iGEM 2022 — Team dut-china

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

## What should this page contain?

---

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

---

* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

{% endblock %}

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

![Engineering Success Image](https://static.igem.wiki/teams/4176/wiki/images/es.png)

Engineering Success

[Overview](#to-1)
[Cycle 1 *galK* gene inactivation](#to-2)
[Cycle 2 Construction and validation of the "strainer"](#to-3)
[Cycle 3 Protein modification](#to-4)
[Cycle 4 Insertion of the long gene fragment](#to-5)
[Cycle 5 Fermentation for IPA production](#to-6)

Overview

Iterative design is an approach to a cyclical process based on the principles of
synthetic biology design:
design, build, test, and learn. In the iGEM competition, we focused on engineering thoughts and
aimed to improve
the foundations and applications of our designs through the iterative process. In the process of
carrying out
this cycle, we strove to be close to the user needs, solve the pain point of efficiency
improvement of the gene
editing tools at this stage, and improve our project in both versatility and stability.
Therefore, we believe
that engineering success should exist in all the aspects of the project design, build, test and
optimization,
and this year we mainly focused on the build and optimization of the purification system.

Cycle1 *galK* gene inactivation

Research: Which kind of gRNA and donor DNA is a good start for the
construction of genome editing
method?

Our project aims to improve the gene editing efficiency by constructing a
purification system (strainer). The
describe of the strainer method can be found in home page. Gene inactivation is a
frequently-used step in the
strain construction. Thus, we use it as a good start for our method design and construction. The
*galK*
gene is one of the popular targeting gene in *E. coli* because the cells with the
inactivation of
*galK* gene will be observed as white colonies on the MacConkey agar. Moreover, the
wild-type control
will be observed as red colonies on the MacConkey agar. To this end, we designed four gRNAs (in
the EC21 to EC24
plasmids) targeting on the different position of the *galK* gene and related donor DNAs
contained stop
codon (TAA). In addition, each gRNA with its related donor DNA is constructed in the same
editing plasmid, which
is good for genome editing using gRNA libraries.

Design: How can we screen an editing plasmid for gene inactivation that can
be used for the construction
and optimization of the "strainer" method?

In the first iteration of the design, we used two *E. coli* strains (EC85
and EC88) with the plasmid
harboring lambda red system and different CRISPR proteins (Fig. 1) as host strains to test our
four editing plasmids. Then, we can compare the editing efficiencies using four editing plasmids
with two
different Cas protein (MAD7 and AsCas12a). The editing plasmid with lowest editing efficiency
can be used for
the design and construction of the third plasmid of strainer system.

![img](https://static.igem.wiki/teams/4176/wiki/images/es-1.png)

**Fig.
1.**
Transformation of EC21, EC22, EC23, and EC24 plasmids into *E. coli* which
contains Cas and lambda red system respectively.

Build: How do we construct the strain with the *galK* gene
inactivation?

We firstly prepared the electrocompetent *E. coli* MG1655 cells, and used
electroporation to introduce
the plasmid harboring lambda red system and different CRISPR proteins. The resulting strains are
EC85 and EC88,
which grow on the plates with Chloramphenicol. Then, we prepared the electrocompetent EC85 and
EC88 cells for
the *galK* gene inactivation. During the competent cell preparation, we added 0.2%
arabinose as an
inducer for Cas system, and culture cells using 42 degree 15 min to induce the lambda red system
before chilling
the cells (the step starting the competent cell preparation). Then, we transformed the four
editing plasmids
into EC85 and EC88 using electroporation, and cultured the cells on the MacConkey Agar with 1%
galactose.

Test: How do we characterize the results of the *galK* gene
inactivation?

The colonies observed as white color were the strains with the *galK*
gene inactivation. The colonies
observed as red color were the wild-type strains (Fig. 2). The editing efficiency was
calculated using the formula as follows:

![img](https://static.igem.wiki/teams/4176/wiki/images/es-2.png)

**Fig.
2.** Two
MacConkey agar plates. One is full of the red colonies (the unedited cells), the
other is full of the white colonies (the edited cells).

