iGEM 2023 — Team zhejiang-united

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Laboratory Notebook

**8.17:**

1. Preparation
of LB medium

2. Production
of gRNA-lacZ PCR system

3. Agarose
gel electrophoresis

4. PCR products were recovered using a DNA extraction kit and the
concentration was determined

5. Nanondrop
concentration measurement

**8.18:**

1. Demethylation
of gRNA-lacZ

2. Inactivation
of *Dpn*I

3. PCR
product recovery

4. Nanodrop
to measure concentration

5. Preparation of PCR system for Cas9-OP and ColE1

6. Transformation
of *Escherichia
coli* DH5a
competent cells

**8.19:**

1. Select
monoclonal identification and shake evenly for verification

2. Preparation
of PCR system

3. Agarose
gel electrophoresis

4. Monoclonal
colonies were transferred for culturing

**8.20:**

1. Amplification
of homologous arms through PCR

2. Plasmid
extraction (gRNA-gntT)

3. Electrophoretic
detection of extraction results

4. PCP product
recovery

**8.21:**

1. Expanded
culture and induction of p15A-Cas9

2. Homologous recombination ColE1-Cas9 plasmid

3. Agarose
gel electrophoresis

4. PCR
product recovery

5. Measure the
concentration of product recovery results by nanodrop concentration measurement

**8.22：**

1. Extraction
of crude protein by ultrasonic crushing

2. Protein
purification

3. Configuration
of LB liquid culture medium.

**8.23：**

1. Configure
the protein gel and electrophoresis

2. Extract
the plasmid

3. Measure
the
concentration of plasmid extraction

4. Run
SDS-PAGE

**8.24**

1. Analysis
of SDS-PAGE results

2. Bradford
method for protein concentration (standard curve section)

3. Draw a standard curve

4. PCR
products lambda-Red plastic recycling

**9.****16**

1. PCR system for cas9-op and 15a fragments

2. PCR products were recovered using a DNA extraction kit and the
concentration was determined

3. Homologous recombination 15A-Cas9-op plasmid

4. Nanondrop
concentration measurement

5. Transformation of Escherichia coli DH5a competent
cells

**9.1****7**

1. Select
monoclonal identification and shake evenly for verification

2. Preparation
of PCR system

3. Agarose
gel electrophoresis

4. Monoclonal
colonies were transferred for culturing

**9.1****8****:**

1. Expanded
culture and induction of p15A-Cas9-op

2. Extract the plasmid

3. PCR
product recovery

4. Measure the concentration of plasmid extraction

5. Prepare Nissle 1917 competent cells

**9.****20****：**

1. Extraction
of crude protein by ultrasonic crushing

2. Protein
purification

3. Run SDS-PAGE

4. Bradford
method for protein concentration

5. PCR amplifies the upstream homology arm and downstream homology
arm of gnTt and lacZ, and overPCR connects the homology arms.

**9****.****21****:**

1. Analysis
of SDS-PAGE results

2. Nissle 1917 Electroporation with 15A-Cas9-op and 15A-Cas9, respectively

**9****.****22****:**

1. Nissle 1917 Electroporation with sgRNA and homology arms, respectively

**9****.****25/26****:**

1. Nissle 1917 Editing efficiency test

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Overview

Nissle 1917 is an
*E.
coli* strain with probiotic
properties, but for certain applications, it is necessary to knock out specific genes[1]. Since CRISPR-Cas systems
have high target DNA specificity and programmability, they can serve as gene editing
tools[2]. The development and
optimization of such tools can facilitate the construction of engineered cell lines for diverse production
needs while accelerating the exploration of biological systems. However, unwanted escapers that do not
undergo the intended editing frequently arise during CRISPR-Cas-mediated microbial genome editing,
reducing editing efficiency[3].

Therefore, this project aims
to construct a highly efficient CRISPR-Cas9-based genome editing tool for
*E.
coli* Nissle 1917 by
increasing Cas9 copy number and codon
optimization, lowering escape rates to improve editing efficiency, and providing a reference for other
genome editing tools. Herein, we constructed two Cas9 systems to test the gene editing
efficiency in *E. coli* Nissle 1917,
one of which was designed by Prof. LI QI[3],
and the other was redesigned by codon optimization.

