iGEM 2022 — Team ykpao

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block page\_content %}

# Notebook

## August

### 11th

- Culture *E. coli*

![](https://static.igem.wiki/teams/4304/wiki/notebook/t-ykpao-notebook-01.jpg)

### 12th

- Cas12a protein expression:

* Addition of IPTG
* Test OD600 value of pET-28a integrated *E. coli* BL21(DE3)

- pUC57 plasmid extraction:

* Measure the concentration using Nanodrop

PCR of cagA, ipaH, invA, 16S\_ pUC57 plasmids

![](https://static.igem.wiki/teams/4304/wiki/notebook/t-ykpao-notebook-02.jpg)

### 13th

- Cas12a protein extraction:

* Ultrasound lysis
* Nickel affinity purification

- Gel electrophoresis of cagA, ipaH, invA, and 16S plasmids:

* Extraction of target DNA fragments
* Measure the concentration using Nanodrop

![](https://static.igem.wiki/teams/4304/wiki/notebook/t-ykpao-notebook-03.png)

### 14th

- Cas12a protein electrophoresis,SDS-PAGE

![](https://static.igem.wiki/teams/4304/wiki/notebook/t-ykpao-notebook-04.jpg)

- cagA, ipaH, invA, and 16S DNA in vitro transcription

- sgRNA purification

![](https://static.igem.wiki/teams/4304/wiki/notebook/t-ykpao-notebook-05.jpg)

### 15th

- Incubation of sgRNA and Cas12a proteins

- Test the reaction system using (cagA, ipaH, invA, and 16S) pUC57 plasmid

* ssDNA was included in the system and the result will be based on fluorescent intensity

- Test the reaction system using oligo DNA (cagA, ipaH, invA, and 16S)

* verify using gel electrophoresis after PCR

![](https://static.igem.wiki/teams/4304/wiki/notebook/t-ykpao-notebook-06.jpg)

### 16th

- Incubation of sgRNA and Cas12a proteins

- Test reaction system using oligo DNA (cagA, ipaH, invA, and 16S)

* prove using gel electrophoresis after PCR

- Test reaction system using (cagA, ipaH, invA, and 16S) pUC57 plasmid

* ssDNA was included in the system and the result will be based on fluorescent intensity

![](https://static.igem.wiki/teams/4304/wiki/notebook/t-ykpao-notebook-07.jpg)

### 17th

- Test resulting system (cagA, ipaH, invA, and 16S) pUC57 plasmid

* ssDNA was included in the system and the result will be based on fluorescent intensity detected by multiscan ascent

![](https://static.igem.wiki/teams/4304/wiki/notebook/t-ykpao-notebook-08.jpg)

- Test the reaction system using bacteria culture

* Bacteria lysis
* Test PCR results using gel electrophoresis
* Add ssDNA into the system and test using multiscan ascent

![](https://static.igem.wiki/teams/4304/wiki/notebook/t-ykpao-notebook-09.png)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block page\_content %}

# Engineering

## Introduction

*Helicobacter pylori* infection has been identified as a major carcinogenic factor causing gastric
cancer, so
its detection has also been included in a common item in physical examination. Early detection and effective
treatment of *H. pylori* infection can effectively reduce the incidence of a series of gastric diseases,
such as
gastric ulcers and gastric cancer. Based on CRISPR pathogenic microbial detection technology was developed in
recent years, and was applied to a series of pathogenic microorganism detection, has the advantages of being
fast and efficient, so the development of CRISPR rapid detection of *H. pylori* can effectively prevent
or find
*H. pylori* infection as soon as possible, so as to reduce the incidence of gastric cancer.

In our project, we applied FnCas12a to develop an in vitro detecting platform. We synthesized four genes as
targets and designed sgRNAs according to their DNA sequences. Then we mixed the sgRNAs, FnCas12a protein, and
corresponding plasmid, and detected if FnCas12a could be used for *H. pylori* detection. We also designed
a
reporter system to measure the activity of FnCas12a. Therefore, our product will provide a candidate protein
for *H. pylori* detection.

## How we design our plasmid

Because the T7 promoter and T7 RNA polymerase have strong ability in translation and usually be used as
protein expression, we choose pET28a-vector and *E. coli* BL21(DE3), to express our target protein
FnCas12a. To
achieve this, we designed the DNA sequences of FnCas12a to be inserted into the pET28a vector and transformed
the recombinant plasmid into *E. coli* BL21(DE3) for protein expression.