Learn: Which editing plasmid for the *galK* gene inactivation that can
be
used for the construction and
optimization of the "strainer" method?

We compared the editing efficiency using different editing plasmid for the
*galK* gene inactivation. When
using the editing plasmid EC21, the editing efficiency in EC85 and EC88 are both lowest compared
to other
editing plasmids (Fig. 3). Thus, we use the plasmid EC21 as the template for the design and
construction of the third plasmid of strainer system.

![img](https://static.igem.wiki/teams/4176/wiki/images/es-3.png)

**Fig.
3.** The
gene inactivation experiments. (A) The CFU and editing efficiency after
transformation of the four gRNA plasmids into EC85 strain, which is MG1655 with pM7. (B) The
CFU and editing
efficiency after transformation of the four gRNA plasmids into EC88 strain, which is MG1655
with pAs.

Cycle 2 Construction and validation of the "strainer"

Research: What is the principle of the "strainer" we built?

The previous studies have demonstrated that the SOS response is the first DNA
repair system described in *E.
coli*. The transcription of genes such as *lexA*, *sula*, *umuD*
and *recA*
are regulated by the SOS response. Thus, we can use the promoters of these genes to start the
transcription of
gRNAs targeting on the plasmid harboring a counter-selection markers if there is a
double-stranded DNA breaks
(DSBs) on the genome. When the *sacB* plasmid is cured by CRISPR/Cas system, the strain
with successful
recombineering can survival in the media with sucrose (Fig. 4). The strain without DSBs, still
has the plasmid harboring *sacB* gene, cannot survival in the media with the sucrose.
This is why we can
increase the overall editing efficiency.

![img](https://static.igem.wiki/teams/4176/wiki/images/es-4.png)

**Fig.
4.**
Transformation of the recombinant plasmids into EC85 separately.

Design: How did we design the "strainer"?

Firstly we designed a second plasmid harboring *sacB* gene in the EC85 or
EC88, and used Trc inducible
promoter to control its expression. Then, we designed another gRNA that can be inserted in the
EC21 plasmid.
This gRNA target on the second plasmid harboring *sacB* gene, and use the promoter of
genes such as
*lexA*, *sula*, *umuD* and *recA*.

Build: How can we construct and validate the "strainer"?

We ordered the second plasmid harboring the *sacB* gene with Trc inducible
promoter. Then, we prepared the
electrocompetent EC85 and EC88 cells and transformed the second plasmid into EC85 and EC88. We
also ordered four
DNA fragments contained the same spacer targeting on the second plasmid with the promoter of
*lexA*,
*sula*, *umuD* and *recA* genes. Then, we constructed these new editing
plasmids by Gibson
assembly using EC21 as template.

We firstly prepared the electrocompetent cells for the *galK* gene
inactivation using the
strain EC85 with
*sacB* plasmid. During the competent cell preparation, we added 0.2% arabinose as an
inducer for Cas
system, and culture cells using 42 degree 15 min to induce the lambda red system before chilling
the cells (the
step starting the competent cell preparation). Then, we transformed the new editing plasmid
(LexA) with the
*lexA* promoter into the strain EC85 with *sacB* plasmid using electroporation,
and cultured the
cells on the MacConkey Agar with 1% galactose and different concentration of sucrose.

Test: Which condition will be good for the "strainer" system?

We firstly tested the new editing plasmid LexA using *lexA* gene promoter
in EC85 to validate the
"strainer" method. At a 0.01% sucrose concentration, the highest CFU/μg and the highest editing
efficiency was
found in the experiment. The editing efficiency using LexA plasmid is 25% higher than the
control without
"strainer" system. However, the CFU/μg decreased significantly compared to the control group
that did not use
"strainer". In addition, we used the 0.01% sucrose to tested another three new editing plasmids.
All of them
showed that the editing efficiency is 25% higher than the control without "strainer" system
(Fig.
5A).

Learn: What does we learn from the validation of the "strainer"?

