iGEM 2022 — Team shbs-banz

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{% block title %}Notebook{% endblock %}
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Notebook

Wet Lab - ATCG Daily Events Summarizing

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Jul 10th

Introduction between members and project’s ideas sharing
Design of team’s name, logo, and peripheral products
Establishment of the official account

![](https://static.igem.wiki/teams/4411/wiki/t-shbs-banz-notebook01.jpg)

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Jul 11th

Break

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Jul 12th

Laboratory Safety Training
Learning how to use laboratory tools: pipette, PCR machine, shaker, sterilizer
Learning basic techniques: how to configure reagents and LB medium; practice the slab scribing method
Incubate strain contains pET28A plasmid.

![](https://static.igem.wiki/teams/4411/wiki/t-shbs-banz-notebook02.gif)

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Jul 13th

PCR to add TSD DNA fragment to pUC19 plasmid, gel electrophoresis and gel extraction, transform the PCR amplicons
into E. coli DH10b competent cells and coat on LB solid medium plate named pUC19-TSD.

![](https://static.igem.wiki/teams/4411/wiki/t-shbs-banz-notebook03.jpg)

pET28a plasmid extraction

![](https://static.igem.wiki/teams/4411/wiki/t-shbs-banz-notebook04.png)

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Jul 14th

Agarose gel preparation, gel electrophoresis
pUC19-TSD plasmid extraction and send for Sanger sequencing
Inoculate DH10b into LB culture medium, incubate at 37℃, 200rpm overnight

![](https://static.igem.wiki/teams/4411/wiki/t-shbs-banz-notebook05.jpg)

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Jul 15th

PCR to amplify different lengths of Kana gene-containing fragments
PCR to amplify DNA fragments containing Ampicillin
Gel electrophoresis and gel extraction
Electrocompetent Cell Preparation

![](https://static.igem.wiki/teams/4411/wiki/t-shbs-banz-notebook06.png)

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Jul 16th

In vitro system reaction with the casposase and recycle the DNA pool
Electronic shock Transformation to introduce the recycled DNA pool into competent cells, incubate at 37℃ overnight

![](https://static.igem.wiki/teams/4411/wiki/t-shbs-banz-notebook07.jpg)

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Jul 17th

Calculate the number of colonies of the transformed strain.
Inoculate the single strain into LB liquid culture medium.

![](https://static.igem.wiki/teams/4411/wiki/t-shbs-banz-notebook08.png)

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Jul 18th

Plasmids extraction and send for Sanger sequencing.
Filming Promotion Video

![](https://static.igem.wiki/teams/4411/wiki/t-shbs-banz-notebook09.jpg)

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Jul 19th

Wiki composing
Data collection

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{% block title %}Engineering Success{% endblock %}
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Engineering

Introduction & Motivation

CRISPR-Cas9 is the most commonly used gene editing tool in laboratories nowadays. This technique shows excellent
simplicity and efficiency. Reversely, the flaw of it as the high potential rate of off-targeting is also
non-negligible. Spacers are short sequence segments similar to phage or DNA, and it’s being used in the CRISPR-Cas9
technique to enable the insertion of an exogenous DNA sequence into part of the internal genes system. To decrease
the off-targeting rate of CRISPR-Cas9 and enhance this gene editing tool into a more precise one, we chose the
casposons as a gene editing tool and decided to change the spacer used in this technique into pUC19-TSD plasmids
according to casposons. By this means, we would receive a gene editing tool that’s more reliable and safer to use
(Figure 1).

![](https://static.igem.wiki/teams/4411/wiki/t-shbs-banz-engineering-success01.png)
Figure 1. The working principle of CRISPR and casposons

Experimental Principle and Design

In our project, we use casposons to achieve gene insertion. For easy detection, we chose the resistance gene,
Kanamycin, as the target gene and set up a precise gene editing system in vitro.

a) pUC19-TSD Plasmids construction

In order to insert the TIR sequence into the pUC19 plasmid (Figure 2A), we use PCR to amplify the target DNA
fragment and inserted the amplicons into DH10b competent cells. Inoculated a single colony into LB (Amp) culture
medium, extracted the plasmid, and send to the company for Sanger sequencing (Figure 2B). As shown in the sequencing
result, we successfully constructed the plasmid.

