iGEM 2023 — Team xmu-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 %}

![](https://static.igem.wiki/teams/4907/wiki/background/loading.png)

# . NoteBook .

Dive into our iGEM 2023 adventure with XMU-China! It's not just a science notebook; it's a tale of sweat,
laughter, mentor wisdom, and the unbeatable thrill of teamwork. Our journey wasn't just about cells and
sequences; it was about passion, courage, and the relentless pursuit of science.

Last December to January

- XMU-China started a new round of recruitment!

- We shared synthetic biology knowledge and lots of excellent projects in iGEM
with college students in different majors. Each one who participates and completes the selection is the
BEST!

Met in February

- Our team was formally founded. This year, we got fourteen fresh faces on
board!

- By chance, we learned that extreme weather had been threatening agricultural
production in recent years. At this point, the project theme about anti-icing and anti-drought came into
our view. We were then divided into two groups, focusing on preventing frost and combating drought
issues respectively. We held meetings and discussed every weekend about everyone’s progress and solved
problems together.

April-May

- We learned the fundamental knowledge of molecular cloning and laboratory
qualities. After that, we were trained for molecular cloning operations, which helped us to design
experiments rationally.

- We optimized and constructed the sequences of the needed genes.

- We completed the original hypothesis testing experiments needed, then
consulted professors for advice. With their help, we gradually refined our project.

Ambition in March

- During the first-ever meetup of XMU-China 2023 friends, we celebrated a
fellow member's birthday. 242 days before Grand Jamboree, we made a golden prize wish.

![](https://static.igem.wiki/teams/4907/wiki/notebook/ambition-fig1.jpg)

April

- Brainstroming! Project design was gradually
improved.

Interview with Prof. Jianwei Lyu

- We interviewed Prof. Lyu at school of life science in Xiamen University. He
laid emphasis on the significance of biosafety to us.

May

- We learned the fundamental knowledge of molecular
cloning and laboratory qualities. After that, we were trained for molecular cloning operations, which
helped us to design experiments rationally.

- We drafted the original hypothesis testing experiments needed under
instruction.

May 6

- Naiads at its very inception.

![](https://static.igem.wiki/teams/4907/wiki/notebook/first-fig1.jpg)

June 9

- We proved that the two cryptic plasmid are in
Nissle 1917, and then we started to carry out CRIPSR/Cas9...(didn't know that this would be a great
torture in the next few months, SHIFT).

June 16

- Our very first group photo!

![](https://static.igem.wiki/teams/4907/wiki/notebook/first-fig2.jpg)

Interview with Prof. Dai Wang

- Breakthrough in biosynthesis of composite water
retaining agent! We finally confirmed to achieve control of the microbial composition by adjusting
antibiotic ratios. And Prof. Wang gave us a lot of advice on the design of HGT (horizontal gene
transfer) prevention as well.

July 2

- The birth of our goddess.

![](https://static.igem.wiki/teams/4907/wiki/notebook/first-fig3.png)

July 2

- Happy!!! The production of TmAFP is improved
significantly because changing the expressed strain from BL21 (DE3) to BL21 Star (DE3).

- Very luckily, we eliminated pMUT1. FANG was so happy. But she didn't know the
power of pMUT2.

July 15

- First successful characterization experiment within the team! Thanks to BBa\_K4907132, thanks to the
Oxford Cup!

Water Resources Bureau

- We held a discussion with personnel from the Water Resources Bureau, where we gained insights into
existing methods for drought monitoring and prevention, further enhancing our project's background
knowledge.

July 28

- Our team had been successfully assigned to the new village this year: Agriculture!

July 31

- After countless mutations, VSW-3 RNAP (BBa\_K4907114) is finally built!

Busy August

August 5

- The third time purification, we finally got a
satisfying protein gel map of CBM. TT was over-excited because it took her a month to do the damn job!

August 9

- Engineering Success! After adding a linker between Kp-SP and GFP, the fusion
protein emit fluorescence finally!

Xinfeng and Mount Wuyi field research

We went our separate ways - one heading to Xinfeng,
Jiangxi for soil frost damage research, and the other to Mount Wuyi, Fujian for soil drought research,
ultimately enhancing our project's background data. Surprise! A local farmer uncle mentioned that we
might be able to spread AFPs and water-retaining agents on crop leaves, providing us with an incredibly
new idea for our project implementation.