Firstly, the "strainer" system significantly improves the gene editing efficiency
compared to the control without
"strainer" system. Secondly, the CFU/μg is still 96% lower than that of the control even using
0.01% sucrose
(Fig. 5B). To this end, we should further improve CFU/μg by designing and modifying SacB
protein to reduce its toxicity.

![img](https://static.igem.wiki/teams/4176/wiki/images/es-5.png)

**Fig.
5.**
Construction and validation of the "strainer". (A) The editing efficiency and CFU/ug of
all the four new editing plasmids under 0.01% sucrose condition. (B) The editing efficiency
and CFU/ug in the
different sucrose concentrations.

Cycle 3 Protein modification

Research: How to attenuate toxicity by engineering SacB protein?

The lethal mechanism of the sacB toxicity gene is that the protein expressed by
the sacB gene (levansucrase) can
catalyze the hydrolysis of sucrose into glucose and fructose, and polymerize fructose into
levan. The large
accumulation of levan in *E. coli* will kill the cells. The S164 forms a hydrogen bond
with the
nucleophilic agent D86 and the 4-OH of the fructose group, and S164 is important to ensure the
position
stabilization of D86 (Fig. 6). If we design some mutation in S164 site, the mutant could reduce
the hydrolysis rate, and then the toxicity of SacB protein will reduced.

![img](https://static.igem.wiki/teams/4176/wiki/images/es-6.png)

**Fig.
6.** The
first layer means that the amino acid shown in the figure is the closest layer to
the substrate (sucrose), and the distance between all amino acids and the substrate is less
than 3.5 A. W85,
D86, W163, R246, D247, E342 are completely conservative in GH68 family.

Design: How to design a SacB mutation that could decrease its toxicity for
our "strainer"
system?

The position of the carboxyl group of D86 is restricted by hydrogen bonding. We
speculate that the S164T mutation
with an additional -CH3 group would change the orientation of the-OH and would effectively form
new hydrogen
bonds. If hydrolysis rate reduce, the toxicity of SacB protein will reduced. Thus, we test S164T
mutation using
Molecular dynamics (MD) simulations. The results showed that This mutation breaks the delicate
balance of the
ternary catalytic amino acid with the ligand (Fig. 7). Therefore, we speculate that it will
lead to reduced cytotoxicity. To this end, we sought to apply S164T mutation in the wet-lab
experiment.

![img](https://static.igem.wiki/teams/4176/wiki/images/es-7.png)

**Fig.
7.**
Molecular docking of SacB Wt (A) (PDB ID 10YG, green) and variant S164A(B) (POB ID 6PWQ.
cyan) using α-glucose as ligand.

Build: How to construct the SacB\_S164T mutation?

We followed the instruction of Mut Express II Fast Mutagenesis Kit V2 to design
the primer. Then, we used the
following PCR conditions to construct the SacB\_S164T mutation. Thirdly, we followed the
instruction of Mut
Express II Fast Mutagenesis Kit V2 to construction the SacB\_S164T mutant.

Table 1 PCR
condition

![img](https://static.igem.wiki/teams/4176/wiki/images/es-t1.png)

We prepared the electrocompetent cells for the galK gene inactivation using the
strains EC85 and EC88 with SacB
or SacB\_S164T mutation. During the competent cell preparation, we added 0.2% arabinose as an
inducer for Cas
system, and culture cells using 42 degree 15 min to induce the lambda red system before chilling
the cells (the
step starting the competent cell preparation). Then, we transformed the editing plasmid (LexA)
with the lexA
promoter into the strain using electroporation, and cultured the cells on the MacConkey Agar
with 1% galactose
and different concentration of sucrose.

Test: How to evaluate SacB\_S164T mutation?

The results showed that the editing efficiency of SacB\_S164T mutant (in EC85) is
25% higher than the control
without "strainer" system. The CFU/μg of SacB\_S164T mutant (in EC85) increased 3-fold compared
to the original
"strainer" system while still keep high editing efficiency (Fig. 8A).

