained
.

Cleavage
2x

buffer
 is prepared with 40mM Bis-Tris, 100mM NaCl, 2mM
DTT, and 2mM MnCl2 concentrations. Among them, Bis-Tris (pH6.0) and NaCl suitable pH and NaCl
concentration for enzyme cleavage, MnCl2 provides the Mn2+ inducer while DTT maintains the oxidation
state of Mn2+ cations. Next, a 10ul system is used to
test
 for cleavage of non-pt BL21 DNA and pt B7A DBA
by enzyme Sga. The system composition is demonstrated in Table 3.2. (unit: ul)

Table 3.2 Cleavage system (unit: ul)

![](https://static.igem.wiki/teams/4959/wiki/results/results-table02.png)

Enzyme cleavage is followed by enzyme digestion.
Protein K is used to digest the enzyme Sga, avoiding potential influence in the following
ag
a
rose
 gel electrophoresis (AGE). The samples are run
on AGE; the results are shown in Figure 3.3. We expect to see cleavage only on ptDNA while no successful
cleavage on non-ptDNA

![](https://static.igem.wiki/teams/4959/wiki/results/result-3-3.png)

Figure 3.3 AGE
r
esult of BL21 &
B
7A Nucleic Acid Cleavage

A
s shown in Figure 3.3, the non-pt BL21 DNA group
presents no significant sign of cleavage on 0 to 80 protein concentration levels with normal chain
length > 15000 bp, while presents a relatively small amount of cleavage on 160 protein concentration
levels with chain lengths around and less than 15000 bp. In contrast, the pt B7A DNA group presents
normal chain lengths >15000 bp on 0 to 2.5 level while a relatively large amount of successful
cleavage on 50 to 160 level (with significantly larger amount on 40 to 160 level).

I
n short, on the 0 to 2.5 level, both groups showed no
sign of cleavage; on the 5 to 20 level, only the ptDNA group showed successful cleavage, and on the 40
to 160 level, the ptDNA group showed a significantly larger amount of cleavage compared to non-ptDNA.
T
herefore, the phosphorothioate-dependent specific
cleavage of the target enzyme is confirmed.

The following are the results of our second round of
experiments. In this round of experiments, we successfully obtained the results of ASP and
SVA.

![](https://static.igem.wiki/teams/4959/wiki/results/result-3-4.png)

Figure 3.4 Nucleic acid cleavage result
s
 of BL21 & B7A

(A)Result of Asp ;(B) Result of Sva

Like the previous analysis, the
phosphorothioate-dependent specific cleavage of the target Asp
and
 Sva enzymes is confirmed.

# **4. Conclusion**

  In conclusion, in the first round of
experiments, we successfully completed the plasmid construction and functional testing of Sga. All our
validations proved the reliability and accuracy of the results. Most importantly, the functionality and
specificity of our product, sulfur modification-dependent restriction enzyme, were
confirmed.

Admittedly
, we
did not
 complete the tests for Sva and Asp in the first
round. This could be due to a number of reasons. For Sva, samples containing elution buffer did not show
results in the SDS
-
PAGE after nickel column purification. This may be due
to the protein being denatured and placed into a lower pH solution. However, in the case of Asp, we
actually found the target protein in the concentrated SDS
-
PAGE but had to abandon testing it because the protein
concentration was too low.

Leveraging the learnings from our initial experiments, our second attempt
proceeded far more seamlessly. Following the established protocol, we successfully obtained results
for both Sva and Asp. However, it's important to recognize that our achievements thus far are
incremental. While our current findings affirm the direction of our research, certain aspects of our
results warrant refinement. The need for repeated trials remains paramount to bolster data accuracy.

Moving forward, once we are confident in the precision of our existing
experimental data, we intend to supplement our research with further functional data related to our
primary product: the sulfur-modification-dependent restriction enzyme.

{% endblock %}