iGEM 2023 — Team pinghe

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![](https://static.igem.wiki/teams/4926/wiki/engineering.png)

Introduction

In this project, we are
committed to providing consumers with a rapid and reliable solution to quickly detect the presence of
Staphylococcus aureus and Pseudomonas aeruginosa in their daily lives. This application extends
beyond cosmetics. Leveraging the state-of-the-art CRISPR/Cas12a gene editing technology, we have
cost-effectively transcribed Cas12 protein in vitro and synthesized sgRNA to guide Cas12 protein cleavage.
Combined with gene fluorescence probes, we have successfully achieved rapid detection of specific
pathogenic gene segments on simulated Escherichia coli, serving as a tool to identify Staphylococcus
aureus. The fluorescence intensity observed by the naked eye can determine negativity, positivity, and
approximate bacterial quantities. This system is user-friendly and easy to use.

**Protein Cas12a expression**

First of all, we need Cas12a protein and plasmid pET28a-FnCas12a (BBa\_K4304007, provided by iGEM22\_YkPaO, SubCat
Academy) was obtained from the company laboratories. We
transformed the pET28a-FnCas12a plasmid into E. coli BL21(DE3) and
cultured overnight. Then we transferred the medium into 300mL fresh LB culture medium. When the OD600 was
around 0.6, we added IPTG to induce FnCas12a expression at 16℃ for 12 hours. Then
FnCas12a was extracted and purified by His-tag.
The size of protein FnCas12a is about 130kDa. The result of SDS-PAGE
showed that the size of the FnCas12a protein is between 100kDa and 160kDa. So
the protein FnCas12a was successfully expressed.

![](https://static.igem.wiki/teams/4926/wiki/engineering/1.png)

Figure 1. SDS-PAGE analysis of FnCas12a protein

Cycle 1

**Construct
design****and build****1**

pUC57-femA:
BBa\_K4926002

We designed a plasmid containing femA. The DNA fragment is from the genome
of Staphylococcus
aureus. In order to construct plasmid, we let the
company synthesize the DNA fragment femA, which was inserted into pUC57 plasmid.

![](https://static.igem.wiki/teams/4926/wiki/engineering/1-5.png)

We transformed the
pUC57-femA into the E.coli DH5α. We obtained the E.coli DH5α with target fragments of femA
(Fig 2)., Then we performed TAE agarose gel electrophoresis to verify that the plasmid presented in the E.coli
DH5α. As shown in Figure 3, we got lanes which length were same with femA.

![](https://static.igem.wiki/teams/4926/wiki/engineering/2.png)

Figure
2. pUC57-femA in DH5α

![](https://static.igem.wiki/teams/4926/wiki/engineering/3.png)

Figure
3.  Bacteria PCR of femA.

**Function test
1**

![](https://static.igem.wiki/teams/4926/wiki/engineering/4.png)

Figure
4. Graph
of efficiency comparison of sgRNAs

We used T7 in vitro
transcription kit to obtain three different
sgRNA of femA. In
order to verify whether Cas12a could recognize and cleave the DNA fragments, we mixed Cas12a, sgRNA
of femA and
the plasmids. We tested the fluorescence of
the sgRNA after incubation. In the graph, the vertical axis represents the
relative intensity, which means the change in the intensity of the fluorescence. Faster decreasing slope
means higher efficiency in uncoiling the plasmid. Since all the sgRNA shows decreasing slope, all of them
successfully react with the plasmids, which means our experiment succeed. The fem-sg3 decreased fastest
with highest change in slope among the same types, which means it has highest efficiency within three
sgRNA.

![](https://static.igem.wiki/teams/4926/wiki/engineering/5-1.png)
![](https://static.igem.wiki/teams/4926/wiki/engineering/5-2.png)
![](https://static.igem.wiki/teams/4926/wiki/engineering/5-3.png)

Figure5:
 Relative fluorescence intensity of different sgRNA for femA

(represent
the sensitivity of different sgRNA)

Besides,
we test the reaction of the sgRNA in different concentration of bacteria liquid. By
this, we can find the minimum detectable concentration, and get the sensitivity of our test
kit. The
higher gradient means the higher efficiency of
cleaved the
plasmid
containing the target sequences.

