iGEM 2019 — Team JiangnanU_China

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# Team:JiangnanU China/Notebook

JiangNan

[![](https://static.igem.org/mediawiki/2019/3/34/T--JiangnanU_China--Logo-Web_version.jpg)JiangnanU\_China](https://2019.igem.org/Team%3AJiangnanU_China)

* Project
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  + [Description](https://2019.igem.org/Team%3AJiangnanU_China/Description)
  + [Design](https://2019.igem.org/Team%3AJiangnanU_China/Design)
  + [Safety](https://2019.igem.org/Team%3AJiangnanU_China/Safety)
  + [Demonstration](https://2019.igem.org/Team%3AJiangnanU_China/Demonstrate)
* Experiments
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  + [Protocol](https://2019.igem.org/Team%3AJiangnanU_China/Experiments)
  + [Parts](https://2019.igem.org/Team%3AJiangnanU_China/Parts)
  + [Improvement](https://2019.igem.org/Team%3AJiangnanU_China/Improve)
  + [Notebook](https://2019.igem.org/Team%3AJiangnanU_China/Notebook)
  + [Results](https://2019.igem.org/Team%3AJiangnanU_China/Results)
  + [Measurement](https://2019.igem.org/Team%3AJiangnanU_China/Measurement)
* Modeling
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  + [Model](https://2019.igem.org/Team%3AJiangnanU_China/Model)
* Human Practices
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  + [Integrated Human Practices](https://2019.igem.org/Team%3AJiangnanU_China/Human_Practices)
  + [Public Engagement](https://2019.igem.org/Team%3AJiangnanU_China/Public_Engagement)
  + [Meetup](https://2019.igem.org/Team%3AJiangnanU_China/Human_Practices#meetup)
* Team
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  + [Team Members](https://2019.igem.org/Team%3AJiangnanU_China/Team)
  + [Collaborations](https://2019.igem.org/Team%3AJiangnanU_China/Collaborations)
* Acknowledgements
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  + [Attributions](https://2019.igem.org/Team%3AJiangnanU_China/Attributions)
  + [Sponsors](https://2019.igem.org/Team%3AJiangnanU_China/Sponsors)
* Medal
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  + [Medal Requirements](https://2019.igem.org/Team%3AJiangnanU_China/Medals)
  + [Awards](https://2019.igem.org/Team%3AJiangnanU_China/Awards)

**iGEM Notebook**

**Overview**

[![back](https://static.igem.org/mediawiki/2019/0/09/T--JiangnanU_China--host_liubianxing.png)

View all](#phage)

**1.Construction of recombinant plasmid and recombinant bacteria with report gene**

27/02/2019

Morning:

*Escherichia coli* (10/50 mL) containing the pET28a plasmid was cultured, and the pET28a plasmid was extracted using
a kit.

Afternoon:

The plasmid extracted in the morning was digested. The effect after the first digestion was not good. After
reflection, it was considered that we are not familiar with the experimental operation. The ethanol was not
dried during the rubber recovery process, and it was not carried out after the addition of the elution
buffer. The recovery efficiency caused by maintaining at room temperature is lowered, so a second digestion
is carried out to obtain a linear plasmid, and gel recovery is performed.

28/02/2019

Morning:

The *gfp* gene was obtained by PCR and recovered by gel.

The *gfp* gene was homologously recombined with the restriction enzyme pET28a plasmid to construct a recombinant
vector.
The pET28a plasmid was digested for later use.

01/03/2019

The experiment was temporarily stopped due to the lack of competent cells in the laboratory, and the team
members made a reflection.

Evening:

Since all of the previously extracted pET28a plasmids were used for enzymatic cleavage, *Escherichia coli* (10/50
mL) containing the pET28a plasmid was re-cultured, and plasmid extraction was performed the next day.

02/03/2019

Morning:

The pET28a plasmid was extracted using a kit.

The constructed recombinant vector was transferred to competent cells by transformation, and plated, and
cultured at 37℃ inversion.

Evening:

Preliminary observation found that the bacteria in the transgenic culture may be Bacillus subtilis, ready to
be re-transformed.

**2.Safety training of phage experiments and discussion of human practice**

15/03/2019

Afternoon:

The team will hold a group discussion to clarify the experimental precautions. For instance, all the
laboratory apparatus must be sterilized before used in the next step.
And discuss the HP content.