Cycle 1: Editing efficiency test of p15A-Cas9-λ-Red (BBa\_K4876011) in E. coli Nissle 1917

**Design 1:**

The original Cas9 encoding plasmid has a
lower copy of pSC101 origin. We selected the higher copy p15A origin to increase Cas9 expression. This allows
the retention of functional Cas9 during host editing to
ensure normal CRISPR-Cas system operation, reducing escape rates and improving editing efficiency. In
addition, the incorporation of the λ-Red recombination system can enhance editing efficiency, so we
constructed the p15A-Cas9-λ-Red plasmid (Figure
1).

![](https://static.igem.wiki/teams/4876/wiki/engineering/1.png)

**Figure****1****Engineering****frame of
the p15A-****Cas9****-λ-Red plasmid.**

**Build 1:**

To
construct p15A-Cas9-λ-Red,
we amplified p15A and Cas9-λ-Red
using primers with homology arms and recombined them using a cloning kit to generate p15A-Cas9-λ-Red.
After transformation, single colonies grew on LB plates. Colony PCR and sequencing verified the correct
construction of the p15A-Cas9-λ-Red
plasmid (Figure 2).

![](https://static.igem.wiki/teams/4876/wiki/engineering/2.jpg)

**Figure****2****The construction results of
p15A-****Cas9****-λ-Red.**

**(****A)** **Colony PCR results. (B) Sequencing
results.**

**Test 1:**

To
verify Cas9 expression
in *E. coli* Nissle
1917, we transformed the constructed p15A-Cas9-λ-Red
plasmid. After inducing expression, we lysed the cells by sonication and purified Cas9. The
results showed that we successfully induced Cas9 protein
expression (Figure 3).

![](https://static.igem.wiki/teams/4876/wiki/engineering/3.jpg)

**Figure****3****The SDS-PAGE result of****p15A-****Cas9****-λ-Red****protein****expression****.**

Subsequently,
we tested the induction of Cas9 expression
by different concentrations of L-arabinose. The results showed that with higher concentrations of
L-arabinose, the expression of Cas9 was
higher, suggesting that we could control the level of Cas9 by
regulating the addition of L-arabinose (Figure 4).

![](https://static.igem.wiki/teams/4876/wiki/engineering/4.jpg)

**Figure****4****Results of****Cas9****protein induction by
L-arabinose.**

**(****A) Standard curve of BSA
concentration.****(B)** **The curve of** **Cas9****concentration.**

We
electroporated p15A-Cas9-λ-Red
along with gRNA-gntT/lacZ and gntT/lacZ-HR homology arms into*E. coli* Nissle
1917. After 16 h growth, colony PCR identified transformants. As shown in Figure 5,
p15A-Cas9-λ-Red
achieved 100% knockout of *gntT* and
80% for *lacZ* in
*E. coli* Nissle
1917.

![](https://static.igem.wiki/teams/4876/wiki/engineering/5.jpg)

**Figure****5****Editing efficiency results of****Cas9****in E. coli Nissle 1917.**

**Learn****1****:**

The
use of a higher copy origin in *E. coli* Nissle
1917 effectively increased the Cas9 copy
number and increased the editing efficiency to 100% for the *gntT* gene.
However, the editing efficiency of the *lacZ* gene
was only 80%, suggesting that there is room for improvement.

Cycle 2: Editing efficiency test of p15A-Cas9-op-λ-Red (BBa\_K4876010) in E. coli Nissle 1917

**Design 2:**

The
*Cas9* gene
used in this project is from *Streptococcus*.
Due to species differences, some rare *E. coli* tRNAs
may be heavily used in the original sequence, causing slow host growth. Codon optimization avoids rare
tRNA usage so bacteria can grow faster while expressing the same product. We named the
codon-optimized Cas9 as Cas9-op and constructed the
p15A-Cas9-op-λ-Red
plasmid (Figure
6).

![](https://static.igem.wiki/teams/4876/wiki/engineering/6.png)

**Figure****6****Expression frame of the
p15A-****Cas9****-op-λ-Red plasmid.**

**Build 2:**

To
construct p15A-Cas9-op-λ-Red, we amplified p15A and Cas9-op-λ-Red
fragments using primers with homology arms and recombined them using a cloning kit. Results showed
successful amplification and construction verified by colony PCR and sequencing (Figure 7).