At
the point that relative fluorescence intensity gets 1000000, human eyes could identify the change in the
light intensity. Therefore, the line of relative fluorescence intensity of different sgRNA that get the
1000000 in least time would represent the most effective sgRNA. We find out that sgRNA1 in femA group in
GbcA group is the most sensitive sgRNA.

**L****earn 1**

Our experimental results
showed that our DETECTR system is reliable and our product can be used to detect
Staphylococcus aureus. When the target gene femA was detected by the sgRNA, Cas12a was activated and
cleaved them. We verified the efficiency and sensitivity of the sgRNA by gel
electrophoresis and using fluorescent probes. We found that Cas12a could recognize and cleave the target
gene effectively. And sgRNA was sensitive.

Cycle 2

**Construct
design****and build****2**

pUC57-GbcA:
BBa\_K4926003

We designed a plasmid containing gbcA. The DNA fragment is from the genome of Pseudomonas aeruginosa to imitate the
bacteria. In order to construct our plasmid, we let the
company synthesize the DNA fragment GbcA, which was inserted into pUC57 plasmid.

![](https://static.igem.wiki/teams/4926/wiki/engineering/6.png)

Figure
6. pUC57-GbcA in  DH5αα

Same with pUC57-femA, we
let the company synthesize the fragment and verify the plasmid presented in the E.coli DH5α by
electrophoresis. As shown in Figure 6 and 7, we got the bacteria containing the plasmid and lanes which
length were same with GbcA.

![](https://static.igem.wiki/teams/4926/wiki/engineering/7.png)

Figure
7.  Bacteria PCR of GbcA.

**Function test
2**

![](https://static.igem.wiki/teams/4926/wiki/engineering/8.png)

Figure
8. Graph
of efficiency comparison of sgRNAs

We
also used T7 in vitro transcription kit to obtain the sgRNA of GbcA. We mixed Cas12a, sgRNA and the
plasmid. We tested the fluorescence of
the sgRNA after incubation. We can see all the two sgRNA shows decreasing slope, all of them successfully
react with the plasmids, which means our experiment succeed. The gbc-sg1RNA has highest efficiency.

![](https://static.igem.wiki/teams/4926/wiki/engineering/9-1.png)
![](https://static.igem.wiki/teams/4926/wiki/engineering/9-2.png)

Figure
9:  relative fluorescence intensity of different sgRNA for GbcA

(represent
the sensitivity of different sgRNA)

Besides,
we test the reaction of the sgRNA in different concentration of bacteria liquid containing
pUC57-gbcA. We
find out that the sgRNA2 in GbcA group is the most sensitive sgRNA.

**Learn2**

This system also could
be used to detect the Pseudomonas aeruginosa. When gbcA was detected by the sgRNA, Cas12a was
activated and cleaved them. We could detect the bacteria by gel electrophoresis and using fluorescent probes.

On the whole, we have innovatively applied CRISPR/Cas
technology to our diagnostic kit, expanding the scope of its application. Combined with simple heating
steps, our kit allows for fluorescence readouts that are easily observable. In conclusion, our product is
highly feasible and addresses many of the pain points and challenges associated with existing mainstream
detection methods.  It offers a new and promising solution for microbial detection.

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![](https://static.igem.wiki/teams/4926/wiki/experiments.png)

1. **PCR**

**Prepare:**

l Materials

|  |  |
| --- | --- |
| Reagent | Volume/mass |
| Template plasmid | 4μL |
| PrimeSTAR Max Premix | 100μL |
| Primer-F | 8μL |
| Primer-R | 8μL |
| ddH2O | To 200μL |

\*PrimeSTAR
Max Premix includes DNA polymerase, dNTP and buffer.