We also used the same condition to test SacB\_S164T mutant in EC88. The results
showed that the editing efficiency
using "strainer" system with SacB\_S164T mutant is 4-fold higher than the control, although the
CFU/μg of
SacB\_S164T mutant (in EC88) is still 71% lower than the control without "strainer" system (Fig.
8B).

We also used the same condition to test SacB\_S164T mutant in EC88. However, the
results showed that the toxicity
of the modified SacB protein was low, and the screening effect served by the lower concentration
of sucrose
chosen at this point was no longer obvious, so we further optimized the sucrose concentration
and found that
testing the mutants at 0.1% concentration conditions, the CFU/μg using the "strainer"
system with the
SacB\_S164T mutant was 2-fold higher than the control, although the SacB\_ S164T mutant (in EC88)
still had a
lower CFU/μg than the control without the "strainer" system.

![img](https://static.igem.wiki/teams/4176/wiki/images/es-8a.png)
![img](https://static.igem.wiki/teams/4176/wiki/images/es-8b.png)

**Fig.
8.**
Improve the CFU/μg of "strainer" method with SacB mutant. (A) The CFU/μg and editing
efficiency of the control group, SacB and SacB\_S164T under 0.01% sucrose condition in EC85.
(B) The CFU/μg and
editing efficiency of the control group, SacB and SacB\_S164T under 0.1% sucrose and 0.2%
sucrose condition in
EC88.

Learn: How toxic is the SacB mutation?

These results showed that our strainer system works as a good purification system
to remove the unedited cells,
and SacB\_S164T mutant is less toxicity than the original SacB.

Cycle 4 Insertion of the long gene fragment

Research: Why do we do the long gene fragment insertion work?

The "strainer" method improved the overall editing efficiency by removing the
unedited cells. We have previously
validated the feasibility of improving editing efficiency by introducing the
"strainer" method for
screening in the single gene editing, but we sought the "strainer" to potentially
serve as a universal
method for cell factory construction. Therefore, we applied the "strainer" to a large
fragment gene
editing process to verify the universality of the system.

Design: How to compare the efficiency improvement of the "strainer" method
for the long gene fragment
editing?

*SS9* is a safe locus in the *E.coli* genome, where we can insert
long gene fragment without strong
effect on the growth of *E.coli*. Therefore, we chose this region to verify the
"strainer" for
long DNA fragment insertion experiment. We chose the CRISPR/Cas9 gene editing method as the
control group and
used CRISPR/Cas9 to construct the "strainer" method as the experimental group, because the
CRISPR/Cas9 is also
commonly used in the genome editing field. We introduced the isopropanol (IPA) production
pathway (~5000 bp)
into the strains using both methods to compare the efficiency of long gene fragment insertion.

Build: Introduction of IPA fragments into EC86 strain using two methods.

We firstly constructed EC86 strain which is MG1655 with the plasmid harboring
lambda red system and Cas9 protein.
Then, we introduced the plasmid with sacB\_S164T into the EC86. Thirdly, we introduced the
gRNA\_SS9 plasmid and
the genome fragment of the IPA pathway into host strain using two methods. The strains after
transformation were
spread on the LB plates, and then using colony PCR to verify the efficiency of long gene
fragment insertion.

Test: How to know that the long gene fragment was inserted successfully?

We selected 30 colonies that were labeled and added to the reaction solution for
colony PCR, and performed colony
PCR under the reaction conditions shown in the table 2. Then we observed the running gel
results, and a band at
the 600 bp position was considered as a successful import.

Table.2
Colony PCR reaction conditions.

![img](https://static.igem.wiki/teams/4176/wiki/images/es-t2a.png)
![img](https://static.igem.wiki/teams/4176/wiki/images/es-t2b.png)

Learn: What do the statistic comparisons show?