August 12

- We try try try, we fail fail fail.
The reason for the three-month-long experimental failures in constructing and characterizing the part
BBa\_K4907145 turned out to be unbelievably stupid - the promoter was installed backward.

August 13

- Amazingly, just when I was about to give up VSW-3 RNAP, it worked. Was it
meant to be?

August 22

- Family who knows……Misfortunes never come alone. After a dramatic three-month
saga of construction and characterization of hrp genes, brace for this epic twist – the sequences used
were dead wrong, god!

August 27

- Finally, after two months of trials and failures,
pMUT2 had been eliminated!!! (roaring). Omg relB is so powerful ahhhhhhh!

August 30

- Construction of BBa\_K4907140 is finished.

New September

September 10

- First operation of FermentX!

![](https://static.igem.wiki/teams/4907/wiki/notebook/sept-fig1.png)

September 13

- Promotion Video, it's done.

September 10-September 20

- BBa\_K4907123 were constructed and characterized
successfully which only took ONE time.

September 23

- We completely abandon the gene circuit building
of parE because of its hypertoxicity after struggling for three months...

September 24

- Xiang’an said goodbye to Siming, one last time.

![](https://static.igem.wiki/teams/4907/wiki/notebook/ending-fig1.jpg)

September 30

- After trying five protocols with no success, TT
is so desperate for
J04450\_pSB3C5 to grow in ECNP.

Sprint in October!

October 12

- Wiki FREEZE!!!

End in November

November 2-November 5

- Wish we all find the gem! In PARIS! End of
competition, infinite in friendship.

read me ↑

![](https://static.igem.wiki/teams/4907/wiki/footer-icon/xmuchina.png)

Xiamen University

![](https://static.igem.wiki/teams/4907/wiki/footer-icon/place.png)

Xiamen University, Fujian, China
No.422, Siming South Road, Fujian, P.R.China 361005

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{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4907/wiki/background/loading.png)

[Overview](#section1)

[Functional KpSP-GFP protein](#section2)

[Eliminating
the cryptic plasmids](#section3)

![](https://static.igem.wiki/teams/4907/wiki/background/sidebar1.png)

# . Engineering Success .

## **Overview**

This year, XMU-China focused on the problem of soil frost and soil drought, endeavored to tackle and had
solved problems during our engineering process, generating expected results.

Countless efforts were made to ensure our design can function to attain our goals. Among them, the two
sections “obtaining a functional KpSP-GFP protein” and “harnessing pEcCas/pEcgRNA system to eliminate the
cryptic plasmids of *E. coli* Nissle 1917” are typically worth mentioning. All the related data are recorded
below. Hopefully, they will make some contribution to the iGEM community.

## **Section 1: Obtaining a functional KpSP-GFP protein**

### **Introduction**

The antifreeze proteins (AFPs) are utilized to solve frost in our design. Since AFPs function directly on
the surface of the ice, whatever thermal hysteresis (TH) or ice recrystallization inhibition (IRI), it is
necessary that AFPs should be secreted from the cytoplasm of our engineered bacteria to the extracellular
circumstance. Hence, we hope to realize it by using a signal peptide from *Kocuria* sp.3-3, or KpSP, which
directs target proteins to the extracellular circumstance.

### **Cycle 1: Using KpSP-GFP to test the secretion function of KpSP**

#### **Design**

KpSP, found from *Kocuria* sp.3-3, has shown remarkable function to secret target proteins to the
extracellular circumstance through the Sec pathway in a previous work (1). To firstly verify the function of
KpSP, we fused it directly with GFP and then obtained KpSP-GFP to see whether GFP was secreted or not.

#### **Build**

There were four parts assembled into the vector: promoter ([BBa\_I0500](http://parts.igem.org/Part%3ABBa_I0500)), RBS ([BBa\_B0034](http://parts.igem.org/Part%3ABBa_B0034)), KpSP-GFP coding
sequence, and terminator ([BBa\_B0015](http://parts.igem.org/Part%3ABBa_B0015)). We got the constructed plasmid successfully and transformed it into *E.
coli* BL21(DE3), then the positive transformants were selected through colony PCR (Fig. 1) and sequencing.

![](https://static.igem.wiki/teams/4907/wiki/engineering-success/yyn/fig-1.png)

**Fig. 1 Agarose gel electrophoresis of the colony PCR products
of I0500-B0034-KpSP-*gfp*-B0015\_pSB1C3.** Target bands (2494 bp) can be observed at the position between 2000
bp and 3000 bp.