|  |  |
| --- | --- |
| Apparatus | Amount |
| PCR thermal cycler | 1 |
| Pipette | 1 |
| Microcentrifuge tube | 3 |

l **Procedure**

1. Configurate
the PCR system. Add s into tube

2. Divide
200 μL
system to 4\*50 μL
system

3. Put
4 tubes into PCR thermal cycler.

4.Set
PCR program 95℃ 5
min；95℃ 30
s，55℃ 30
s，72℃ 30，Cycles
72℃ 10
min 4℃∞。

**Agarose Gel
Electrophoresis**

l Materials

|  |  |
| --- | --- |
| Reagents | Volume/mass |
| Agarose | 1g |
| TAE Buffer | 100mL |
| Ts-GelRed | 10μL (10000X) |
| DNA Loading Buffer | 10X in template |
| Marker | 5μL |
| Template | -- |

|  |  |
| --- | --- |
| Apparatus | Amount |
| Conical flask | 1 |
| Pipette | 1 |
| Weighing paper | 1 piece |
| Electronic balance | 1 |
| Microwave oven | 1 |
| Mold for creating gel | 1 |
| Electrophoresis chamber | 1 |
| UV transilluminator | 1 |
| Scalpel | 1 |

l Procedure

**Making
gel**

1. Use
a balance and a digital scale to get the exact amount of reagent that we need.

2. Add
all the reagents into a conical
flask

3. Place
the conical
flask into
microwave oven until the reagents in the conical
flask turn
clear.

4. Placed
the beaker under water and slowly shook it so that the every part of the mixture can cooled down
evenly.

5. Add
10μL nuclei
acid dye by using a pipette( be careful as it is poisonous)

6. Put
the gel into the mold and insert a comb into the gel. Wait until the gel is solidified.

**Electrophoresis**

1. Take
out the solidified gel and place it in the electrophoresis chamber.

2. Add
the loading buffer to the sample.

3. Add
5μL marker and samples in the hole of the gel.

4. Start
the electrophoresis and wait for about 30 minutes.

5. After
the agarose gel electrophoresis done, using UV light to detect the DNA ladder and cut down the gel with
DNA,

6. Use
Gel doc to take the photo of DNA stripe

2. **Recycle the****gel**

l **Goal:** purify
the DNA sequence that we needed for latter experiment.

l **Material**

Reagent

agarose
gel

Binding buffer

deionized
water: 20µl

wash
solution:500µl

b.
Apparatus

pipette

pipette
tips

metal
bath instrument

l **Procedures**

1.Add
agarose gel and binding buffer into
one microcentrifuge tube.

2.
Place the tube in water bath at
50°C for 10 minutes.

3.
Transfer the solution into the spin column.

4.
Centrifuge the column at 12000rpm for 1 minute.

5.
Add 700μL wash buffer into the spin column and centrifuge.

6.
Repeat steps 5 for 2 times.

7.
Centrifuge the tube at 12000rpm for 2 minutes

8.
Put the spin column into a new microcentrifuge tube.

9.
Add 20μL water to the column membrane and centrifuge

3. **I****n vitro
transcription of sgRNA**

l **Goal：**synthesize
specific RNA molecules for the following experiment

l **Material**

a. Reagent

DNA
model 2μL

T7
transcription buffer 40μL

T7
transcription enzyme mix 4μL

RNase-free
H2O 34μL

EDTA
solution 500mM

b. Apparatus

pipette

test
tubes

PCR

PCR
tubes

fridge

l **Procedures**

1. Add
2μL DNA model, 40μL T7 transcription buffer, 4 μL
T7 transcription enzyme mix and 34μL
Rnase-free H2O to configurate reaction system.