![](https://static.igem.wiki/teams/4411/wiki/t-shbs-banz-engineering-success02.jpg)
Figure 2. the pUC19-TSD plasmids and sequencing data. A. The pUC19-TSD plasmids, B. the sequencing data mapped
to the plasmid.

b) PCR amplification of target genes

In order to obtain our target genes, we amplified different lengths of the target genes containing the Kanamycin
gene fragment from the pUC19-DONER plasmid. In order to successfully amplify the genes, we use different annealing
temperatures, such as 59℃, 61℃, and 63℃ (Figure 3).

![](https://static.igem.wiki/teams/4411/wiki/t-shbs-banz-engineering-success03.png)
Figure 3. The PCR gel electrophoresis result of the Kana gene fragment. M. DNA marker, 1. The annealing
temperature is 59℃, 2. The annealing temperature is 61℃, 3. 1. The annealing temperature is 63℃

In figure3, a clear and single DNA band at 1kp can be seen, indicating that we
successfully amplified our target genes. We extracted the DNA fragments and stored them at -20℃ for future use.

Casposons functional test

a) In vitro casposons gene-editing system

To verify whether the long fragment gene with TIR sequence could be inserted into the TSD sequence effectively and
correctly, the protein casposase was added for reaction, and the reaction products were recovered. Mixed components
according to the table below, reacted in a metal bath at 37°C for 1h. Add PK enzyme at 37°C for 30min, then 95°C for
10min to terminate the reaction, added isopropanol into the reaction system and discard the supernatant, and
resuspend the pellet with sterile water.

![](https://static.igem.wiki/teams/4411/wiki/t-shbs-banz-engineering-success04.jpg)

b) Screen for TIR-Kan plasmids

We transformed the recycled plasmids pool into E. coli DH10b competent cells, and coat on the LB solid medium plate
containing both Kanamycin and Ampicillin antibiotics, incubated at 37℃ overnight. The next day, we calculated the
number of colonies on the plate (Figure 4).

![](https://static.igem.wiki/teams/4411/wiki/t-shbs-banz-engineering-success05.jpg)
Figure 4. The plates of recombinant plasmids containing strains. NC: pUC19-TSD, PC: pUC19 (Amp plate), 1258bp:
PUC19-TSD-1258bp-TIR-Kan gene

c) Sanger sequencing to amplify the recombinant plasmids

We inoculate the single colony in the LB liquid culture medium (Kana+Amp), extracted plasmids, amplified the
target-gene-containing fragments, and send the company for Sanger sequencing. The returned sequencing comparison
results showed that there were no mutations in the ORF region (Figure 5), and the plasmids were successfully
constructed. So far, we have successfully developed our gene editing system.

![](https://static.igem.wiki/teams/4411/wiki/t-shbs-banz-engineering-success06.jpg)
Figure 5. The sequencing data mapped to the plasmid sequence

As the result shown above, compared with the negative control, we can find that with the casposase in the reaction
system we successfully inserted the Kanamycin gene into the pUC19 plasmid so that the strain could grow on the plate
containing antibiotics. So that we successfully developed a precise gene editing system in vitro, and this system
could be used in future use or even be applied in clinical treatment.

Learn

We have already collected the figures from our experiments. Casposons is a new gene editing tools that can implement
gene insertion precisely. In our project, we set up an in vitro gene editing system with the casposons. From the
result, we can find out that with the casposase protein in this system, we inserted the Kanamycin gene into the
pUC19 plasmid at the TSD site.
Because there are many diseases caused by gene variation, such as gene duplication, gene deletion, gene mutation,
and so on, it is important to develop a gene editing tool to solve these problems at its source. Casposons is one of
the tools nowadays. In the very near future, this tool will be improved and may be used in disease treatment and
bring patients a better life.