#### **Test**

The fluorescence intensity of the bacterial culture and supernatant was measured to test whether KpSP can
function
well to lead to the secretion of GFP or not. In this case, [BBa\_I0500](http://parts.igem.org/Part%3ABBa_I0500)
(*araC*/pBAD, no gene downstream) was set as the negative control,
while [BBa\_K4907100](http://parts.igem.org/Part%3ABBa_K4907100) (for expressing GFP
intracellularly) which
has no signal peptide's coding sequence was also tested to compare the fluorescence intensity.
After being induced by *L*-arabinose for 6 hours, the fluorescence intensity of the three groups was
measured.

![](https://static.igem.wiki/teams/4907/wiki/engineering-success/yyn/fig-2-2.png)

**Fig. 2 RFUGFP/OD600 of the three groups were calculated.**  The relative
fluorescence units (RFU) of bacterial culture subtracted the background fluorescence of growth media,
resulting in the RFUGFP. *p*-value: no significance (ns), 0.0002 (\*\*\*).

Compared to the group expressing GFP intracellularly, no fluorescence was observed in the KpSP-GFP group,
and even the ratio of fluorescence intensity to OD600 of the KpSP-GFP group showed no differences to the
negative control group (Fig. 2), which indicated the fusion protein KpSP-GFP cannot work normally.

#### **Learn**

The result above showed that the fusion of KpSP might have negative effects on the folding or maturation of
GFP, both of which are vital for fluorescent proteins to exhibit fluorescence normally (2).

### **Cycle 2: Using KpSP-GFP added a linker to test the secretion function of KpSP**

#### **Design and Build**

Based on the result of Cycle 1, we added a linker between KpSP and GFP ([BBa\_K4907101](http://parts.igem.org/Part%3ABBa_K4907101)),
for the purpose of improving the folding stability so that both KpSP and GFP can function properly (3). Additionally, to enhance the efficiency of induction,
*E. coli* DH10β; was selected as the expression strain for the following characterization.

![](https://static.igem.wiki/teams/4907/wiki/engineering-success/yyn/fig-3.png)

**Fig. 3 Agarose gel electrophoresis of the colony PCR products of BBa\_K4907101\_pSB1C3.** Target bands (2557
bp) can be observed at the position between 2000 bp and 3000 bp.

#### **Test**

The test method was the same as which in Cycle 1. At this time, the bacteria expressing KpSP-GFP showed
bright fluorescence upon excitation, and the normalized fluorescence intensity was much higher than that of
the negative control (Fig. 4a), which meant the correct folding structure of GFP was retained. Moreover, the
fluorescence intensity ratio of supernatant to the bacterial culture of KpSP-GFP was much higher than that
of those expressing GFP only in cytoplasm (Fig. 4b), representing the good performance of KpSP.

![](https://static.igem.wiki/teams/4907/wiki/engineering-success/yyn/fig-4-2-01.png)

**Fig. 4 Inserting a linker between KpSP and GFP retains the protein's function. a**
RFUGFP/OD600 of the three groups. **b** Comparation of the fluorescence
intensity ratio (supernatant to bacterial culture) between I0500 (set as negative control group)
and KpSP-GFP with a linker. **c** The illustration for the comparison of misfolded KpSP-GFP and
correctly folded KpSP-GFP. *p*-value: <0.0001 (\*\*\*\*).

#### **Learn**

The result indicated that KpSP can function well to direct target proteins out of the engineered bacteria as
expected, which lays the foundation of our project for anti-soil frost. Although the fluorescence was
recovered, the normalized fluorescence intensity of the bacterial culture of KpSP-GFP was still lower than that
of the intracellularly expressed GFP, which meant the negative effects were not eliminated completely. From
another aspect, **the lesson implied an important but easy-to-be-neglected issue between the folding
properties of target proteins and the (potential) transmembrane mechanisms of the implemented secretion
systems** (Fig. 4c), which might be helpful and valuable for other teams or labs when they are considering
ideas about the secretion of recombinant proteins from engineered bacteria.

#### **Conclusion**

Engineering a biological system cannot be easy, we need to pay our all attention to it and move towards
success through experiments and feedback step by step. In the beginning, we found that the KpSP signal
peptide might influence the folding or maturation of GFP negatively when they are directly fused. For
optimizing, **we added a linker between KpSP and GFP, and then validated their function successfully**. Fig. 5
demonstrates how we change the coding sequence of KpSP-GFP fusion protein to get the functional KpSP-GFP protein
through successive iterations of the **DBTL** (Design-Build-Test-Learn) cycle.