2. Transport
the solution to PCR tubes
and put tubes into PCR Amplifier. Keep temperature at 37 degree for 2 hours.

3. Take
out the RNA and add 500mM EDTA solution to inactive T7 RNA polymerase.

4. Get
the RNA and preserve them in -80°C
fridge.

4. **Detection of sgRNA
cleavage by agarose gel electrophoresis**

l **Material**

a. Reagent

dye
2μL

FEM
sample

GBC
sample

marker

b. Apparatus

pipette

PCR
tubes

Agarose
Gel Electrophoresis

l **Procedures**

1. Inject
2μL
of the dye to the FEM and GBC sample in the PCR tubes and then add 1μL
to the control sample.

2. Add
the mixed solution into the glue with the sequence of marker, FEM, control, GBC and control.

3. Start
the electrophoresis.

5. **Preparation of LB
solution**

l **Objective**

To
prepare the basic material for bacteria cultivation

l **Material**

Reagent

Tryptone 2μL

Yeast
Extract

NaCl

Water

Apparatus

conical
flask

PCR
tubes

Agarose
Gel Electrophoresis

Pipette

Weighing
paper

Electronic
balance

l **Procedure**

1. Weigh
10g of Tryptone, 5g Yeast Extract and 10g of NaCl and put them in a 1L conical flask.

2. Add
about 800 ml of deionized water.

3. Add
deionized water to set the volume to 1L.

4. After
aliquot, autoclave at 121°C
for 20 min.

l **Procedure of LB****solid****culture****medium**

1. Add
1.5 g agar powder to 100 ml of LB solution, shake well.

2. Autoclave
at 121°C
for 20 min.

3. When
LB solution cooled to about 50°C,
add the corresponding concentration of antibiotics.

4. Mix
well, pour in a sterile Petri dish, and wait for the agar to solidify before use.

6. **Transformation of
competent cells of E. coli BL21**

l **Material**

Reagent

pUC57-femA

pUC57-gbcA

pET28a-LbCas12a

DH5α
chemically competent cell
200μL

Liquid
LB medium

Solid
LB medium

Apparatus

Petri
dish

Shaker

Pipette

Centrifuge
tube (1.5 mL)

Incubator

Bacterial
culture tube

l **procedure**

1. Prepare
a 42° water bath.

2. Take
200 μL of E.coli DH5a suspension from a -80 °C freezer and thaw on ice.

3. After
thawing the competent states, add plasmid DNA (1-10 ng, and the volume is < 10μL) to the competent
cell suspension, gently rotate the off-center tube to mix the contents, and take an ice bath for 30
min.

4. Incubate
in a water bath at 42°C for 90s and quickly cool on ice for 3min.

5. Add
lmL of LB liquid medium (without antibiotics) to the tube, mix well and culture lh at 37°C with
shaking to recover the bacteria.

6. After
shaking the above bacterial solution, take 100μL on the screening plate containing antibiotics,
place the front side up for 30min, after the bacterial solution is completely absorbed by the medium, put
the dish upside down at 37 °C for 16h-24h.

7. **IPTG formulation +
induction overnight**

**Material**

Reagent

IPTG

Apparatus

Pipette

Centrifuge
tube (1.5 mL)

Incubator

Bacterial
culture tube

l **procedure**

1. Weigh
0.238g IPTG

2. Add
deionized water to 10 mL and dissolve completely.

3. IPTG
solution was sterilized through membrane with a final concentration of 100mM.

4. Induction
concentration: 0.01 mM IPTG, 16 ℃ 180
rpm low temperature induction for 12 h overnight

**Reconnection**

1. Turn
30mL LB liquid medium to six bacterial
culture tubes with 5mL medium per tube.

2. Select
two bacterial colonies on
each of the three LB solid media with transformed *E. coli* on
them (pGEX-6P-1/ pGEX -6P-1-VP1/ pGEX-6P-1-VP1-LTB) and transfer them to the six tubes with one pipette
(range: 0.5-2.5μL).

3. Add
50μL ampicillin to each tube with another pipette (range: 20-200μL).

4. Incubate
the three tubes in the shaker for 5 hours.

**IPTG
induction**

1. Mark
three tubes as the control group and the others as the experimental group.

2. Aspirate
10μL bacteria from each tube, and measure OD600 value.

3. When
OD600 value
reaches 0.2-0.6, add 25μL IPTG to the three experimental tubes.

4. Incubate
the six tubes in the shaker for 3 hours.

8. **SDS****-****PAGE**

l Material

|  |  |
| --- | --- |
| Reagent | Volume |
| ddH2O | -- |
| Loading buffer | -- |
| Running buffer | -- |
| Coomassie brilliant blue staining solution | -- |
| Marker | 10μL |
| SDS-PAGE gel | 1 piece |