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Experiments

1. Preparation of DH10 b competent cells

1.1 Preparation of LB solid medium

1. Required substances: 5g peptone, 2.5g yeast extract, 5g NaCl, 500ml deionized water to prepare LB culture
medium
2. Put the culture medium into a shaker to mix up
3. After shaking, add 10g of agarose (1g of agarose per 100ml)
4. Use of sterilizer: first take out the sterilization frame, then add enough water to the pot, so that the water
surface is slightly below the triangular shelf
5. Put back the sterilization frame and fill it with the items that need to be sterilized. Be careful not to pack
too much so as to prevent the steam flow and affects the sterilization effect. Do not contact the wall of the
triangular flask and the mouth of the test tube to avoid condensation from wetting the paper at the mouth of the
package and penetrating the cotton plug
6. Add the lid, then tighten the two bolts in a symmetrical manner, so that the bolts are in the same situation
(or
the rotating disk on the sterilization pot is tightened), and do not make air leakage
7. Set the sterilization temperature to 121℃ and time to 25 minutes, and open the exhaust valve at the same time,
turn on the power to make the water boil to exclude the cold air in the pot. After the cold air is completely
exhausted (at about 109°C), close the exhaust valve and let the temperature inside the pot rise gradually with the
increase of steam pressure. When the pressure in the pot rises to the desired pressure, the heat source is
automatically controlled to maintain the pressure for the desired time
8. When the required time for sterilization is up, cut off the power supply and let the temperature inside the
sterilizer drop naturally. When the pressure in the pressure gauge drops to 0, open the exhaust valve, loosen the
bolt, open the lid and take out the sterilized items. If you open the exhaust valve when the pressure does not
drop
to 0, the pressure inside the pot would drop suddenly, causing the culture medium inside the container to rush out
of the flask mouth or test tube mouth due to the imbalance of internal and external pressure, resulting in
contamination by cotton plugs staining the culture medium
9. After completing sterilization, wait for the medium temperature to be about 50℃, then move it into the Petri
dish
for cooling

1.2 Coated plate method for culturing competent cells

1. Take an LB solid medium plate (non-antibodies) and place it in a 37°C incubator to preheat
2. Take a tube of Top10 competent cells, burn the inoculation loop to sterilize, and cool it (lean against the
inner wall a few times)
3. Then dip it in Top10 bacterial solution and line on the preheated plate (draw three to five parallel lines
quickly with the inoculation loop dipped in the strain, cauterize the inoculation loop again, cool it, lean
against the blank medium a few times, start drawing lines from the end of the first area, repeat the above steps
in the third area to avoid connecting one and three areas)
4. The plate is inverted at 37℃ and incubated overnight. When the colony grows up, it can be stored at 4℃
5. Inoculate a single colony with a pipette into LB liquid medium (without antibiotics) and incubate overnight at
37°C, 200 rpm
6. Transfer 1ml of the cultured medium into 100 mL fresh LB-liquid medium (without antibiotics)
7. 37°C, 200 rpm, shake until OD600 is 0.3-0.5 (competent cells are suitable for about 0.4)
8. Place the conical flask directly in the ice bath for 15 min
9. Transfer 100 mL of the bacterial solution to two 50 mL centrifuge tubes (pre-cooled in the ice bath)
10. Centrifuge at 4500 rpm, 4°C, for 8 min (cool down the centrifuge in advance)
11. Discard the supernatant and add 2 mL of sterile water to each tube and gently resuspend the cells (dispense an
appropriate amount of sterile water from the ultra-clean table and pre-chill on ice in advance)
12. Centrifuge at 4500 rpm, 4°C, for 8 min
13. Discard supernatant, centrifuge briefly, and aspirate supernatant
14. Add 5 mL of (0.05 M CaCl2 + 15% glycerol) premix to each tube, blow gently, resuspend the organism and bathe
on ice for 45 min. (operate on ice, use a sterilized gun tip on the ultra-clean table)
15. Centrifuge, 4500 rpm, 4°C, 8 min
16. Discard the supernatant and aspirate the liquid.
17. Add 2 mL (0.05 M CaCl2 +15% glycerol) premix into tube ① first, resuspend the bacteria, then transfer to tube
②, then wash tube ① with 1 mL premix, transfer to tube ②, and mix 3 mL of bacteria in tube ②, mix well
18. Dispensation (100 μL per tube), and label the tube with the strain name and freeze at -80℃