![](https://static.igem.wiki/teams/4907/wiki/engineering-success/yyn/fig-5.png)

**Fig. 5 Summary of the different engineering steps completed before reaching the functional KpSP-GFP
protein finally.**

## Section 2: Harnessing pEcCas/pEcgRNA system to eliminate the cryptic plasmids of *E. coli* Nissle 1917

### **Introduction**

For bacterial cellulose (BC) production, after comprehensive consideration, we chose *E. coli* Nissle 1917
(EcN) as our engineered bacteria, which will then be co-cultured with *E. coli* BL21(DE3) for the production
of water-retention material, BC/HA (see [Design](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27design%27%29%20%7D%7D) page for more details). During the project, some
modifications for the chassis EcN, **especially the elimination of endogenous cryptic plasmids**, have been
achieved for the purpose of improving the production of BC.

### **Cycle 1: Construction of a circuit for characterizing the growth rate of *E. coli* Nissle 1917**

#### **Design**

The water-retention material BC/HA that we plan to produce is a cross-linked product of BC and HA, and the
ratio of BC to HA in the product will directly affect the water-holding capacity of the material. In order
to ensure that the BC/HA we produce has the best water retention performance, we decided to regulate the
strain ratio of two engineered bacteria, *E. coli* Nissle 1917 and *E. coli* BL21(DE3), during the process of
co-culture fermentation. Inspired by previous work, we planned to introduce different fluorescent proteins
into the two engineered bacteria respectively and use the ratio of the two fluorescence intensities to
reflect the ratio of the two strains (4). Specifically, we planned to express the red fluorescent protein in
*E. coli* Nissle 1917 and hence constructed a corresponding circuit (Fig. 6).

#### **Build**

There were four basic parts assembled into the vector pSB3C5: promoter ([BBa\_J23100](http://parts.igem.org/Part%3ABBa_J23100)), RBS ([BBa\_B0034](http://parts.igem.org/Part%3ABBa_B0034)), *rfp* coding sequence ([BBa\_K4907037](http://parts.igem.org/Part%3ABBa_K4907037)), and terminator ([BBa\_B0015](http://parts.igem.org/Part%3ABBa_B0015)). We got the constructed plasmid successfully and
transformed it into *E. coli* Nissle 1917.

![](https://static.igem.wiki/teams/4907/wiki/engineering-success/zcf/j23100-rfp-psb3c5.png)

**Fig. 6 Graphic description of the *rfp*-expressing gene circuit.**

#### **Test**

Surprisingly, after several trials, we found that our constructed plasmid, J23100-B0034-*rfp*-B0015\_pSB3C5
could not be successfully transformed to *E. coli* Nissle 1917. At the same time, we also tried to transform
another plasmid into *E. coli* Nissle 1917, including B0034-*hasA*-B0015\_pSB1C3,
J23100-B0034-*gfp*-B0015\_pET-28a(+), pT7-B0034-*rfp*\_pET-28a(+) and J04450\_pSB4A5. We found those plasmids that are
based on the pET-28a(+) or pSB4A5 vector are transformable, while the others are not transformable (Fig.
7).

![](https://static.igem.wiki/teams/4907/wiki/engineering-success/zcf/lb-agar-plate-1917-different-backbone.png)

**Fig. 7 The images of LB agar plate with transformants. a** B0034-*hasA*-B0015\_pSB1C3 in *E. coli* Nissle 1917
(no colonies). **b** J23100-B0034-*gfp*-B0015\_pET-28a(+) in *E. coli* Nissle 1917. **c**
pT7-B0034-*rfp*-B0015\_pET-28a(+) in *E. coli* Nissle 1917. **d** J04450\_pSB4A5 in *E. coli* Nissle 1917.

#### **Learn**

Through the results, we hypothesized that there might be some endogenous plasmids in the *E. coli* Nissle 1917
strain, which have conflictive replication mechanisms with that of the pSB1C3 and pSB3C5 vectors that we are
going to transform into. It is reported that **two constitutive plasmids, pMUT1 and pMUT2, generally exist in
*E. coli* Nissle 1917 and some called them cryptic plasmids** (5). The presence of these two constitutive
plasmids will affect the transformation of exogenous plasmids, *i.e.*, plasmid incompatibility occurs (5). To
better facilitate our subsequent engineering and experimental operations, we decided to explore whether
these cryptic plasmids existed in the *E. coli* Nissle 1917 strain used in our lab or not.