![](https://static.igem.wiki/teams/4876/wiki/engineering/7.jpg)

**Figure****7****The construction
results****of
p15A-****Cas9****-op-λ-Red.**

**(****A)** **Transformation results. (B) Sequencing
results.**

**Test 2:**

Comparing to p15A-Cas9-λ-Red ([BBa\_K4876011](http://parts.igem.org/Part%3ABBa_K4876011)), after
Cas9 codon optimization, p15A-Cas9-op-λ-Red ([BBa\_K4876010](http://parts.igem.org/Part%3ABBa_K4876010))
knocked
out both *gntT* and
*lacZ* at
100% efficiency which is higher than p15A-Cas9 (Figure
8).

![](https://static.igem.wiki/teams/4876/wiki/engineering/8.png)

**Figure****8****Editing efficiency
results of p15A-Cas9 and p15A-Cas9-op in** **E.
coli****Nissle
1917.**

We
further studied the
editing efficiencies of p15A-Cas9-op-λ-Red
for the *gntT* gene
and *lacZ* gene
by adjusting the length of the repair homology arms. The results are shown in Figure 9, for the
*gntT* gene,
the editing efficiencies of the repair homology arms of 100-200 bp were 0, while it reached 100% when the
lengths were extended to 300-500 bp. Similarly, for the*lacZ* gene,
the editing efficiency of the repair homology arm of 100 bp was 0, which increased to 80% for 200-300 bp,
and reached 100% for 400-500 bp.

![](https://static.igem.wiki/teams/4876/wiki/engineering/9.jpg)

**Figure 9 Editing efficiency results
of** **Cas9****-op with different lengths of homology arms
in E. coli Nissle 1917.**

**Learn 2:**

Using
a higher copy origin and codon optimizing Cas9 effectively
improved the editing efficiency of this CRISPR-Cas9 system
on the *gntT* and
*lacZ* genes
in *E. coli* Nissle
1917 to 100%. The
length of the repair homology arm is crucial to the editing efficiency. To ensure high editing efficiency,
the length of the repair homology arm should preferably be more than 300 bp. In the future, we will conduct more experiments to evaluate our
Cas9-op tool on gene editing in order to expand its application for broader contribution.

Reference

[1]
Buddenborg
C., Daudel D., Liebrecht S., et al. Development of a tripartite vector system for live oral immunization
using a gram-negative probiotic carrier [J]. Int J Med Microbiol, 2008, 298(1-2): 105-114.

[2]
Vento
J.M., Crook N., Beisel C.L. Barriers to genome editing with crispr in bacteria [J]. J Ind Microbiol
Biotechnol, 2019, 46(9-10): 1327-1341.

[3]
Li
Q., Sun M., Lv L., et al. Improving the editing efficiency of crispr-Cas9 by
reducing the generation of escapers based on the surviving mechanism [J]. ACS Synthetic Biology, 2023,
12(3): 672-680.

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I.Polymerase chain reaction (PCR)

The
following fragments all use the same PCR amplification system, with the difference being that the
templates are different.

A. Cas9-λ-Red fragment:
Use the plasmid pEcCas9-λ-Redas
a template.

B. Cas9-op-λ-Red fragment:
Use the plasmid pEcCas9-op-λ-Redas
a template.

C. p15A
fragment: Use the synthetic plasmid containing p15A as
a template.

D. ColE1
fragment: Use the synthetic plasmid containing ColE1 as
a template.

E. gntT homology
arm: Use the plasmid pEcgRNA-gntTas
a template.

F. lacZ
homology arm: Use the plasmid pEcgRNA-lacZas
a template.

The
reaction system for PCR amplification is shown
in the table below:

|  |  |
| --- | --- |
| Reagent | Volume (μL) |
| 2´HieffÒPCR Master Mix (With Dye) | 25 |
| primer F (10 μM) | 1 |
| primer R (10 μM) | 1 |
| DNA template | 1 |
| ddH2O | 22 |
| Total | 50 |

The
reaction procedure for PCR amplification are shown in the table below:

|  |  |  |
| --- | --- | --- |
| Step | Temperature ( ℃) | Time (sec) |
| 1 | 98 | 180 |
| 2 | 98 | 30 |
| 3 | 60 | 15 sec/kb |
| 4 | 72 | 120 |
| 5 | 72 | 300 |
| 6 | 4 | ¥ |

Notice:
Repeat step 2 to step 4 for 28 times.