1.3 DNA nucleic acid gel electrophoresis

1. Dissolve agarose and prepare the gel
2. Add an appropriate amount of electrophoresis buffer and note that the volume should not exceed one-third of the
conical flask.
3. Add ethidium bromide to a concentration of 0.5 mg/ml
4. First cool the dissolved agarose gel solution in a 65 ℃ water bath, and DNA nucleic dye
5. Put the comb into the glue to form a sample loading hole. Pour the dissolved agarose gel into the mold and then
solidify at room temperature
6. After the agarose gel solidifies, remove the comb
7. Before starting electrophoresis, add the loading buffer to the DNA sample to be separated. Set the power supply
to the specified voltage. Add enough electrophoresis buffer into the electrophoresis tank until it covers the
surface of the gel. Plug the connector of the electrophoresis tank into the power supply and then turn on the
power supply
8. Remove the electrophoresis tank cover and slowly and carefully add DNA samples. Cover the electrophoresis tank
and check again whether the electrode is plugged into the correct socket of the power supply
9. Turn on the power and start electrophoresis until the dye reaches the appropriate position of the gel
10. Observe the isolated DNA fragments. First, take out the gel from the glue tank and irradiate it with the UV
lamp
11. After electrophoresis, turn off the power and remove the electrophoresis tank cover. Take out the gel from the
electrophoresis tank and drain the excess electrophoresis buffer on the surface. Put the glue tank on a paper
towel and suck up the remaining electrophoresis buffer

1.4 PCR amplification and amplification product recovery

1. Cut down the gel containing the target fragment and calculate the weight difference in the EP tube from adding
the gel by weighing the tube before and after adding the gel
2. Add 600 μl buffer into the tube, place it in a 60 ℃ water bath for 10min, and mix it every 2 minutes to make
the rubber block melt completely
3. Transfer the entire dissolved solution to the adsorbent column and place it at room temperature for 2 min
4. Centrifuge at 8000 rpm for 1 min at room temperature and discard the filtrate in the collection tube
5. Put the adsorption column into the same collection pipe and add 500 μ L wash solution, 8000rpm, centrifuged at
room temperature for 1min
6. Repeat the 4th step
7. Remove the adsorption column, discard the filtrate, put the adsorption column back into the same collection
tube, and centrifuge at room temperature at 12000 rpm for 1 minute
8. Put the adsorption column into a new 1.5 ml centrifuge tube, add 40ml elution buffer in the center of the
column, and place for 2 minutes at room temperature or 37 ℃
9. Centrifuge at 12000rpm for 1 min at room temperature and the liquid in the centrifuge tube is the recovered
product

2. PREPARATION OF PLASMID

2.1 PCR amplification

1. Use a pipette to add reagents in a PCR tube and mix up (which contains enzymes required for PCR; dNTPs; ddH2O;
and Buffer). Next, add the primers into the tube. Afterward, divide the prepared mixture into 2 PCR
tubes.