Through the three repeated experiments of the long gene fragment insertion, 30
samples were detected by colony
PCR each time, and the electrophoresis results were observed to calculate the proportion of the
colonies that
had inserted IPA pathway gene fragments. We found that the efficiency of successfully inserting
long gene
fragment using the CRISPR/Cas9 method was very low, and even there were cases where the
introduction was not
successful at all. In contrast, the use of the "strainer" method effectively improved the
editing efficiency of
the long gene fragment insertion, so that the insertion efficiency is about 30%. (Fig. 9)

![img](https://static.igem.wiki/teams/4176/wiki/images/es-9.png)

**Fig.
9.** The
editing efficiency of long gene fragment insertion by using CRISPR/Cas9 and
"strainer" methods.

Cycle 5 Fermentation for IPA production

Research: What is the effect of "strainer" on IPA production fermentation
after introducing point
mutation?

We constructed the EC61 strain by inserting the IPA production pathway into the
genome in MG1655, and then we
further modified the EC61 strain by introducing the FliA\_R94K mutation that confers isopropanol
resistance.

Design: How to design a controlled trial?

We used the "strainer" and the original CRISPR/Cas methods for the
introduction of FliA\_R94K mutations
into the EC61 strain. The "strainer" method had 20% higher editing efficiency than the
original
CRISPR/Cas method (Fig. 10A). Then we set EC61 as the control group and EC65 as the
experimental group. Three shake flasks were set up in each group to culture and ferment as the
parallel samples,
two of which were sealed with parafilm and one with tinfoil.

Build: How should the fermentation experiments be conducted?

We added 24 mL of SD-8 medium, 1.25 mL of 400 g/L glucose, 20 μL of trace
elements, and antibiotics to each shake
flask (10 μL was taken to measure pH before inoculation to ensure that the pH range was around
6.0 before
inoculation) in the reagent preparation phase of the shake flask fermentations. SD-8 medium
(NH4Cl, 7.0g/liter;
KH2PO4, 7.5g/liter; Na2HPO4, 7.5g/liter; K2SO4, 0.85g/liter; MgSO4·7H2O, 0.17g/liter; trace
elements, 0.8
mL/liter; yeast extract; 10g/liter) containing 2% glucose was used for fermentations. The trace
element solution
contained the following (in grams per liter of 5 M HCl): FeSO4·7H2O, 40.0; MnSO4· H2O, 10.0;
Al2(SO4)3, 28.3;
CoCl2·6H2O, 4.0; ZnSO4·7H2O, 2.0; Na2MoO4·2H2O, 2.0; CuCl2·2H2O, 1.0; and H3BO4, 0.5. For
antibiotic selection,
the concentrations of antibiotics were 100 μg/mL (Ampicillin) and 34 μg/mL (Chloramphenicol). We
took samples by
shaking a dozen times after inoculation, centrifuged and kept the supernatant in the freezer for
HPLC detection,
measured pH, measured sugar concentration and recorded data. The shake flasks were incubated at
37°C and 200 rpm
(after 12 h incubation, the speed could be slightly lowered at 180 rpm).

We found that the pH of EC65 decreased significantly after 12 h, so we added a
small amount of 5M NaOH to keep
the pH of the culture solution at about 6.0; when the result of our sugar measurement was less
than 10 g/L, we
added glucose to make the concentration of glucose in the shake flasks to around 20 g/L, and
then shaked the
flask for a dozen times after the sugar was added. Take the sample and store it in the freezer.

Test: How did we characterize the IPA production of the different strains?

We stored the samples taken during the fermentation process in the freezer. After
completing the 48 h
fermentation process, we used HPLC to detect glucose and IPA concentrations, and finally
calculated the IPA
yield and productivity of the EC61 and EC65 fermentations.

Learn: Comparison of the IPA production of the two strains.

We could see that the isopropanol titer of EC65 was 3-fold higher than that of
EC61 from the result of HPLC
(Fig. 10B). In the process of strain modification for IPA production, the "strainer" method
could significantly accelerate the construction speed of the strains.