**3.Phage preservation and purification of E.coli BL21 genome**

18/03/2019-21/03/2019

Night:

Three bottles of *E.coli*BL21- PET28a (10/50 mL) were cultured and labeled a, b, and c.
The deposited BL21-28a-gfp *E. coli* was transferred to a large bottle for culture, and the next day, IPTG
induction was performed to observe whether fluorescence was generated or not.

22/03/2019

Night:

Morning:

the phage was inserted into a bottle of *E. coli*, and the lysate was observed to be clarified in about 3
hours. The lysate was aspirated for centrifugation, and the glycerol tube was stored in a -70℃ refrigerator.

23/03/2019

The 28a plasmid and the *E. coli* BL21 genome were extracted from the kit.

Glue verification of genomic PCR failed, suspected to be a problem with *E. coli* genome extraction.

27/03/2019

The kit failed to extract the *E. coli* BL21 genome.

The reason for reflection is that the column is used incorrectly, and the oscillating force is insufficient
during the extraction process, resulting in no separation of the supernatant and protein precipitation,
which affects the subsequent extraction results.

28/03/2019

Overnight PCR *E. coli* genome, nucleic acid gel validation.

**4.Discussion of moot court with Professor Hu**

26/03/2019

Teacher Hu Jie in Law school gave us the opinion: Due to the lag of legislation, our national laws currently
contain no relevant laws and regulations on genetic modification.

Legal workers do not understand the field of science and technology and can only promote legislation after
the occurrence of controversial events.

Genetic modification is not a bad thing in the future, but it is not advisable to deprive a small number of
people of their rights because of the interests of most people.

**5.Construction of recombinant plasmid and recombinant bacteria with anti-phage gene**

08/04/2019

Evening: Inoculate BL21-28a *E. coli*.

09/04/2019

Morning:

The plasmid was extracted and the resistance protein gene *abpAB* was amplified by PCR.
Afternoon: Run the glue to verify *abpAB*, glue recovery. Linked to the digested 28a plasmid.
Night:

Carry out the transfer. The plates were incubated overnight.

**6.Verification of anti-phage protein AbpAB**

10/04/2019

Afternoon:

Colony PCR was carried out, and the flask was cultured in a small bottle, then transferred to a
large bottle for cultivation. After 3 hours, IPTG was added to induce.

Night: The plate was cultured, and the phage was added dropwise so that we can observe the infection effect.

11/04/2019

Morning:

prepare the phage solution, transfer the *E. coli* BL21 vial to the large bottle, raise the OD to 0.8, and
add 500 μL of the phage preservation solution to it, shake it at 37℃ until the bacterial solution is lysed
by the phage.

Gene *abpAB* was transferred to two large bottles (added to the final concentration of 100 μL / mL of Amp
antibiotics), one bottle was added with IPTG induction, the other bottle without antibiotics, and both of
the bottles were raised to OD of 0.8, coated.

The 28a-abpAB vial was transferred to two large vials (Kan antibiotic was added at a final concentration of
50 μL/mL), one vial was added with 0.2 mM IPTG, and the other was not added, and the culture OD was 0.8.
Since the T vector expression was slow, 28a-abpAB was used as a control to verify expression after T-vector
ligation.

Pour the plate, two add IPTG, two do not. Four bottles of E. coli were separately plated, and after
culturing for three hours, 1 μL of phage solution was added dropwise to the center of the plate, and the
plate was allowed to stand at 37℃ after standing to dry.

Evening:

The results show that the protein AbpAB has certain anti-phage ability

**7.Obtained transcriptomics data and performed data analysis**

12/04/2019-24/04/2019

The phage was used to infect the Escherichia coli in log phase, and the frozen ice was used for freezing at
5 minutes and 20 minutes, and the sample was sent to the company for mRNA content, thereby obtaining
transcriptomics data and searching for inducible start-up in response to phage. Finally, through data
analysis, we selected two inducible promoters, P*putA* and P*glcF*, which are capable of responding to phage
infection in the early and late stages of phage infection.