2. Put the PCR tube into the PCR machine. Close the lid and set the program.

| Steps | Temperature | Time | Number of cycles |
| --- | --- | --- | --- |
| Initial denaturation | 94 ℃ | 5 min | / |
| Denaturation | 94 ℃ | 30 sec | / |
| Primer annealing | 55 ℃ | 45 sec | 33 |
| Extension | 72 ℃ | 2 min | 33 |
| Final extension | 72 ℃ | 5 min | / |

\*PS: Cycle steps 2 to 4, 34 times.

2.2 PLASMID EXTRACTION (PURIFICATION)

1. Inoculate the bacteria and culture overnight, and centrifuge the cultured medium at 12000rpm for 1 min. Discard
the supernatant
2. Add 500μl of the balance solution to the adsorption column, centrifuge the discard the filtrate
3. Add 250μl of Buffer SP1 from the Diamond Mini Plasmid Kit to the pellet cells. Add 250μl Buffer SP2 and 350μl
Buffer SP3 and mix well.
4. Centrifuge at 12000rpm for 10 minutes, add the supernatant to the adsorption column, and centrifuge at 8000rpm
for 30s.
5. Add 500 mL of wash solution, centrifuge at 9000×g for 30 seconds, and pour off the liquid in the tube (repeat
once)
6. Centrifuge the empty adsorption column at 9000 ×g for 1 minute
7. Put the adsorption column into a new 1.5mL centrifuge tube, add 100 mL of Elution Buffer to the adsorption
membrane, stand at room temperature for 1 minute, and centrifuge for 1 minute.

2.3 HOMOLOGOUS RECOMBINATION

1. In the process of PCR amplification, the terminal homologous sequence of the linearized cloning vector was
introduced at the TSD end and TIR end
2. The assembly enzyme and PCR-amplified products are mixed together so that the insert can be positioned on the
linearized vector. Assembly enzyme catalyzes a homologous recombination reaction to form a recombinant
vector
3. Transform the recombinant plasmids into competent cells and coat on the LB solid medium plate

3. Extraction of target plasmids and detection

3.1 Plasmids extraction without kit

1. Inoculate the transformed strain, incubate at 37 ℃for 12 ~ 16 hours
2. Add 1.5ml bacterial solution into EP centrifuge tube, centrifuge 12000g for 30 seconds, and discard the
supernatant
3. Add 100μl of cooled solution I, resuspend bacterial cells on the vortex oscillator to disperse the cells as
much as possible
4. Add 200μl of newly prepared solution II, quickly reverse the centrifuge tube to mix the contents, and place it
on ice for 3-5min (NaOH in solution II can cleave cells, denature DNA and denature proteins to form a cross-linked
network structure)
5. Add 150μl of solution III, reverse it 6-7 times after capping, mix it evenly, and place it on ice for 2 ~ 3min
(Solution III is a low pH potassium acetate buffer that neutralizes NaOH to renature partially denatured
closed-loop plasmids, while bacterial chromosomal DNA cannot be renatured correctly)
6. Centrifuge 12000 g for 6 min, and transfer the supernatant into another clean EP tube
7. Add 2 times the volume of supernatant (about 1ml) of absolute ethanol, shake and mix well, and place at room
temperature for 2min
8. Centrifuge at 12000g for 10min, the supernatant was discarded, and then washed once with 70% ethanol.
Centrifuge at 12000g for 1min, discard the liquid
9. Add 40ml of sterile distilled water extract containing 20 mg/ml RNase A, place it at room temperature until the
plasmid is completely dissolved (about 8min), store it at - 20 ℃ or directly use it for enzyme digestion

3.2 in vitro reaction system

1. Mix the target gene product with casposase protein in equal proportion. React in a metal bath at 37°C for
1h
2. Add 4uL PK enzyme per tube (PK/0.5M EDTA v/v=1:1)
3. 37℃ metal bath digestion for 30min
4. 95℃ 10min inactivation 100uL/tube isopropanol precipitation 1h
5. Recovery of integration product 100uL DH10b (electrotransfer)/sample
6. Add 1mL SOC medium Recovery for 1h
7. Centrifuge at 4000rpm for 8min, discard 850uL supernatant
8. Blow the remaining well, coat all, and pick monoclonal
9. Plasmid extraction

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{% block title %}Results{% endblock %}
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Results

1. Schematic diagram of the mechanism underlying the casposase mediated TIR integration

In this project, we developed a precise gene-editing system in E. coli named casposons (Figure 1), in order to test
the length of the integrated gene, we chose kanamycin as our reporter system.