We designed another 2 plasmids: 16S, cagA transcription plasmids, and the DNA fragments 16S and cagA are
amplified from the genome of *Helicobacter Pylori*. These two genes’ DNA fragments were inserted into
pUC57
vector.

## How we build our plasmid

In order to construct our plasmids, we let the company synthesize the DNA fragments, FnCas12 was inserted into
the pET28a vector, and the fragments 16S and cagA were inserted into the pUC57 vector. The constructed
plasmids were contained in *E. coli* strains, we streak inoculated them on LB solid medium plates
containing
corresponding antibiotics, and incubate them at 37℃ overnight (Figure 1).

![](https://static.igem.wiki/teams/4304/wiki/engineering/t-ykpao-engineering-01.jpg)

Figure 1. incubate the plasmids containing strains.
(A) Plasmid 1: pUC57-16S plasmid containing strain.
(B) Plasmid 2: pUC57-cagA plasmid containing strain.

## How we test FnCas12a

### a) Protein expression and purification

We transformed the pET28a-FnCas12a expression plasmid into *E. coli* BL21(DE3) competent cells, and
cultured
at 37℃ overnight (Figure 2A). we inoculated a single colony into LB (Kana+) culture medium, incubated
overnight, and then transferred the cultured medium into 1L fresh LB (Kana+) culture medium. We induced the
expression of FnCas12a with IPTG when the OD600 was around 0.6-1.0, and cultured at 16℃ for 12h.
Subsequently, we used nickel affinity purification to purify the acquired Cas12a proteins from other
proteins in *E. coli* (Figure 2B).

![](https://static.igem.wiki/teams/4304/wiki/engineering/t-ykpao-engineering-02.jpg)

Figure 2. Expression and purification of protein FnCas12a.
A. Incubate the plasmid pET28a-FnCas12a containing BL21(DE3).
B. SDS-PAGE electrophoresis gel of Cas12a protein compared to nonspecific protein impurities.

Cas12a protein has a size of 130kDa. The SDS-PAGE electrophoresis result indicates that the Cas12a protein
is present in the solution we collected at 130kDa, and not present in the nonspecific protein impurities.
Thus, Cas12a proteins were expressed and purified with high quality.

Then, we tested the concentration of Cas12a protein by Bicinchoninic Acid Assay (BCA), using SpectraMax i3x
Multi-Mode Microplate Reader with the absorption peak at 562nm (Figure 3).

![](https://static.igem.wiki/teams/4304/wiki/engineering/t-ykpao-engineering-03.jpg)

Figure 3. BCA method standard linear regression line for calculation of protein concentration.

Table 1. Absorbance and calculated protein concentration of Cas12a 1 and Cas12a
2.

|  | Absorbance (L/(g·cm)) | Protein Concentration (µg/ml) |
| --- | --- | --- |
| Cas12a 1 | 0.2057 | 10.9358974 |
| Cas12a 2 | 0.1775 | 7.32051282 |

With this BCA standard curve, we measured the concentration of two samples of Cas12a protein, they are 10.9
µg/mL and 7.32 µg/mL respectively. This result indicated that we obtained a sufficient concentration of
Cas12a protein.

### b) Functional test through recognizing and cutting target genes

In order to verify if FnCas12a we purified could precisely recognize and cut the target DNA sequence, we
developed an in vitro reaction platform. Firstly, we obtained the sgRNAs through an in vitro transcriptional
method and extracted the target sgRNAs fragments. Next, we mixed the purified FnCas12a protein, the sgRNAs,
the corresponding plasmids containing DNA fragments, and the reaction buffer together. Then we incubated the
reaction system at 37°C for 2 hours, and we verified the result by gel electrophoresis (Figure 4).

![](https://static.igem.wiki/teams/4304/wiki/engineering/t-ykpao-engineering-04.jpg)

Figure 4. Gel electrophoresis comparing before and after cleavage of oligo DNA.
M = Marker, NC = Negative Control

After Cleavage is constituted of oligo DNA after cleavage by cas12a protein and sgRNA. In contrast, NC
(negative control) contains oligo DNA before cleavage only. Compared to NC, the oligo DNA band is
significantly diminished after cleavage. This displays that the in vitro cutting experiment is successful.

## How we learn from our project

We have already collected the data from our experiments. The DETECTR technology is based on CRISPR-Cas12a.
When target DNA is detected under the specific sgRNAs, the endonuclease activity of Cas12a is activated and
cleaves the target DNAs. From our result, we can find that the FnCas12a could recognize the target genes and
precisely cleave the target DNA.