**8.Verification of phage-inducible promoters**

25/04/2019-15/05/2019

1. Inoculate three 10 mL vials.

2. Common *E. coli* BL21, BL21 with plasmid, *glcF-rfp* on plasmid, and *E.coli* BL21 with *putA-gfp* recombinant plasmid.
100 μL is connected to a 10 mL Erlenmeyer flask.

3. 37 °C 180 shaker overnight culture

![](https://static.igem.org/mediawiki/2019/1/1c/T--JiangnanU_China--notebook_0.png)

**9.Purification of phage genome**

16/05/2019-

Evening:

Connect the *E. coli* BL21 vial to prepare the reagents needed to extract the phage genome.

17/05/2019-27/05/2019

1. Preparation of phage pure culture. A small bottle of *E. coli* BL21 was transferred to a large bottle, and
the OD was cultured to 0.5-0.6. The solution was inoculated with 1 mL of phage solution for 4-6 hours until
the bacterial solution was clear. The lysate was centrifuged at 10,000 rpm for 10 min, and the supernatant
was a phage pure culture solution.

2. Preparation of phage crude particles. The overnight cultured *Escherichia coli* BL21 was transferred to a
large vial, the inoculum amount was 1%, amplified to a log phase (OD of about 0.4), 5 mL of phage pure
culture solution was added, and cultured at 37℃ for 6-8 hours to obtain a phage lysate. DNase1 and RNaseA
were added to the phage lysate to a final concentration of 5 μg/mL, and the mixture was allowed to stand at
37 °C for 1 h, then NaCl was added to a final concentration of 0.1 mM, and the mixture was dissolved and
dissolved in an ice bath for 1 h (overnight).

3. The overnight phage lysate was centrifuged at 12000 rpm for 20 min. The supernatant was transferred to
another centrifuge tube and PEG 6000 was added to a final concentration of 10% (w/v). After fully
oscillating and dissolving, it was allowed to stand at 4 °C for 1 h, and centrifuged at 12,000 rpm for 20
min to discard the supernatant. The pellet was resuspended in 500 μL TM (or ddH2O) solution and repeatedly
extracted three times with an equal volume of chloroform, and centrifuged at 12,000 rpm for 10 min to remove
PEG 6000 in the resuspension, and finally a crude phage particle extract was obtained.

4. Manual extraction of phage genome
DNase I and RNase A were added to the purified phage particles at a final concentration of 1 μg/mL and
allowed to stand at 37 ° C for 1 h to degrade the DNA or RNA of the remaining host bacteria.
Then EDTA (pH 8.0) was added to a final concentration of 50 mmol/L to terminate DNase I and RNase A
activities.

Add SDS to a final concentration of 0.5%, add proteinase K to a final concentration of 50 μg/mL, mix, and
digest the protein at 56 °C for 2 h.
Add an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1), mix at 10,000 rpm for 10 min, collect
the supernatant

Step 4 was repeated 3 times; the supernatant was collected, an equal volume of chloroform was mixed, and
10000 rpm, 10 min, and the supernatant was collected;

Add 1/10 volume of 3 mol/L NaAc and 2 volumes of ice 95% ethanol and mix at -20 °C for at least 2 hours.
10000 rpm, 10 min, precipitated DNA;

Add 70% ethanol (200 μL) to the pellet, and invert the capped tube several times and centrifuge at 12000 rpm
for 5 min to recover the DNA.

Remove the supernatant, remove the alcohol droplets from the tube wall, and dry the open centrifuge tube for
10 min at room temperature, then resuspend the DNA with TE solution.

Detection by 0.8% agarose electrophoresis (mainly see if the strip is present, whether it is single). The
gel electrophoresis band was very shallow, the DNA concentration after the gel recovery was very low, and
the phage genome was successfully extracted.

**10.Discussion of next step of experiment**

28/05/2019

Regular meetings in the group to discuss the next step. Decided to use the ARTP mutagenesis system to find
new anti-phage genes.