![img](https://static.igem.wiki/teams/4176/wiki/images/es-10a.png)
![img](https://static.igem.wiki/teams/4176/wiki/images/es-10b.png)

**Fig.
10.**
Fermentation for IPA production. (A)
The editing efficiency of FliA\_R94K mutations by using CRISPR/Cas9 and "strainer" methods.
(B) IPA production of
EC61 and EC65 strains at 48 h

{% endblock %}

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

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

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

![Results Image](https://static.igem.wiki/teams/4176/wiki/images/re.png)

Results

[1. *galK* gene inactivation](#to-1)
[2. Construction and validation of the “strainer”](#to-2)
[3. Protein modification](#to-3)
[4. Insertion of the long gene fragment](#to-4)
[5. Fermentation for IPA production](#to-5)

1. *galK* gene inactivation

We firstly prepared the electrocompetent *E. coli* MG1655 cells, and used
electroporation
to introduce
the plasmid harboring lambda red system and different CRISPR proteins. The resulting strains are
EC85 and EC88,
which grow on the plates with Chloramphenicol. Then, we prepared the electrocompetent EC85 and
EC88 cells for
the **galK** gene inactivation. During the competent cell preparation, we added
0.2%
arabinose as an
inducer for Cas system, and culture cells using 42 degree 15 min to induce the lambda red system
before chilling
the cells (the step starting the competent cell preparation). Then, we transformed the four
editing plasmids
into EC85 and EC88 using electroporation, and cultured the cells on the MacConkey Agar with 1%
galactose.

The colonies observed as white color were the strains with the
**galK** gene
inactivation.
The colonies
observed as red color were the wild-type strains (Fig. 1). The editing
efficiency was
calculated using the formula as follows:

![img](https://static.igem.wiki/teams/4176/wiki/images/re-0.png)

![img](https://static.igem.wiki/teams/4176/wiki/images/re-1.png)

**Fig. 1.** Two MacConkey agar plates. One is full of the red colonies (the
unedited
cells), the
other is full of the white colonies (the edited cells).

We compared the editing efficiency using different editing plasmid for the
**galK** gene
inactivation. When
using the editing plasmid EC21, the editing efficiency in EC85 and EC88 are both lowest compared
to other
editing plasmids (Fig. 2). Thus, we use the plasmid EC21 as the template for
the design and
construction of the third plasmid of strainer system.

![img](https://static.igem.wiki/teams/4176/wiki/images/re-2.png)

**Fig. 2.** The gene inactivation experiments. (A) The CFU and editing
efficiency
after
transformation of the four gRNA plasmids into EC85 strain, which is MG1655 with pM7. (B) The
CFU
and editing
efficiency after transformation of the four gRNA plasmids into EC88 strain, which is MG1655
with
pAs.

2. Construction and validation of the “strainer”

We ordered the second plasmid harboring the *sacB* gene with Trc inducible
promoter. Then,
we prepared the
electrocompetent EC85 and EC88 cells and transformed the second plasmid into EC85 and EC88. We
also ordered four
DNA fragments contained the same spacer targeting on the second plasmid with the promoter of
*lexA*,
*sula*, *umuD* and *recA* genes. Then, we constructed these new editing
plasmids by Gibson
assembly using EC21 as template.

We firstly prepared the electrocompetent cells for the **galK**
gene inactivation
using the
strain EC85 with
*sacB* plasmid. During the competent cell preparation, we added 0.2% arabinose as an
inducer for Cas
system, and culture cells using 42 degree 15 min to induce the lambda red system before chilling
the cells (the
step starting the competent cell preparation). Then, we transformed the new editing plasmid
(LexA) with the
*lexA* promoter into the strain EC85 with *sacB* plasmid using electroporation,
and cultured the
cells on the MacConkey Agar with 1% galactose and different concentration of sucrose.

We firstly tested the new editing plasmid LexA using *lexA* gene promoter
in EC85 to
validate the
“strainer” method. At a 0.01% sucrose concentration, the highest CFU/μg and the highest editing
efficiency was
found in the experiment. The editing efficiency using LexA plasmid is 25% higher than the
control without
“strainer” system. However, the CFU/μg decreased significantly compared to the control group
that did not use
“strainer”. In addition, we used the 0.01% sucrose to tested another three new editing plasmids.
All of them
showed that the editing efficiency is 25% higher than the control without “strainer” system
(Fig.
3A).