*H. pylori* was originally isolated from the body of gastric ulcer patients and has been listed as a
well-defined carcinogen. The detection of *H. pylori* has become an important physical examination
project for
the prevention of gastric cancer. This project aims to develop a fast and convenient detection program for H.
pylori based on CRISPR to help people facilitate an efficient detection of *H. pylori* to contribute to
the
prevention of gastric cancer. We believed that we could apply FnCas12a in *H. pylori* detection in the
future.
When the technology developed, it could be easier for people to diagnose *H. pylori*.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block page\_content %}

# Experiments

{% include 'experiments/section-1.html' %}
{% include 'experiments/section-2.html' %}
{% include 'experiments/section-3.html' %}
{% include 'experiments/section-4.html' %}
{% include 'experiments/section-5.html' %}
{% include 'experiments/section-6.html' %}
{% include 'experiments/section-7.html' %}
{% include 'experiments/section-8.html' %}
{% include 'experiments/section-9.html' %}

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block page\_content %}

# Results

## 1. Construction of plasmids

We designed 5 plasmids: the FnCas12 protein expression plasmid, 16S, cagA, ipaH, and invA expression plasmids.
Among them, the DNA fragments 16S and cagA are amplified from the genome of *Helicobacter Pylori*, and
the gene
fragments ipaH and invA are amplified from *salmonella* and *shigella* genomic DNA respectively.

In order to construct our plasmids, we let the company synthesize the DNA fragments, FnCas12 was inserted into
the pET28a vector, and the fragments 16S, cagA, ipaH, and invA were inserted into the pUC57 vector. The
constructed plasmids were contained in *E. coli* strains, we streak inoculated them on LB solid medium
plates
containing corresponding antibiotics, and incubate them at 37℃ overnight (Figure 1).

![](https://static.igem.wiki/teams/4304/wiki/results/t-ykpao-results-01.jpg)

Figure 1. incubate the plasmids containing strains.
(A) Plasmid 1: pUC57-16S plasmid containing strain.
(B) Plasmid 2: pUC57-cagA plasmid containing strain.
(C) Plasmid 3: pUC57-invA plasmid containing strain.
(D) Plasmid 4: pUC57-ipaH plasmid containing strain.

## 2. Extraction of oligo DNA in plasmid

![](https://static.igem.wiki/teams/4304/wiki/results/t-ykpao-results-02.jpg)

Figure 2. 1.5% TAE agarose gel electrophoresis to verify the construction of oligo DNA containing plasmids.

We used TAE agarose gel electrophoresis to testify the presence of oligo DNA in the plasmid by performing PCR
and then doing gel electrophoresis of the amplicons (Figure 2).

Our results show that a band of 200bp to 400bp is present in cagA, 16S, invA, and ipaH, but not in negative
control (NC) lanes. Because oligo DNA has a size between 200bp to 400bp, our result supports the fact that the
plasmids contain desired oligo DNA. The four plasmid transformations were successful.

## 3. Expression and purification of Cas12a protein

We transformed the pET28a-FnCas12a expression plasmid into *E. coli* BL21(DE3) competent cells, and
cultured at
37℃ overnight (Figure 3A). we inoculated a single colony into LB (Kana+) culture medium, incubated overnight,
and then transferred the cultured medium into 1L fresh LB (Kana+) culture medium. We induced the expression of
FnCas12a with IPTG when the OD600 was around 0.6-1.0, and cultured at 16℃ for 12h. Subsequently, we
used
nickel affinity purification to purify the acquired Cas12a proteins from other proteins in *E. coli*
(Figure
3B).

![](https://static.igem.wiki/teams/4304/wiki/results/t-ykpao-results-03.jpg)

Figure 3. Expression and purification of protein FnCas12a.
A. Incubate the plasmid pET28a-FnCas12a containing BL21(DE3).
B. SDS-PAGE electrophoresis gel of Cas12a protein compared to nonspecific protein impurities.

Cas12a protein has a size of 130kDa. The SDS-PAGE electrophoresis result indicates that the Cas12a protein is
present in the solution we collected at 130kDa, and not present in the nonspecific protein impurities. Thus,
Cas12a proteins were expressed and purified with high quality.