**11.Analysis of mutant strain to obtain anti-phage genes and verified them**

28/05/2019-28/7/2019

The mutant strain of *E. coli* BL21 was obtained by ARTC (Atmospheric and room temperature plasma) mutagenesis
system, and the mutant strain and the phage were mixed and cultured for 12 h, the temperature was 37 ° C,
the shaking speed was 180 rpm, and the culture solution was coated on the culture solution. The culture dish
was cultured at 37 ° C for 24 hours at a constant temperature. A single colony capable of growing a single
colony on the plaque was a mutant strain capable of resisting phage infection. All single colonies were
inoculated into 10 mL of LB medium at 37 ° C, and the rotation speed was 180. Incubate at 180 rpm for 12
hours, then apply 100 μL of the culture solution to the plate, and add 1 μL of the phage solution to the
center of the plate. If the plaque appears after 37 ° C culture overnight, the mutant strain is eliminated.
When no plaque appeared, the plate was streaked, and single colony re-culture and plate verification were
carried out. Through 10 rounds of culture and plate verification, the mutation site and anti-phage traits
were consolidated, and 8 mutant strains were finally screened.

The whole genome sequencing of eight mutant strains was carried out to obtain the mutant genes shared by
several mutant strains: *nuoE*, *yhjH*, *rzpD*, *gntR*, and these four genes were ligated to the pET-28a plasmid
with lactose operon respectively. The substrate microorganism *E. coli* BL21 was induced by IPTG with a final
concentration of 0.5 mmol/L. The induction temperature was 37 °C for 12 h. During the period, samples were
taken at 0 h, 4 h, 6 h, 8 h, 10 h. Its OD600, compared with the E. coli growth curve containing the empty
plasmid, and the model determined that *gntR* had the least effect on the growth of *E. coli*.

After incubation for 12 h, 100 μL of the culture solution was applied to a Petri dish containing a final
concentration of 0.5 mmol/L, and then 1 μL of the phage solution was added dropwise to the center of the
culture dish, and cultured at 37 ° C for 12 hours at a constant temperature to observe plaques. Transparency
to determine which resistance gene is better, the results show that *gntR* is best against phage.

**12.Constructed recombinant bacteria with antimicrobial peptide and verified it**

29/07/2019-15/08/2019

Construction and effect verification of antimicrobial peptide P-1 (BBa\_K628000), induced engineering
bacteria containing antimicrobial peptides by IPTG, and observed growth.

**13.Construction of respond circuit and characterized it**

16/08/2019-19/09/2019

We ligated the combined resistance protein genes（*antP1-anpAB*, *antP2-gntR*） and the kill switch P-1
（BBa\_K628000）after the latent phage-inducible promoter and the burst-inducible promoter, and performed
the phage infection assay on the LB agar plate, and got very good resistance.

In addition, we inoculated *E. coli* BL21 and recombinant *E. coli* BL21 -pET28a-P*putA*-*abpAB*-*gntR*-P*glcF*-P-1
in LB liquid medium to raise the logarithmic growth phase, i.e. OD 0.6-0.8 . Than the fresh phage
solution was inoculated at the same time and culture was continued for 1-2 h. As a result, it was found
that the recombinant grew well.

**14.Standardized all our components**

19/09/2019-05/10/2019

Standardize all components. The person is responsible for learning to use an electron microscope and taking
pictures of the phage.

![](https://static.igem.org/mediawiki/2019/a/a5/T--JiangnanU_China--footer_1.png)


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:JiangnanU China/Experiments

JiangNan

[![](https://static.igem.org/mediawiki/2019/3/34/T--JiangnanU_China--Logo-Web_version.jpg)JiangnanU\_China](https://2019.igem.org/Team%3AJiangnanU_China)