II.Agarose gel electrophoresis

1. Preparation
of agarose gel

1) Add
100 mL 1× TAE buffer and 1 g agarose to an flask, and place it in a microwave oven on high for 2
min.

2) Take
it out after the solution turns transparent and shake it evenly.

3) Add
2 μL nucleic acid dye GelRed to
the molten agarose.

4) Assemble
the gel tray and insert comb into mold.

5) Pour
the liquid agarose into the mold, avoiding air bubbles.

6) Wait
for solidification of the gel at room temperature.

7) Carefully
remove comb and transfer solidified
gel to electrophoresis tank.

8) Completely
submerge gel in 1´ TAE
buffer.

2. Loading
samples

1) Add
DNA
loading buffer
into samples to dye them blue, then load into wells sequentially.

2) Add
10 μL of Trans15K DNA Marker into a lane.

3. Electrophoresis

1) Place
wells at the negative electrode, and cover tank.

2) Turn
on the power, and run the agarose gel at 180 V for 20 min.

4. Visualizing
DNA bands

1) Turn
off power, and remove gel.

2) Place
the gel under the Ultra Slim LED Illuminator to visualize the results.

3) Take
out the gel, and put it under gel-imaging system to save data.

III.Gel extraction

1. Excise
the DNA band of interest under UV light using a clean scalpel.

2. Transfer
the gel slices into centrifuge tubes, and
measure the weight of the gel slices.

3. Add
3 times of sample volume of Buffer B2 into the centrifuge tubes, and mix until gel completely
dissolved.

4. Transfer
the solution into
the absorption column, and centrifuge at 9000 ×*g* for
30 sec. Pour off the filtrate in
the collection tube.

5. Add
500 μL of Wash Solution and centrifuge at 9000 ×*g* for
30 sec. Pour off the filtrate in the collection tube.

6. Repeat
wash step.

7. Centrifuge
the empty column at 9000 ×*g* for
1 min.

8. Place
the absorption column into a clean 1.5 mL centrifuge tube, add 15-40 μL Elution Buffer to the center of
the absorption membrane, and centrifuge at 9000 ×*g* for
1 min. Store the DNA solution in the tube.

IV.Homologous recombination of fragments and vectors

For
the recombinant plasmids p15A-Cas9-λ-Red,
p15A-Cas9-op-λ-Red, and
ColE1-Cas9-op-λ-Red,
since the upstream and downstream primers used previously for fragment amplification contained homology
arms, the following fragments can be homologously recombined using the ClonExpress II One Step Cloning
Kit:

|  |  |  |
| --- | --- | --- |
| Plasmid | Fragment 1 | Fragment 2 |
| p15A-Cas9 | Cas9-λ-Red | p15A |
| p15A-Cas9-op | Cas9-op-λ-Red | p15A |
| ColE1-Cas9-op | Cas9-op-λ-Red | ColE1 |

The
homologous recombination reaction system is as follows:

|  |  |
| --- | --- |
| Reagent | Volume (μL) |
| Fragment 1 | X |
| Fragment 2 | Y |
| 5× CE II Buffer | 4 |
| Exnase II | 2 |
| ddH2O | to 20 |

\* X/Y
is calculated according to the formula according to the manual.

Incubate
at 37 °C for 30 min; cool to 4 °C or place on ice immediately.

V.Ligation of recombinant plasmids pEcgRNA-gntT and pEcgRNA-lacZ

1. Plasmid
pEcgRNA was digested with *Bsa*I
to generate linearized pEcgRNA carrying the overhangs 5′-TAGT-3′ and
5′-AAAC-3′.

The
digestion system is shown in the table below:

|  |  |
| --- | --- |
| Reagent | Volume (μL) |
| pEcgRNA | 20 |
| *Bsa*Ⅰ | 1 |
| Buffer (CutSmart) | 5 |
| ddH2O | 23 |
| Total | 50 |

The
enzyme digestion system is incubated at 37 ℃ for 3 h.