|  |  |
| --- | --- |
| Apparatus | Amount |
| Pipette | 1 |
| Microcentrifuge tube(1.5mL) | 1 |
| Electrophoresis chamber | 1 |
| Box | 1 |
| Microwave oven | 1 |

l Procedure

**Electrophoresis**

1. Transfer
bacteria liquid to microcentrifuge tubes.

2. Centrifuge
the tubes and
discard the supernatant.

3. Add
ddH2O
and loading buffer to the tube.

4. Place the
SDS-PAGE gel into electrophoresis
chamber and add
running buffer to it.

5. Add
marker and samples to the hole of the gel

6. Start
running.

**Staining**

1. Clean
the gel deionized
water 3 times after running.

2. Put
the gel into an appropriate amount of Coomassie Brilliant Blue dyeing solution, microwave until close to
boiling or just boiling, and stop heating immediately.

3. Then
shake on a room temperature shaker for 30 minutes under the condition that the temperature of the dyeing
solution is high.

4. Pour
out the staining solution.

5. Add
an appropriate amount of decolorizing solution to ensure that the dyeing solution can fully cover the
gel.

6. Heat
the microwave oven until it is close to boiling or just boiling and stop heating immediately.

7. Then
shake on the shaker for 30min under the condition that the temperature of the decolorization solution is
high. Relatively clear bands of protein are usually observed at this point.

8. Change
the fresh decolorization solution, decolorize for 2h, until the blue background is basically
removed.

9. Take
photos and take samples.

9. **Cas12a-crRNA
complex in assembly:**

**Material**

Reagent

Cas12a

50nM
sgRNA

NEBuffer2.1

Apparatus

Pipette

PCR
tube

PCR
amplifier

l **Procedure**

1. 50nM
Cas12a + 50nM sgRNA + NEBuffer2.1

2. Incubate
in a 20μL
system at 37°C
for 10min. According to the concentration calculation, the above is added to the PCR tube, expanding to
the 50ul system.

3. Centrifuge
the mixture for 30 sec.

4. Incubate
the solution in a 37°C
gradient thermal cycler for 10 min.

5. Add
about 100ng of oligo DNA fragments to be cut to the Cas12a-crRNA complex, incubate at 37 °C
for 2 hours, and inactivate the protein at 95 °C
for 5min.

6. This
is followed by 2.5-3% agarose gum identification: sgRNA, oligo DNA, oligo DNA(after cutting)

 **10.De****tection of sgRNA
efficiency by ssDNA fluorescent probes**

Material

 Reagent

 Cas12a

 50nM
sgRNA

 NEBuffer2.1

 Apparatus

 Pipette

 PCR
tube

PCR
amplifier

l **Procedure**

1. 50nM
Cas12a + 50nM crRNA + NEBuffer2.1

2. Incubate
in a 20μL
system at 37°C
for 10min

3. Configure
Cas12A-crRNA complex negative control (no incubation required):Add 38.5 μl
of reaction buffer to the microtube without protein and sgRNA; Add 0.5 μl
of the corresponding plasmid and 3 μl
of ssDNA fluorescent probe

4. In
each centrifuge tube, add the corresponding plasmid and ssDNA as recorded in the table above.

5. Denature
the Cas12a protein by placing the centrifuge tubes in a gradient thermal cycler and incubating at
37°C
for 2 h and then at 95°C
for 5 min.

6. Measure
the ssDNA fluorescence value

11. **Bacterial
lysis**

**Material**

 Reagent

 Lysis
buffer

 Apparatus

 Pipette

centrifuge
tubes

l **Procedure**

1. Aspirate
1 ml of bacterial solution into a centrifuge tube and centrifuge at 8,000 rpm for 1 min. Discard the
supernatant.

2. Add
50 μL
of bacterial lysis buffer to all centrifuge tubes.

3. Place
all tubes in a water bath and heat at 80°C
for 10 min.

4. Centrifuge
all tubes again at 8,000 rpm for 2 min.

5. Measure
the concentration and dilute to different concentrations of bacterial liquid.

DETECER
tests the sensitivity of different concentrations of bacterial solutions.