* Project
  + ![  ](https://static.igem.org/mediawiki/2019/3/31/T--JiangnanU_China--chosen_bar.jpg)
  + [Description](https://2019.igem.org/Team%3AJiangnanU_China/Description)
  + [Design](https://2019.igem.org/Team%3AJiangnanU_China/Design)
  + [Safety](https://2019.igem.org/Team%3AJiangnanU_China/Safety)
  + [Demonstration](https://2019.igem.org/Team%3AJiangnanU_China/Demonstrate)
* Experiments
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  + [Protocol](https://2019.igem.org/Team%3AJiangnanU_China/Experiments)
  + [Parts](https://2019.igem.org/Team%3AJiangnanU_China/Parts)
  + [Improvement](https://2019.igem.org/Team%3AJiangnanU_China/Improve)
  + [Notebook](https://2019.igem.org/Team%3AJiangnanU_China/Notebook)
  + [Results](https://2019.igem.org/Team%3AJiangnanU_China/Results)
  + [Measurement](https://2019.igem.org/Team%3AJiangnanU_China/Measurement)
* Modeling
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  + [Model](https://2019.igem.org/Team%3AJiangnanU_China/Model)
* Human Practices
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  + [Integrated Human Practices](https://2019.igem.org/Team%3AJiangnanU_China/Human_Practices)
  + [Public Engagement](https://2019.igem.org/Team%3AJiangnanU_China/Public_Engagement)
  + [Meetup](https://2019.igem.org/Team%3AJiangnanU_China/Human_Practices#meetup)
* Team
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  + [Team Members](https://2019.igem.org/Team%3AJiangnanU_China/Team)
  + [Collaborations](https://2019.igem.org/Team%3AJiangnanU_China/Collaborations)
* Acknowledgements
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  + [Attributions](https://2019.igem.org/Team%3AJiangnanU_China/Attributions)
  + [Sponsors](https://2019.igem.org/Team%3AJiangnanU_China/Sponsors)
* Medal
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  + [Medal Requirements](https://2019.igem.org/Team%3AJiangnanU_China/Medals)
  + [Awards](https://2019.igem.org/Team%3AJiangnanU_China/Awards)

**Protocol**

![](https://static.igem.org/mediawiki/2019/d/d1/T--JiangnanU_China--protocol_0.png)

[Welcome to click here to get our team protocol.](https://static.igem.org/mediawiki/2019/2/2a/T--JiangnanU_China--protocol.pdf)

![](https://static.igem.org/mediawiki/2019/a/a5/T--JiangnanU_China--footer_1.png)

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:JiangnanU China/Results

JiangNan

[![](https://static.igem.org/mediawiki/2019/3/34/T--JiangnanU_China--Logo-Web_version.jpg)JiangnanU\_China](https://2019.igem.org/Team%3AJiangnanU_China)

* Project
  + ![  ](https://static.igem.org/mediawiki/2019/3/31/T--JiangnanU_China--chosen_bar.jpg)
  + [Description](https://2019.igem.org/Team%3AJiangnanU_China/Description)
  + [Design](https://2019.igem.org/Team%3AJiangnanU_China/Design)
  + [Safety](https://2019.igem.org/Team%3AJiangnanU_China/Safety)
  + [Demonstration](https://2019.igem.org/Team%3AJiangnanU_China/Demonstrate)
* Experiments
  + ![  ](https://static.igem.org/mediawiki/2019/3/31/T--JiangnanU_China--chosen_bar.jpg)
  + [Protocol](https://2019.igem.org/Team%3AJiangnanU_China/Experiments)
  + [Parts](https://2019.igem.org/Team%3AJiangnanU_China/Parts)
  + [Improvement](https://2019.igem.org/Team%3AJiangnanU_China/Improve)
  + [Notebook](https://2019.igem.org/Team%3AJiangnanU_China/Notebook)
  + [Results](https://2019.igem.org/Team%3AJiangnanU_China/Results)
  + [Measurement](https://2019.igem.org/Team%3AJiangnanU_China/Measurement)
* Modeling
  + ![  ](https://static.igem.org/mediawiki/2019/3/31/T--JiangnanU_China--chosen_bar.jpg)
  + [Model](https://2019.igem.org/Team%3AJiangnanU_China/Model)
* Human Practices
  + ![  ](https://static.igem.org/mediawiki/2019/3/31/T--JiangnanU_China--chosen_bar.jpg)
  + [Integrated Human Practices](https://2019.igem.org/Team%3AJiangnanU_China/Human_Practices)
  + [Public Engagement](https://2019.igem.org/Team%3AJiangnanU_China/Public_Engagement)
  + [Meetup](https://2019.igem.org/Team%3AJiangnanU_China/Human_Practices#meetup)
* Team
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  + [Team Members](https://2019.igem.org/Team%3AJiangnanU_China/Team)
  + [Collaborations](https://2019.igem.org/Team%3AJiangnanU_China/Collaborations)
* Acknowledgements
  + ![  ](https://static.igem.org/mediawiki/2019/3/31/T--JiangnanU_China--chosen_bar.jpg)
  + [Attributions](https://2019.igem.org/Team%3AJiangnanU_China/Attributions)
  + [Sponsors](https://2019.igem.org/Team%3AJiangnanU_China/Sponsors)
* Medal
  + ![  ](https://static.igem.org/mediawiki/2019/3/31/T--JiangnanU_China--chosen_bar.jpg)
  + [Medal Requirements](https://2019.igem.org/Team%3AJiangnanU_China/Medals)
  + [Awards](https://2019.igem.org/Team%3AJiangnanU_China/Awards)

**Results**

In order to make *E. coli* in the laboratory resistant to phage infection, this year our team
first
found components that responded to phage infection through transcriptome analysis.
Then we found components that could make the bacteria resistant to phage infection through
literature search and mutagenesis screening.