Firstly, the “strainer” system significantly improves the gene editing efficiency
compared to the
control without
“strainer” system. Secondly, the CFU/μg is still 96% lower than that of the control even using
0.01% sucrose
(Fig. 3B). To this end, we should further improve CFU/μg by designing and
modifying SacB
protein to reduce its toxicity.

![img](https://static.igem.wiki/teams/4176/wiki/images/es-5.png)

**Fig.
3.**
Construction and validation of the "strainer". (A) The editing efficiency and CFU/ug of
all the four new editing plasmids under 0.01% sucrose condition. (B) The editing efficiency
and CFU/ug in the
different sucrose concentrations.

3. Protein modification

We followed the instruction of Mut Express II Fast Mutagenesis Kit V2 to design
the primer.
Then, we used the
following PCR conditions to construct the SacB\_S164T mutation. Thirdly, we followed the
instruction of Mut
Express II Fast Mutagenesis Kit V2 to construction the SacB\_S164T mutant.

Table 1 PCR condition

![img](https://static.igem.wiki/teams/4176/wiki/images/re-5.png)

We prepared the electrocompetent cells for the *galK* gene inactivation
using the strain
EC85 and
EC88. Then, we
transformed the plasmid harboring SacB\_S164T mutation into EC85 and EC88.

We prepared the electrocompetent cells for the *galK* gene inactivation
using the strains
EC85 and
EC88 with SacB
or SacB\_S164T mutation. During the competent cell preparation, we added 0.2% arabinose as an
inducer for Cas
system, and culture cells using 42 degree 15 min to induce the lambda red system before chilling
the cells (the
step starting the competent cell preparation). Then, we transformed the editing plasmid (LexA)
with the lexA
promoter into the strain using electroporation, and cultured the cells on the MacConkey Agar
with 1% galactose
and different concentration of sucrose.

The results showed that the editing efficiency of SacB\_S164T mutant (in EC85) is
25% higher than
the control
without “strainer” system. The CFU/μg of SacB\_S164T mutant (in EC85) increased 3-fold compared
to the original
“strainer” system while still keep high editing efficiency (Fig. 4A).

We also used the same condition to test SacB\_S164T mutant in EC88. The results
showed that the
editing efficiency
using “strainer” system with SacB\_S164T mutant is 4-fold higher than the control, although the
CFU/μg of
SacB\_S164T mutant (in EC88) is still 71% lower than the control without “strainer” system
(Fig.
4B).

We also used the same condition to test SacB\_S164T mutant in EC88. However, the
results showed
that the toxicity
of the modified SacB protein was low, and the screening effect served by the lower concentration
of sucrose
chosen at this point was no longer obvious, so we further optimized the sucrose concentration
and found that
testing the mutants at 0.1% concentration conditions, the CFU/μg using the "strainer"
system with the
SacB\_S164T mutant was 2-fold higher than the control, although the SacB\_ S164T mutant (in EC88)
still had a
lower CFU/μg than the control without the "strainer" system.

![img](https://static.igem.wiki/teams/4176/wiki/images/re-6.png)
![img](https://static.igem.wiki/teams/4176/wiki/images/re-7.png)

**Fig. 4.** Improve the CFU/μg of “strainer” method with SacB mutant. (A) The
CFU/μg
and editing
efficiency of the control group, SacB and SacB\_S164T under 0.01% sucrose condition in EC85.
(B)
The CFU/μg and
editing efficiency of the control group, SacB and SacB\_S164T under 0.1% sucrose and 0.2%
sucrose
condition in
EC88.

These results showed that our strainer system works as a good purification system
to remove the
unedited cells,
and SacB\_S164T mutant is less toxicity than the original SacB.