12. **Bacteria
collection**

**Material**

 Apparatus

 Pipette

 centrifuge
tubes

l **Procedure**

1. The
whole bacteria are centrifuged at 4 degree with 5000g X 5mins (Balance: 2 tubes with 59.8g; 2 tubes with
62.2 g)

2. Remove
the supernatant and retain pellet.

3. Add
10ml resuspension solution, make sure the pellets were dissolved and fell off from the test tube
wall.

4. The
pellet was sonicated for 20mins at 4 degrees with 300w, 10s/10s.

5. centrifuged
it again at 4 degrees, 16000g X 25mins.

6. The
rest of the supernatant was transferred into a whole new clean tube and was kept in the ice to maintain
the temperature of 4 degree.

12. **Ni-chelating
affinity purification**

**Material**

 Reagent

his-tag
purification resin

non-denaturing
lysate

affinity
column

non
denatured washing solution

 Apparatus

 Pipette

 centrifuge
tubes

1. Add
1ml 50% his-tag purification resin (mixed well) into the supernatant.

2. centrifuged
at 4 degree (1000g X 10s), Ni beads turns from blue into yellow.

3. Add
0.5ml non-denaturing lysate and shake it in ice (4 degree) for 2 hours

4. Transfer
the solution into the affinity column.

5. 1ml
non denatured washing solution is added to wash the non-relevant materials off. This process was repeated
three times.

6. 0.5
ml non-denatured elution solution is added to get our target protein. This process was repeated for four
times so we get 4 samples.

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![](https://static.igem.wiki/teams/4926/wiki/results.png)

Overview

Many cosmetics that
contain high water content and abundant nutrients are susceptible to microbial contamination.Staphylococcus aureus
and Pseudomonas aeruginosa are two types of pathogenic bacteria that can be found in certain
cosmetics.

We designed a swift and
straightforward detector to detect femA and GbcA, which were expressed in
Staphylococcus aureus and Pseudomonas aeruginosa, respectively. This detector is based on the
CRISPR/Cas12a-based DETECTR system, which is fast, accurate and easy to operate.

1.PCR amplification and plasmid construction

We designed and ordered three plasmids, pUC57-femA,
pUC57-GbcA and pET-28a(+)-FnCas12a-6His. from other company laboratories. Genes femA and GbcA are
amplified from the genome of Staphylococcus aureus and Pseudomonas aeruginosa respectively. In order to
construct our plasmids, we let the company synthesize the DNA fragments, femA and GbcA.
And these two fragments were inserted into pUC57 plasmid. pET-28a(+)-FnCas12a-6His
was obtained from the company laboratories.

We transformed the
pUC57-femA and pUC57-GbcA into the E.coli DH5α.As shown in Figure 1, we obtained the E.coli
DH5α with target fragments of femA and GbcA. To
verify whether these two plasmids presented in the E.coli DH5α, we performed
TAE agarose gel electrophoresis. As shown in Figure 2, we got lanes which length were same with
femA (1105bbp) and GbcA (646 bp). And we confirmed
that these two plasmids were in the E.coli DH5α. (Figure 2).

![](https://static.igem.wiki/teams/4926/wiki/results/1.png)

Figure 1.
pUC57-femA/pUC57-GbcA in DH5α

![](https://static.igem.wiki/teams/4926/wiki/results/2.png)

Figure
2.  Bacteria PCR of femA and GbcA.

2.In vitro transcription of sgRNA

The corresponding
genes of
sgRNA of femA and GbcA were obtained by PCR
(isothermal amplification) as DNA templates. We verified that the
sgDNA is the one we
planned to target by checking the bp length (Figure 3).

![](https://static.igem.wiki/teams/4926/wiki/results/3.png)

Figure
3. DNA fragment of femA and GbcA in target sgRNA

We then
used T7 in vitro transcription kit to obtain the
sgRNA of femA and GbcA. We also checked whether the sgRNA underwent a successful transcription by taking
its DNA template as negative control. As shown in figure 4, since sgRNA of both femA and GbcA is under
100bp, we confirmed that the T7 in vitro
transcription was successful.