Then, we tested the concentration of Cas12a protein by Bicinchoninic Acid Assay (BCA), using SpectraMax i3x
Multi-Mode Microplate Reader with the absorption peak at 562nm (Figure 4).

![](https://static.igem.wiki/teams/4304/wiki/results/t-ykpao-results-04.jpg)

Figure 4. BCA method standard linear regression line for calculation of protein concentration.

Table 1. Absorbance and calculated protein concentration of Cas12a 1 and Cas12a 2.

|  | Absorbance (L/(g·cm)) | Protein Concentration (µg/ml) |
| --- | --- | --- |
| Cas12a 1 | 0.2057 | 10.9358974 |
| Cas12a 2 | 0.1775 | 7.32051282 |

With this BCA standard curve, we measured the concentration of two samples of Cas12a protein, they are 10.9
µg/mL and 7.32 µg/mL respectively. This result indicated that we obtained a sufficient concentration of Cas12a
protein.

## 4. Cleavage experiment - cleavage of oligo DNA

In order to verify if FnCas12a we purified could precisely recognize and cut the target DNA sequence, we
developed an in vitro reaction platform. Firstly, we obtained the sgRNAs through an in vitro transcriptional
method and extracted the target sgRNAs fragments. Next, we mixed the purified FnCas12a protein, the sgRNAs,
the corresponding plasmids containing DNA fragments, and the reaction buffer together. Then we incubated the
reaction system at 37°C for 2 hours, and we verified the result by gel electrophoresis (Figure 5).

![](https://static.igem.wiki/teams/4304/wiki/results/t-ykpao-results-05.jpg)

Figure 5. 1.5% Agarose gel electrophoresis comparing before and after cleavage of oligo DNA.
M = Marker, NC = Negative Control

After Cleavage is constituted of oligo DNA after cleavage by Cas12a protein and sgRNA. In contrast, NC
(negative control) contains oligo DNA before cleavage only. Compared to NC, The oligo DNA band is
significantly diminished after cleavage. This displays that the in vitro cutting experiment is successful.

## 5. Variation of fluorescence intensity with the concentration of oligo DNA.

To assess if our Cas12a-based system worked well, we designed a reporter system by ligating a 6-FAM at the 5’
terminal of the target DNA probes. Then we measured the fluorescence intensity using SpectraMax i3x Multi-Mode
Microplate Reader every time the oligo DNA concentration increased, with the excitation of 485nm and emission
wavelength of 507nm.

![](https://static.igem.wiki/teams/4304/wiki/results/t-ykpao-results-06.jpg)

Figure 6. Fluorescence intensity at different concentrations of oligo DNA in simulated bacteria.
NC = negative control

The time between oligo DNA mixed with Cas12a protein and sgRNA system and measuring a sharp change in
fluorescence intensity within 10 minutes. As shown in the graph, as the concentration of oligo DNA increased
from 0 ng/μL to 4 ng/μL, the fluorescence intensity of the ssDNA fluorescent probes of the ipaH, invA, cagA,
and 16S systems also increased significantly. The fluorescence intensity of a system with higher oligo DNA
concentration is always higher than the fluorescence intensity of a system with lower oligo DNA concentration.
Also, our results showed that the fluorescence intensity of the oligo DNA represent group is similar to or
higher than the negative control (Figure 6).

Thus, the results indicate that our fluorescent detection experiment is successful. Cas12a protein and sgRNA
can recognize and cut the oligo DNA probes and the fluorescence emission can be detected.

## 6. Amplifying oligo DNA sequences by LAMP

![](https://static.igem.wiki/teams/4304/wiki/results/t-ykpao-results-07.jpg)

Figure 7. 1.5% TAE agarose gel electrophoresis to verify the amplified oligo DNA fragments.

The lamp is a novel nucleic acid isothermal amplification method, which is widely used in the rapid detection
of bacteria, pathogenic microorganisms, parasites, and viruses. Compared with the currently widely used
real-time qPCR technology, it requires four to six template-specific primers and DNA Polymerase with strand
displacement activity, and the amplification is completed within 1 h under isothermal conditions; The
corresponding detection dye can determine whether there is an amplification product. In order to make our
detection kit more suitable for home portable use, we tested amplified oligo DNA with the lamp method, which
is used as an alternative to the PCR amplification method. The experimental results show that there is our
target band at the position of 500bp, indicating that the scheme of amplifying oligo DNA by the lamp method is
feasible. This provides strong experimental data support for our development of portable detection kits.

{% endblock %}