### **Cycle 2: Confirmation that the *E. coli* Nissle 1917 strain we used contains pMUT1 with pMUT2**

#### **Design and Build**

![](https://static.igem.wiki/teams/4907/wiki/engineering-success/zcf/pmut1-pmut2.png)

**Fig. 8 The maps of the two endogenous plasmids. a** The map of pMUT1 with primer binding sites and
restriction site of *Nde* I. **b** The map of pMUT2 with primer binding sites and restriction site of *Eco*R I.

We got the sequences of two plasmids from NCBI (pMUT1: [NZ\_MW240712](https://www.ncbi.nlm.nih.gov/nuccore/NZ_MW240712.1), pMUT2: [NZ\_CP023342](https://www.ncbi.nlm.nih.gov/nuccore/NZ_CP023342)) and designed the
corresponding primers according to the previous work (6). Meanwhile, we searched for suitable restriction
sites on the plasmid in order to linearize them and confirm the size of the plasmids (Fig. 8).

**Table 1 The primer used for verifying pMUT1 and pMUT2**

| Primer | Sequence (5′→3′) | Notes |
| --- | --- | --- |
| pM1S2chk\_F | GAATAGGGTGACACTGGCGCC | Forward primer for pMUT1 |
| pM1S2chk\_R | CCAGATGGCATTGTAACAGACTTCTC | Reverse primer for pMUT1 |
| pM2S3chk\_F | GATAAAACTATCAACTCACCGTCTTG | Forward primer for pMUT2 |
| pM2S3chk\_R | GCCGTTGGTCTTTACTGATTTTAAG | Reverse primer for pMUT2 |

#### **Test**

Firstly, we extracted the plasmids from *E. coli* Nissle 1917 and performed PCR using the designed primers
(Table 1), followed by agarose gel electrophoresis. At the same time, we performed restriction digest of the
extracted plasmids (pMUT1 at the *Nde* I site and pMUT2 at the *Eco*R I site). The bands' sizes were consistent
with the predicted ones generated from the known maps of pMUT1 and pMUT2, whatever the results of PCR (Fig.
9a) or digestion (Fig. 9b), which indicated that the *E. coli* Nissle 1917 we used did contain those two
constitutive plasmids, pMUT1 and pMUT2.

![](https://static.igem.wiki/teams/4907/wiki/engineering-success/zcf/pmut-verification1.png)

**Fig. 9 Agarose gel electrophoresis of the products. a** Agarose gel electrophoresis of the PCR products.
Target bands of pMUT1 (left, 212 bp) and pMUT2 (right, 210 bp) can be observed at the position between 100
bp and 250 bp. **b** Agarose gel electrophoresis of the restriction digest products. Target bands of pMUT2
(5514 bp) can be observed at the position between 4000 bp and 7000 bp. Target bands of pMUT1 (3173 bp) can
be observed at the position between 3000 bp and 5000 bp.

#### **Learn**

From the experimental results, we confirmed that the *E. coli* Nissle 1917 strain in our lab contained the
plasmids pMUT1 and pMUT2. In order to facilitate our subsequent experimental manipulations, we decided to
eliminate these two plasmids.

### **Cycle 3: Elimination of pMUT1 using the pEcCas/pEcgRNA system**

#### **Design**

The pEcCas/pEcgRNA system (Fig. 10a) was modified from pCas/pTargetF, a tool that has been shown to have
higher genome editing efficiency in *E. coli* (7). In this system, pEcCas ([Addgene number: 73227](https://www.addgene.org/73227/)) harbors a
sequence that expresses the Cas9 protein, while the sgRNA sequence expressed by pEcgRNA ([Addgene number: 166581](https://www.addgene.org/166581/))
will bind to the Cas9 protein to form a surveillance complex. Once the surveillance complex finds
the target sequence, it cleaves the DNA double strand to cause DSB (double strand break) (8, 9). Considering
the performance of pEcCas/pEcgRNA system and higher elimination efficiency of pMUT1 reported by previous
research (6, 10), we therefore performed pMUT1's elimination at first. The sgRNA sequence, or specifically
N20 sequence, will be designed to target a specific sequence on pMUT1 in this case (Fig. 10b).