![](https://static.igem.wiki/teams/4411/wiki/t-shbs-banz-result01.jpg)
Figure.1 Schematic representation of the kanamycin resistance screen to detect integration near the leader-TSD
junction(Wang X, et al, 2021).

a) Constructed TSD-containing plasmid

In order to insert the TIR sequence into the pUC19 plasmid (Figure 2A), we use PCR to amplify the target DNA
fragment and inserted the amplicons into DH10b competent cells. Inoculated a single colony into LB (Amp) culture
medium, extracted the plasmid, and send to the company for Sanger sequencing (Figure 2B). As shown in the sequencing
result, we successfully constructed the plasmid.

![](https://static.igem.wiki/teams/4411/wiki/t-shbs-banz-result02.png)

Figure 2. the pUC19-TSD plasmids and sequencing data.
A. The pUC19-TSD plasmids,
B. the sequencing data mapped to the plasmid.

b) PCR amplification of target genes

In order to obtain our target genes, we amplified different lengths of the target genes containing the Kanamycin
gene fragment from the pUC19-DONER plasmid (Figure 3A). In order to successfully amplify the genes, we use different
annealing temperatures, such as 59℃, 61℃, and 63℃ (Figure 3B).

![](https://static.igem.wiki/teams/4411/wiki/t-shbs-banz-result03.png)

Figure 3. Different lengths of target genes.
A. the template plasmid containing Kanamycin gene,
B. different lengths of target DNA fragments.

c) In vitro casposons gene-editing system

To verify whether the long fragment gene with TIR sequence could be inserted into the TSD sequence effectively and
correctly, the protein casposase was added for reaction, and the reaction products were recovered. Mixed components
according to the table below, reacted in a metal bath at 37°C for 1h. Add PK enzyme at 37°C for 30min, then 95°C for
10min to terminate the reaction, add isopropanol into the reaction system and discard the supernatant, and resuspend
the pellet with sterile water.

![](https://static.igem.wiki/teams/4411/wiki/t-shbs-banz-result04.png)

d) Screen for TIR-Kan plasmids

We transformed the recycled plasmids pool into E. coli DH10b competent cells, and coat on the LB solid medium plate
containing both Kanamycin and Ampicillin antibiotics, incubated at 37℃ overnight. The next day, we calculated the
number of colonies on the plate (Figure 4).

![](https://static.igem.wiki/teams/4411/wiki/t-shbs-banz-result05.jpg)

Figure 4. The plates of recombinant plasmids containing strains.
NC: PUC19-TSD;
PC: PUC19 (Amp plate);
1258bp: PUC19-TSD-1258bp-TIR-Kan gene;
2151bp: PUC19-TSD-2623bp-TIR-Kan gene;
3292bp: PUC19-TSD-3292bp-TIR-Kan gene;
3452bp: PUC19-TSD-3452bp-TIR-Kan gene.

Because the colonies on the plate are too intensive to calculate, we resuspended the colonies with LB culture
medium, incubated at 37℃ for 30min, diluted them 8 times, coated them on the LB (Kana+Amp) solid medium plates and
incubated them at 37℃ overnight (Figure 5). The next day, we calculated 1/4 area of the plate of the number of
colonies (Figure 6). Because the colonies on the plate are too intensive to calculate, we resuspended the colonies
with LB culture medium, incubated at 37℃ for 30min, diluted them 8 times, coated them on the LB (Kana+Amp) solid
medium plates and incubated them at 37℃ overnight (Figure 5). The next day, we calculated 1/4 area of the plate of
the number of colonies (Figure 6).