[![back](https://static.igem.org/mediawiki/2019/0/09/T--JiangnanU_China--host_liubianxing.png)

View all](#phage)

Phage Isolation

We added 1 μL of phage-infected fermentation broth to a plate containing *E. coli* BL21.

After proper culture for a period of time, plaque appeared on the plate (Fig.1).

We isolated the phages from the plate and photographed them using a projective electron microscope (Fig.
2).

We finally determined that the T4 phages infected our fermentation broth by sequencing the genome of the
phages.

![](https://static.igem.org/mediawiki/2019/thumb/2/23/T--JiangnanU_China--zhang.new.png/1200px-T--JiangnanU_China--zhang.new.png)

**Selection of Inducible Promoters**

1.Selection

In order to screen inducible promoters, we first made the one-step growth curve of phages (Fig. 3).
After that, we selected two time points of phage infection for 5min （in the latent period of phage
infection）and phage infection for 20min （in the burst period of phage infection）through the one-step
growth curve of phage.
By analyzing transcriptome data，we selected an inducible promoter P*putA* (Fig.4) for 5 min and an inducible
promoter P*glcF* (Fig.5) for 20 min.

![](https://static.igem.org/mediawiki/2019/e/e2/T--JiangnanU_China--results_3.png)![](https://static.igem.org/mediawiki/2019/e/e8/T--JiangnanU_China--results_1.png)

2. Characterization

In *E. coli* BL21, we connected the green fluorescence gene *gfp* with the inducible promoter P*putA*
for 5 min
(Fig.6) and the red fluorescence gene *mCherry* with the inducible promoter P*glcF* for 20 min
(Fig.7) in
our genetic circuits.
After infecting the bacteria with phages for the corresponding time, we observed that the infected cells
gave off green and red fluorescence respectively.

![](https://static.igem.org/mediawiki/2019/b/be/T--JiangnanU_China--results_0.png)

3. Anti-phage Parts

When we had components that responded to phage infection, we searched for anti-phage parts through
literature search and mutagenesis screening.

3.1 Anti-phage part from literature

Through literature search, we found a resistant protein AbpAB that can resist T4 phage. Protein AbpAB can inhibit the replication and late gene expression of phage, which resulted in blocking of phage propagation. AbpAB have no effect on the bacterial growth(Fig8.) which is important to the industry. However, protein AbpAB didn’t work well as we expected(Fig 9.).

![](https://static.igem.org/mediawiki/2019/2/28/T--JiangnanU_China--abpAB.png)

3.2. Anti-phage part from mutation screening

To get more efficient phage resistant parts, we used the ARTP(Atmospheric and room temperature plasma)
mutagenesis system to obtain a large number of mutant strains. Then we co-cultured the mutant strains
with the phage and screened the mutant strains that could resist phage infection. In the screening
process, we continuously verified their resistance, eliminated the bacterial strains with degraded
resistance and retained the ones with excellent resistance. Then we obtained 4 mutant strains which were
resistant to phage and four key mutation sites (*nuoE*, *yhjH*, *rzpD*, and *gntR*) through comparation of
genome (Fig. 10). Resistance tests on these key sites were performed respectively(Fig 11.). According to
the advice of corporate stakeholders, if the Genetic Modified (GM) strain is to be applied in industry,
our components cannot have a great influence on bacterial growth. Since it is not possible to directly
see from the figure which component has the least influence on the growth of the bacteria, we use the
Grey Relation Analysis（GRA） method to analyze the growth curve（Fig 12.） of the bacteria connecting the
various components. We used the Entropy Weight Method (EWM) to determine the weight of each growth point
to select the most similarly modified strain (the highest correlation), which is the component that has
the least impact on bacterial growth. At the same time, after consulting the industry experts, we
revised the weights according to the experts' recommendations to evaluate the components again, making
them more in line with the real situation of production, that is, the most suitable components for
industrial production. Using GRA's two evaluations of the four components at different weights, we
selected the component *gntR*.(Fig 13.)