![](https://static.igem.wiki/teams/4907/wiki/engineering-success/zcf/cripsr-mechanism-2.png)

**Fig. 10 A graphic description of eliminating the cryptic plasmids in *E. coli* Nissle 1917 by harnessing
pEcCas/pEcgRNA system. a** A graphic description of the pEcCas and pEcgRNA. **b** A graphic description of pMUT1
elimination using pEcCas/pEcgRNA system. **c** A graphic description of pMUT2 elimination using pEcCas/pEcgRNA
system.

#### **Build**

We first transformed the pEcCas plasmid into *E. coli* Nissle 1917. After successful transformation, the
bacteria were cultivated and made into electrocompetent cells. Meanwhile, we designed the N20 sequence
targeting pMUT1 on the online server ([CRISPR](http://crispor.tefor.net/)) and constructed the pEcgRNA plasmid targeting pMUT1 according
to the methods of the original paper(7). Finally, we transformed the constructed pEcgRNA-pMUT1
plasmid into *E. coli* Nissle 1917 harboring pEcCas by electroporation. (For more details, please see the
protocol: **Harnessing the pEcCas/pEcgRNA system for the elimination of cryptic plasmids in *E. coli* Nissle
1917**)

#### **Test**

**Table 3 The primer used for verifying the existence of pMUT1**

| Primer | Sequence (5′→3′) | Notes |
| --- | --- | --- |
| pMUT1\_F | ATGAGCTATGCGATAACTTTAACTGTGAAGC | Forward primer for pMUT1 |
| pMUT1\_R | ATACTTTGCCAGTCCAAGCTGGGAT | Reverse primer for pMUT1 |

The transformants were randomly picked and verified *via* colony PCR. Next, we extracted the plasmids of those
colonies that showed no target bands of colony PCR, and performed another PCR to verify whether the pMUT1
was really eliminated or not (primers are listed in Table 3). Luckily, there is one colony showing no bands
after the second round of PCR, which indicated that the elimination of pMUT1 was achieved (Fig. 11, Lane
1).

![](https://static.igem.wiki/teams/4907/wiki/engineering-success/zcf/pmut1-successful-elimination.png)

**Fig. 11 Agarose gel electrophoresis of the plasmid PCR products.** Lane 1, **no** target bands of pMUT1 (1079
bp) can be observed at the position between 1000 bp and 2000 bp. Lane 2, target bands of pMUT1 (1079 bp)
can be observed at the position between 1000 bp and 2000 bp.

Then the pEcCas/pEcgRNA plasmids of the chosen colony were cured, followed by the preparation of EcN ΔpMUT1
chemically competent cells. Subsequently, we tested whether I0500-B0034-*tmafp*-B0015\_pSB1C3 (Fig. 12) and
J23100-B0034-*gfp*-B0015\_pSB3C5 can be transformed into EcN ΔpMUT1 or not, and finally found that the plasmid
based on pSB3C5 backbone was still not transformable.

![](https://static.igem.wiki/teams/4907/wiki/engineering-success/zcf/ecn-pmut1-1c3.png)

**Fig. 12 The image of LB agar plate.** I0500-B0034-*tmafp*-B0015 \_pSB1C3 in EcN ΔpMUT1.

#### **Learn**

After the elimination of pMUT1, pSB1C3-bone plasmids could be transformed into *E. coli* Nissle 1917, which
indicated that pSB1C3 might have a similar replication mechanism with pMUT1, and this was also consistent
with the conclusion of the previous work (10). Due to the failure of transforming the pSB3C5-bone plasmids,
we hypothesized that the presence of pMUT2 would still affect the transformation of the exogenous plasmid, and
therefore we continued our work on the elimination of pMUT2 from EcN ΔpMUT1.

### **Cycle 4: Elimination of pMUT2 using the pEcCas/pEcgRNA system**

#### **Stage 1**

#### **Design and Build**

Similarly, we designed the N20 sequence and constructed the pEcgRNA-pMUT2 plasmid (Table 4), and then
transformed it into EcN ΔpMUT1 harboring pEcCas (Fig. 10c).