![](https://static.igem.wiki/teams/4411/wiki/t-shbs-banz-result06.jpg)

Figure 5. The plates of diluted recombinant plasmids containing strains.

![](https://static.igem.wiki/teams/4411/wiki/t-shbs-banz-result07.png)

Figure 6. The number of recombinant plasmids containing strains.

As a result, we can find that when the length of inserted gene is around 3.5k, we still achieved gene-editing with
casposons. What’s more, as the length of the inserted gene increased, the number of colonies decreased. However,
casposons is still an excellent tool we could use in future research for gene editing.

e) Sanger sequencing to amplify the recombinant plasmids

We inoculate the single colony in the LB liquid culture medium (Kana+Amp), extracted plasmids, amplified the
target-gene-containing fragments, and send the company for Sanger sequencing. The returned sequencing comparison
results showed that there were no mutations in the ORF region (Figure 7.), and the plasmids were successfully
constructed. So far, we have successfully developed our gene editing system.We inoculate the single colony in the LB
liquid culture medium (Kana+Amp), extracted plasmids, amplified the target-gene-containing fragments, and send the
company for Sanger sequencing. The returned sequencing comparison results showed that there were no mutations in the
ORF region (Figure 7.), and the plasmids were successfully constructed. So far, we have successfully developed our
gene editing system.

![](https://static.igem.wiki/teams/4411/wiki/t-shbs-banz-result08.png)
Figure 7. The sequencing data mapped to the plasmid sequence

2. Integration Ampicillin with casposons

a) Construct Ampicillin expression plasmid

We amplified the Ampicillin DNA fragment and promoter from template DNA and extracted it from the gel, and stored it
at -20℃ for future use. In order to successfully amplify the genes, we use different annealing temperatures, such as
57℃ and59℃ (Figure 8). And as shown in the figure, there were two clear bands at 1kb can be seen, which means we
correctly amplified the target gene fragment.

![](https://static.igem.wiki/teams/4411/wiki/t-shbs-banz-result09.png)
Figure 8. Gel electrophoresis diagram.

b) In vitro casposons gene-editing system

Mixed components according to the table below, reacted at 37℃ for 1h in the metal bath; 4uL PK enzyme (PK/ 0.5m EDTA
V/V =1:1) was added to each tube, and digested in the metal bath at 37℃ for 30min and inactivated at 95℃ for 10min,
added isopropanol into the reaction system and discard the supernatant, and resuspend the pellet with sterile water.

![](https://static.igem.wiki/teams/4411/wiki/t-shbs-banz-result10.png)

c) Screen for TIR-Amp plasmids

We transformed the recycled plasmids pool into E. coli DH10b competent cells, and coat on the LB solid medium plate
containing both Kanamycin, Ampicillin antibiotics, and IPTG, incubated at 37℃ overnight. The next day, we calculated
the number of colonies (Figure 9).

![](https://static.igem.wiki/teams/4411/wiki/t-shbs-banz-result11.png)

Figure 9. The plates of recombinant plasmids containing strains.
A. pET28a + Amp DNA fragment transformed strain,
B. pET28a +Amp DNA fragment + casposase transformed strain.

From the result, compared with the negative control, we can find that with casposase in the reaction system we
successfully inserted the Ampicillin gene into the pET28a plasmid so that the strain could grow on the plate.

Reference

Wang X, Yuan Q, Zhang W, Ji S, Lv Y, Ren K, Lu M, Xiao Y. Sequence specific integration by the family 1 casposase from Candidatus Nitrosopumilus koreensis AR1. Nucleic Acids Res. 2021 Sep 27;49(17):9938-9952. doi: 10.1093/nar/gkab725. PMID: 34428286; PMCID: PMC8464041.

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