4. Insertion of the long gene fragment

We firstly constructed EC86 strain which is MG1655 with the plasmid harboring
lambda red system
and Cas9 protein.
Then, we introduced the plasmid with sacB\_S164T into the EC86. Thirdly, we introduced the
gRNA\_SS9 plasmid and
the genome fragment of the IPA pathway into host strain using two methods. The strains after
transformation were
spread on the LB plates, and then using colony PCR to verify the efficiency of long gene
fragment insertion.

We selected 30 colonies that were labeled and added to the reaction solution for
colony PCR, and
performed colony
PCR under the reaction conditions shown in the table 2. Then we observed the running gel
results, and a band at
the 600 bp position was considered as a successful import.

Table.2 Colony PCR reaction conditions.

![img](https://static.igem.wiki/teams/4176/wiki/images/re-8.png)

![img](https://static.igem.wiki/teams/4176/wiki/images/re-9.png)

Through the three repeated experiments of the long gene fragment insertion, 30
samples were
detected by colony
PCR each time, and the electrophoresis results were observed to calculate the proportion of the
colonies that
had inserted IPA pathway gene fragments. We found that the efficiency of successfully inserting
long gene
fragment using the CRISPR/Cas9 method was very low, and even there were cases where the
introduction was not
successful at all. In contrast, the use of the “strainer” method effectively improved the
editing efficiency of
the long gene fragment insertion, so that the insertion efficiency is about 30%. (Fig.
5)

![img](https://static.igem.wiki/teams/4176/wiki/images/re-10.png)

**Fig. 5.** The editing efficiency of long gene fragment insertion by using
CRISPR/Cas9 and
“strainer” methods.

5. Fermentation for IPA production

We added 24 mL of SD-8 medium, 1.25 mL of 400 g/L glucose, 20 μL of trace
elements, and
antibiotics to each shake
flask (10 μL was taken to measure pH before inoculation to ensure that the pH range was around
6.0 before
inoculation) in the reagent preparation phase of the shake flask fermentations. SD-8 medium
(NH4Cl, 7.0g/liter;
KH2PO4, 7.5g/liter; Na2HPO4, 7.5g/liter; K2SO4, 0.85g/liter; MgSO4·7H2O, 0.17g/liter; trace
elements, 0.8
mL/liter; yeast extract; 10g/liter) containing 2% glucose was used for fermentations. The trace
element solution
contained the following (in grams per liter of 5 M HCl): FeSO4·7H2O, 40.0; MnSO4· H2O, 10.0;
Al2(SO4)3, 28.3;
CoCl2·6H2O, 4.0; ZnSO4·7H2O, 2.0; Na2MoO4·2H2O, 2.0; CuCl2·2H2O, 1.0; and H3BO4, 0.5. For
antibiotic selection,
the concentrations of antibiotics were 100 μg/mL (Ampicillin) and 34 μg/mL (Chloramphenicol). We
took samples by
shaking a dozen times after inoculation, centrifuged and kept the supernatant in the freezer for
HPLC detection,
measured pH, measured sugar concentration and recorded data. The shake flasks were incubated at
37°C and 200 rpm
(after 12 h incubation, the speed could be slightly lowered at 180 rpm).

We found that the pH of EC65 decreased significantly after 12 h, so we added a
small amount of 5M
NaOH to keep
the pH of the culture solution at about 6.0; when the result of our sugar measurement was less
than 10 g/L, we
added glucose to make the concentration of glucose in the shake flasks to around 20 g/L, and
then shaked the
flask for a dozen times after the sugar was added. Take the sample and store it in the freezer.

We stored the samples taken during the fermentation process in the freezer. After
completing the
48 h
fermentation process, we used HPLC to detect glucose and IPA concentrations, and finally
calculated the IPA
yield and productivity of the EC61 and EC65 fermentations.

We could see that the isopropanol titer of EC65 was 3-fold higher than that of EC61 from the
result of HPLC
(Fig. 6). In the process of strain modification for IPA production, the
“strainer” method could
significantly accelerate the construction speed of the strains.

![img](https://static.igem.wiki/teams/4176/wiki/images/re-11.png)

**Fig. 6.** IPA production of EC61 and EC65 strains at 48 h

{% endblock %}