**Table 4 The primer and N20 sequence used in constructing pEcgRNA-pMUT2**

| Sequence (5′→3′) | Notes |
| --- | --- |
| ATTACCTGATCCTTCCGCAG | N20 sequence |
| TAGTATTACCTGATCCTTCCGCAG | Top strand sgRNA (pMUT2) with overhang sequence |
| AAACCTGCGGAAGGATCAGGTAAT | Bottom strand sgRNA (pMUT2) with overhang sequence |

#### **Test**

**Table 5 The primer used for verifying the existence of pMUT2**

| Primer | Sequence (5′→3′) | Notes |
| --- | --- | --- |
| 8F | AAAGAGTCCTTAGCACCCGC | Forward primer for pMUT2 |
| 8R | GGTGCGGTGTCAAGAAAGTT | Reverse primer for pMUT2 |

![](https://static.igem.wiki/teams/4907/wiki/engineering-success/zcf/pmut2-exist.png)

**Fig. 13 Agarose gel electrophoresis of the plasmid PCR products.** Target bands of pMUT2 (547 bp) can be
observed at the position between 500 bp and 700 bp.

#### **Learn**

Consistent with most of what has been noted in previous research, the elimination of pMUT2 often requires
a lot of effort (6, 10). Since the pMUT2 plasmid could not be eliminated after many times of trials and
failures, we decided to give up our current scheme and search for other feasible approaches. It was reported
that the *relE-relB* toxin-antitoxin system is naturally present on pMUT2, and the introduction of the
expression cassette of *relB* into the plasmid harboring the sgRNA designed to target the relaxase coding
sequence on pMUT2 would contribute to eliminating pMUT2 (6). We therefore decided to follow this experience
for pMUT2's elimination.

#### **Stage 2**

#### **Design and Build**

We obtained the sequence of *relB* and the N20 sequence targeting the relaxase coding sequence on pMUT2 from
the article (6). Next, we amplified the fragment comprising the coding sequence of *relB* from pMUT2 and
performed overlap-extension PCR (OE-PCR) by using the amplified fragments and the synthesized upstream
untranslated sequences as templates to obtain the expression cassette of *relB*. Subsequently, we cloned the
expression cassette of *relB* into pEcgRNA-relaxase that was constructed before (Table 6) *via* Gibson assembly
to obtain the construction of the final sgRNA-harboring plasmid. (For more details, please see the protocol:
**Harnessing the pEcCas/pEcgRNA system for the elimination of cryptic plasmids in *E. coli Nissle* 1917**)

#### **Test**

In previous trials of validations, we picked the colonies and then performed colony PCR, followed by plasmid
extraction and another round of PCR. However, at this time, we found that the primers we designed were not
specific enough, and hence they can bind to pEcCas and pEcgRNA, leading to false-positive results.
Therefore, we decided to implement agarose gel electrophoresis of the extracted plasmids directly instead of
colony PCR, after curing the pEcgRNA-relaxase plasmid **harboring the expression cassette of *relB***. The
extracted plasmids from the cured transformants showed no bands of pMUT2 (Fig. 14), which validated the
elimination of pMUT2.

![](https://static.igem.wiki/teams/4907/wiki/engineering-success/zcf/pmut2-elimination-successful.png)

**Fig. 14 Agarose gel electrophoresis of the extracted plasmid.** No target bands of pMUT2 (5514 bp) can be
observed at the position between 5000 bp and 7500 bp. Lane 1, the strain contains only the bands of pEcCas
but no bands of pMUT2. Lane 2, pEcCas: 14605 bp. Lane 3, pEcgRNA-relaxase (comprising *relB*): 2369 bp. Lane
4, the mixture of pMUT1 and pMUT2, target bands of pMUT2 (5514 bp) can be observed at the position between
5000 bp and 7500 bp.

After that, we tried to transform J23100-B0034-*gfp*-B0015\_pSB3C5 into the *E. coli* Nissle 1917 strain that
contained no endogenous plasmids (EcNP for short), and the results showed that the pSB3C5-bone plasmid could
be successfully transformed eventually (Fig. 15).

![](https://static.igem.wiki/teams/4907/wiki/engineering-success/zcf/ecnp-gfp-psb3c5.png)

**Fig. 15 The image of LB agar plate.** J23100-B0034-*gfp*-B0015\_pSB3C5 in EcNP.

#### **Learn**

We successfully eliminated pMUT2 and the experimental results showed that there is no obstacles for
transforming the plasmids we want, which would facilitate our downstream experimental manipulations. In
addition, since we have already obtained wild type EcN, EcN ΔpMUT1 that eliminated pMUT1, as well as the
EcNP that eliminated both pMUT1 and pMUT2, we were interested in the growth rate and the ability to express
proteins of the three strains, and hence we carried out the experiments of next stage.