![](https://static.igem.org/mediawiki/2019/7/73/T--JiangnanU_China--result_5.png)

![](https://static.igem.org/mediawiki/2019/c/c8/T--JiangnanU_China--11.png)
![](https://static.igem.org/mediawiki/2019/c/c0/T--JiangnanU_China--anti-phage-data2.png)

![](https://static.igem.org/mediawiki/2019/1/16/T--JiangnanU_China--result_7.png)![](https://static.igem.org/mediawiki/2019/3/31/T--JiangnanU_China--result_8.png)

3.3. Cascade of protein AbpAB and GntR

First, resistant proteins AbpAB and GntR were verified by SDS-PAGE（Fig 14.）Then，We connected gntR with
abpAB in pET-28a plasmid , and transformed them into *E. coli* BL21. We co-expressed *abpAB* and *gntR*, and
subsequently obtained a recombinant strain that is resistant to phage（Fig 15.）. The result showed that
two proteins works better together.

![](https://static.igem.org/mediawiki/2019/a/a8/T--JiangnanU_China--result_9.png)![](https://static.igem.org/mediawiki/2019/5/59/T--JiangnanU_China--result_10.png)

4. Assembly and application

In order to prevent the phage from escaping the attack of our resistant protein, we connected a kill
switch P-1 ([BBa\_K628000](http://parts.igem.org/Part%3ABBa_K628000)) with the 20 min induction promoter(Fig 16).

![](https://static.igem.org/mediawiki/2019/a/ac/T--JiangnanU_China--result_11.png)

We inoculated *E. coli* BL21 and recombinant *E. coli* BL21-pET28a-P*putA*-*abpAB*-*gntR*-P*glcF*-P-1 in LB liquid
medium to raise the logarithmic growth phase, i.e. OD 0.6-0.8 . Then the fresh phage solution was
inoculated at the same time and continuous cultured for 1-2 h. As a result, it was found that the
recombinant grew well.(Fig 17.).

![](https://static.igem.org/mediawiki/2019/d/d5/T--JiangnanU_China--result_12.png)

In addition, we cooperated with [NINGXIA EPPEN BIOTECH CO.,LTD](http://nxyp.xafangao.com) to carry out
small-scale and pilot test fermentation experiments of resistant strain in the special fermentation
laboratory of Jiangnan University. This ensured that our experiments were controllable without any phage
and engineered bacteria leaking.

We transferred the constructed plasmid into an engineering bacteria strain producing γ-aminobutyric acid
(GABA).

The fermentation laboratory is subjected to UV irradiation and ozone fumigation prior to formal
fermentation to remove phage that may be present. The *E. coli* BL21-pET28a-P*putA*-*abpAB*-*gntR*-P*glcF*-P-1, *E.
coli* BL21-pET28a-P*putA*-*abpAB* and the control （*E. coli* BL21） were added T4 phage after six hours of
culture, and the fermentation was continued for 10 hours. During the fermentation, the OD was measured,
and the effects of the phage on the three were observed.

Then we used whole-cell transformation with a combination of resistant strain *E. coli*
BL21-pET28a-P*putA*-*abpAB*-*gntR*-P*glcF*-P-1, and we got a good whole-cell transformation ability of the
resistant strain (Figure 18).

From the results, our resistant composite part has great advantages in the production of γ-aminobutyric
acid and are not threatened by T4 phage. The productivity of γ-aminobutyric acid is 278.3 g/L, and the
molar conversion rate is really high, reaching 98.4%(Table 1.), which means that the circuit we built
can be used in production without any impact (Figure 19).

![](https://static.igem.org/mediawiki/2019/7/7a/T--JiangnanU_China--result_13.png)![](https://static.igem.org/mediawiki/2019/thumb/8/83/T--JiangnanU_China--fajiao.png/1200px-T--JiangnanU_China--fajiao.png)[![back](https://static.igem.org/mediawiki/2019/2/24/T--JiangnanU_China--host_back.png)](#head)

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