2. Single-strand
oligonucleotides comprising 4 nt of the overhangs and 20 nt of target-specific sequences (*gntT* or
*lacZ*)
were annealed to form dsDNA.

The
annealing system is shown in the table below:

|  |  |
| --- | --- |
| Reagent | Volume (μL） |
| Oligonucleotides 1 | 5 |
| Oligonucleotides 2 | 5 |
| T4 ligase buffer (10×) | 5 |
| Total | 35 |

3. The
reaction mix was incubated at 95 °C for 5 min, and the temperature was then gradually reduced
by 5–10 °C/min. The reaction was finally held at 16 °C for 10 min.

4. The
annealed dsDNA was then diluted 200-fold and 1 µL of the diluted DNA was ligated to
1 µL of *Bsa*I-linearized
pEcgRNA in a mixture of T4 ligase buffer and T4 ligase for 1 h at 16 °C.

VI.Transformation of recombinant plasmids to E. coli DH5α

1. Add
5 μL of plasmids into 50 μL of *E. coli* DH5α
competent cells, respectively. Incubate
the mixture on
ice for 30 min.

2. Heat
shock at 42 °C for 90 sec, and put the mixture to the ice for 4-5
min.

3. Add
the LB culture into the tubes to 1 mL, and shake the tubes at 37 ℃, 220 rpm for 30-60 min.

4. Centrifuge
it at 9000 ×*g* for
1 min, remove 800
μL of supernatant, and resuspend the cells in the remaining LB culture.

5. Spread
50 μL cells onto LB agar plates containing spectinomycin
or chloromycetin
and
incubate overnight at
37 ℃.

VII.PCR identification of transformants

1. Place
50 μL of Lysis Buffer for Microorganism to Direct PCR in a sterilized microtube. Pick a single colony
with a sterilized toothpick or lance tip, place it in the microtube, and stir a few times before removing
it.

2. After
heat denaturation at 80 °C for 15 min, centrifuge at low speed and take 1~5 μL of supernatant after
lysis as PCR template.

The
PCR reaction system is shown in the table below:

|  |  |
| --- | --- |
| Reagent | Volume (μL) |
| Lysis supernatant | 1 |
| Premix Taq (Ex Taq Version 2.0) | 10 |
| Forward Primer (10 μM) | 1 |
| Reverse Primer (10 μM) | 1 |
| sterilized water | 7 |

The
PCR reaction condition is shown in the table below:

|  |  |  |
| --- | --- | --- |
| Step | Temperature (˚C) | Time (sec) |
| 1 | 94 | 600 |
| 2 | 94 | 30 |
| 3 | 55 | 30 |
| 4 | 72 | 120 |
| 5 | 72 | 300 |
| 6 | 4 | ¥ |

Notice:
Repeat step 2 to step 4 for 28 times.

3. Take
positive transformants and add 3 mL LB culture medium containing spectinomycin
or chloromycetin,
shake bacteria, and incubate at 37 ℃ at 220 rpm for 12-16 h.

4. Inoculate
the positive transformants in 3 mL LB culture medium containing spectinomycin or chloromycetin at a 37 ℃
shaker with 220 rpm for 12-16 h.

5. Extraction
the plasmids from overnight cultures.

VIII.Plasmid extraction (Plasmid Mini-PREPS Kit)

1. Equilibrate
the adsorption column

1) Add
500 μL of Buffer S to the adsorption column and place a collection tube below it.

2) Centrifuge
at 12,000 ×*g* for
1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the
collection tube.

2. Transfer
the *E. coli* culture into
a 2 mL centrifuge tube, centrifuge at 12,000 ×*g* for
2 min to collect the bacteria. Discard the supernatant and retain the precipitate.

3. Add
250 μL of Buffer SP1 to the 2 mL centrifuge tube, and thoroughly suspend the precipitate.

4. Add
250 μL of Buffer SP2, and gently invert the tube 5-10 times to mix the solution.

5. Add
350 μL of Buffer SP3 and mix the solution by gently inverting the tube 5-10 times.

6. Centrifuge
at 12,000 ×*g* for
5-10 min. Transfer 700 μL of the supernatant to the adsorption column, centrifuge at 8,000 ×*g* for
30 sec, and discard the filtrate in
the collection tube.