![](https://static.igem.wiki/teams/4926/wiki/pinghe-001.jpg)

Figure 4. femA and GbcA sgRNA and DNA template

3.Cas-12a Protein Expression and Purification

We transformed the
pET-28a(+)-FnCas12a-6His expression
plasmid into *E.
coli* BL21(DE3)
and cultured
overnight.
Then we transferred the medium into 300mL fresh LB culture
medium. When the OD600 was around 0.6, we added IPTG to induce FnCas12a
expression at 16℃ for 12
hours.
Then FnCas12a was extracted and purified by His-tag.

As shown in Figure
5, the size of the LbCas12a protein is
between 100kDa and 160kDa. Hence we can see that
the protein was successfully induced to express.

![](https://static.igem.wiki/teams/4926/wiki/results/5.png)

Figure
5. SDS-PAGE analysis of LbCas12a
protein

4.Function Validation

![](https://static.igem.wiki/teams/4926/wiki/results/6.png)

Figure6.
Graph
of efficiency comparison of sgRNAs

After the
testing of the fluorescence of
the sgRNA after incubation, we draw this graph to show the efficiency of the different sgRNAs. In the
graph, faster decreasing slope means higher efficiency in uncoiling the
plasmid. Since all the sgRNA shows decreasing slope, all of them successfully react with the plasmids,
which means our experiment succeed. The gbc-sg1RNA and the fem-sg3 decreased fastest with highest change
in slope among the same types, which means they have highest efficiency.

Besides,
we tested the
reaction of the sgRNA in different concentration of bacteria liquid containing plasmids pUC57-femA and pUC57-GbcA. By
this, we can find the minimum detectable
concentration, and get the sensitivity of our test kit.

Below in Figure 7 and Figure 8 are the
graphical representations of
the sensitivities of the five sgRNA: The vertical axis represents the
relative intensity, which means the change in the intensity of the fluorescence. Therefore, the higher
gradient means the higher efficiency of
cleaved the
plasmid containing the target sequences.

![](https://static.igem.wiki/teams/4926/wiki/results/7-1.png)
![](https://static.igem.wiki/teams/4926/wiki/results/7-2.png)
![](https://static.igem.wiki/teams/4926/wiki/results/7-3.png)

Figure7:
 Relative
fluorescence intensity of different sgRNA for femA

(represent
the sensitivity of different sgRNA)

![](https://static.igem.wiki/teams/4926/wiki/results/8-1.png)
![](https://static.igem.wiki/teams/4926/wiki/results/8-2.png)

Figure8:
 Relative
fluorescence intensity of different sgRNA for GbcA

(represent
the sensitivity of different sgRNA)

At
the point that relative fluorescence intensity gets 1000000, human eyes could identify the change in
the light intensity. Therefore, the line of relative fluorescence intensity of different sgRNA
that get the 1000000 in least time would represent the
most effective sgRNA. And by analyzing the 1ul and 2ul, we can find the sensitivity of each sgRNA. After
the analyzing, we find out that sgRNA1 in femA group and the sgRNA2 in GbcA group is the most sensitive
sgRNA.

In
conclusion, each sgRNA has their own advantages and disadvantages. Therefore, we would not focus on any
single type of sgRNA to make our product. We will try to mixed these sgRNA together in a specific
concentration ratio to get the highest efficiency and sensitivity in detect the pathogen.

5.Future Plan

There are multiple pathogens that will lead to the infection in
the cosmetics.    In this project, we only designed the detector of two kinds of most
usual pathogens.    In the future, we can design more sgRNA for a larger detection range
in our test kit.    Besides, we can test different concentrations and combinations of the
sgRNA and find the highest efficiency for sgRNA.    Also, we will use other instruments
like Cas13 and Cas 9 proteins for other pathogens in addition to the bacteria.
   Additionally, we can also design a test kit that can be applied to other conditions
like food testing, water testing, or even air testing.    As a result, with improved or
multiple solutions for detecting, our test kit can be more useful and convenient.

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