7. Add
500 μL of Wash Solution, centrifuge at 9,000 ×*g* for
30 sec, and discard the filtrate in
the collection tube.

8. Repeat
wash step.

9. Centrifuge
the empty column at 9,000 ×*g* for
1 min.

10. Place
the column into a clean 1.5 mL centrifuge tube, and add 25 μL of Elution Buffer to the center of the
adsorption membrane. Incubate
for 1 min at room temperature,
centrifuge at 9,000 ×*g* for
1 min, and keep the DNA solution in the tube.

IX.Electroporation of Cas9 plasmids, pEcgRNA plasmids, and homology arms into E. coli Nissle 1917
competent cells

1. Cultivate
the *E. coli* Nissle
1917 strain overnight in LB medium at 37 °C, 200 rpm/min.

2. Inoculate
50 mL LB medium on the second day at 1% inoculation volume.

3. When
the OD600of
the freshly inoculated culture reaches 0.6-0.8, ice-bath the culture for 20 min to fully cool.

4. Centrifuge
the culture in 50 mL centrifuge tubes at 4000 rpm/min and 4 °C for 10 min. Discard the supernatant and
quickly return the tubes to ice.

5. Resuspend
the cell pellet in ice-cold sterile ddH2O
or 10% glycerol by pipetting up and down on ice. Centrifuge again at 4000 rpm/min and 4 °C for 10 min.
Discard the supernatant and quickly return the tubes to ice.

6. Repeat
the wash step 1-2 times.

7. According
to the number of transformation samples, add ice-cold sterile deionized water to the tubes at 100 μL
per sample, and resuspend the cell pellet.

8. Transfer
100 μL of resuspended cells per sample into electroporation cuvettes on ice.

9. Add
approximately 100 ng of Cas9 plasmids
(p15A-Cas9-λ-Red,
p15A-Cas9-op-λ-Red, and
ColE1-Cas9-op-λ-Red)
to be transformed, mix well, and ensure the cells-plasmid mixture is between the two metal electrodes
inside the cuvette.

10. Wipe
the exterior metal electrodes of the cuvette dry and electroporate at 1.8 kV, 200 Ω, 25 μF.
Immediately add 900 μL LB without antibiotics and resuspend by pipetting. Transfer the cuvette contents
into a 1.5 mL tube.

11. Recover
the electroporated cells by shaking at 37 °C for 1 h. Plate on LB agar with chloramphenicol and
incubate overnight at 37 °C.

12. Pick
transformants and culture overnight at 37 °C, 200 rpm/min in liquid LB with chloramphenicol.

13. Inoculate
chloramphenicol-containing LB medium on the second day at 1% inoculation volume. Add L-arabinose to a
final concentration of 10 mM.

14. Repeat
steps 1-12 to electrotransform pEcgRNA (pEcgRNA-gntT and pEcgRNA-lacZ) and homology arms into competent
cells containing the Cas9 plasmid.

X.Protein disruption

1. Transfer
*E. coli* Nissle
1917 culture to 50 mL centrifuge tubes and centrifuge at 8000 rpm for 5 min. Discard supernatant and pool
all cell pellets.

2. Add
15 mL bacterial protein extraction buffer (100 mM NaCl, 10 mM EDTA, pH 8.0) to each tube. Resuspend
pellets by pipetting up and down.

3. Place
tubes in a rack, submerge probe tip of ultrasonic cell disruptor into tube. Position tube and close door.
Turn on power and sonicate each tube with 5 cycles of 2 sec on, 2 sec off at 80 W power.

4. Centrifuge
at 8000 rpm for 5 min after 4 treatments.

5. Carefully
collect supernatant as crude protein extract into new tubes and store at 4 °C.

XI.Protein purification

1. Column
assembly

Fix
the column on iron stand. Unscrew top adaptor and seal bottom outlet with a clamp. Add deionized water to
remove air bubbles. Prepare 2-5 mL slurry of 20% ethanol-equilibrated Chelating Sepharose Fast Flow resin
and pack into column with a glass rod against inner wall. Open outlet to let resin settle. Resin volume
2-5 mL. Reattach top adaptor and seal inlet/outlet with tubing.

2. System
setup

(1)
Peristaltic pump: Turn on the power, press start, and set flow rate to 15 rpm (2 mL/min) with arrow keys.
Fill the tubing with deionized water.

(2)
Connect pump outlet tubing to column inlet. Connect column outlet to UV monitor inlet. Connect UV monitor
outlet to the fraction collector.

(3)
UV monitor: Turn on power, adjust ABS sensitivity knob and zeroing knob.

(4)
Fraction collector: Turn on power, reset, set tube position via manual control, then set collection
parameters (*e.g.* 120
sec per tube). Press auto to begin timed collection.

(5)
Record UV absorbance data on computer to generate chromatograms.

3. Column
equilibration

(1)
Wash ethanol-preserved column with 5× bed volumes of deionized water to remove ethanol.

(2)
Pass 2× bed volumes of 0.2 M NiSO4 through
column. Observe phenomena.

(3)
Wash with 10× bed volumes of deionized water. Equilibrate with 5× bed volumes of 0.1 M PBS
with 5 mM imidazole.

4. Sample
loading

First,
take 1 mL from 20 mL lysate supernatant as total protein control before chromatography. Load clarified
lysate supernatant onto column at 2 mL/min flow rate. Collect 1 mL fractions, with highest UV peak as
flowthrough. Wash with equilibration buffer until UV absorbance plateau is reached. Load 15 mL
sample.

5. Washing

Wash
with 20× bed volumes of PBS with 15 mM imidazole (pH 8.0) to remove weakly bound contaminants.
Collect flowthrough and analyze.

6. Elution

Elute
bound proteins with 10 mL PBS containing 500 mM imidazole. Collect 1 mL fractions and identify the tube
with the highest UV absorbance peak.

XII.Detection of protein expression

1. Prepare
BSA standards of known concentrations.

2. Add
standards and protein samples to a 96-well plate.

3. Add
Bradford reagent to each well.

4. Incubate
plate at room temperature for 5 min.

5. Measure
the absorbance at 595 nm.

6. Subtract
the blank and plot the standard curve.

7. Compare
sample absorbances to the standard curve to determine protein concentrations of Cas.

XIII.Editing efficiency test

After
the transformants to be cultured were incubated for 16 h, the growing transformants were identified by
colony PCR, and those with bands matching the expected size were the successful edited transformants. By
comparing the knockdown efficiency of the bacteria using the new plasmid with the knockdown efficiency of
the control group using the original plasmid, it can be known that the editing rate is improved.

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Overview

Nissle
1917 is an*E.
coli* strain
with probiotic properties, but for certain applications, it is necessary to knock out specific genes.
Since CRISPR-Cas systems have high target DNA specificity and programmability, they can serve as gene
editing tools. However,
unwanted escapers that do not undergo the intended editing frequently arise during CRISPR-Cas-mediated
microbial genome editing, reducing editing efficiency.

Therefore,
this project aims to construct a highly efficient CRISPR-Cas9-based
genome editing tool for *E. coli* Nissle
1917 by increasing Cas9 copy number and conducting
Cas9 codon
optimization, lowering escape rates to improve editing efficiency, and providing a reference for other
genome editing tools.

Our
experimental results mainly include:

(1) Construction
of Cas9 plasmids,
gRNA plasmids, and homology
arms.

Ø Cas9:
p15A-Cas9-λ-Red (p15A-Cas9),
p15A-Cas9-op-λ-Red
(p15A-Cas9-op),
pColE1-Cas9-op-λ-Red
(pColE1-Cas9-op)

Ø gRNA:
gRNA-gntT,
gRNA-lacZ

Ø Homology
arms: gntT-HR, lacZ-HR

(2) Inducing protein
expression of Cas9.

(3)
Editing efficiency tests.

We
compared our re-designed Cas9-op tool with the “old” Cas9 tool[1] which
was designed by Prof. Li in order to evaluate the gene editing efficiency on *E. coli* Nissle
1917.

![](https://static.igem.wiki/teams/4876/wiki/results/figure-1.png)

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