### **Cycle 5: Testing the performance of EcN, EcN ΔpMUT1, EcNP**

#### **Design and Build**

We planned to express red fluorescent protein in three strains, EcN, EcN ΔpMUT1 and EcNP, so as to compare the growth rate and protein expression level of these three strains by comparing the OD600 and the fluorescence intensity. Thus, we reconstructed a gene circuit J23100-B0034-*rfp* on pET-28a(+).

There were four basic parts assembled into the vector, pET-28a(+): promoter ([BBa\_J23100](http://parts.igem.org/Part%3ABBa_J23100)), RBS ([BBa\_B0034](http://parts.igem.org/Part%3ABBa_B0034)), and *rfp* coding sequence ([BBa\_K4907037](http://parts.igem.org/Part%3ABBa_K4907037)). We got the constructed plasmid ([BBa\_K4907037](http://parts.igem.org/Part%3ABBa_K4907037))\_pET-28a(+) successfully and transformed it into EcN, EcN ΔpMUT1 and EcNP, respectively.

#### **Test**

We conducted OD600 and fluorescence intensity measurements on the three strains which harbored J23100-B0034-*rfp*\_pET-28a(+).

![](https://static.igem.wiki/teams/4907/wiki/engineering-success/zcf/1917-rfp-comparison.png)

**Fig. 16 Characterizations of the growth rate and protein expression level of three different *E. coli* Nissle 1917 strains. a.** The growth curve of EcN, EcN ΔpMUT1 and EcNP harboring BBa\_K4907147\_pET-28a(+). **b.** The fluorescence intensity normalized to OD600 of EcN, EcN ΔpMUT1 and EcNP harboring BBa\_K4907147\_pET-28a(+) as time progressed.

#### **Learn**

The growth rate of EcNP containing no endogenous plasmids was faster than the other two strains (Fig. 16a), while the expression level of fluorescent protein showed no significant differences among the three strains (Fig. 16b), which indicated the elimination of pMUT1 and pMUT2 might have little contribution to improve the expression of heterogenous proteins in *E. coli* Nissle 1917.

### **Conclusion**

Firstly, we found plasmid incompatibility during our experiments, then, leveraging the pEcCas/pEcgRNA
system with several rounds of attempts and modifications of protocols, **we finally eliminated the two
cryptic plasmids**, pMUT1 and pMUT2, from the *E. coli* Nissle 1917 strain we used. We found
that **EcNP had a higher growth rate but a similar protein expression level compared to the wild
type EcN**. The elimination of the cryptic plasmid increases the space for the exogenous plasmids,
which undoubtedly provides a better performing engineered bacteria for our subsequent experimental
operations as well as our project. Additionally, **it was the first time that the pEcCas/pEcgRNA
system which is commonly used for genome editing was implemented and demonstrated to eliminate
endogenous plasmids**, which would extend the boundary of applications of this pEcCas/pEcgRNA
system. Here, we have organized the protocol for the whole plasmid elimination workflow,
with an expectation to offer valid help to other teams or labs which are using or want to use
*E. coli* Nissle 1917 for various synthetic biology applications and contribute
to the iGEM community.

All in all, engineering a biological system cannot be easy,
we need to put our heart into it and move towards success through experiments and
feedbacks step by step. Here, we have tried to demonstrate how **we harnessed the pEcCas/pEcgRNA
system to eliminate the cryptic plasmids of *E. coli* Nissle 1917** through successive iterations of
the **DBTL** (**D**esign-**B**uild-**T**est-**L**earn) cycle (Fig. 17).

![](https://static.igem.wiki/teams/4907/wiki/engineering-success/zcf/engineering-success-summary.png)

**Fig. 17 Summary of the different engineering steps completed before reaching the final goal of eliminating pMUT1 and pMUT2 in *E. coli* Nissle 1917.**

## **Reference**

1. Y. Cui *et al*., Efficient secretory expression of recombinant proteins in Escherichia coli with a novel actinomycete signal peptide *Protein Expression Purif.* **129**, 69-74 (2017).
2. T. D. Craggs, Green fluorescent protein: structure, folding and chromophore maturation. *Chem. Soc. Rev.* **38**, 2865-2875 (2009).
3. C. J. Crasto, J.-a. Feng, LINKER: a program to generate linker sequences for fusion proteins. *Protein Eng. Des. Sel.* **13**, 309-312 (2000).
4. J. Gutiérrez Mena, S. Kumar, M. Khammash, Dynamic cybergenetic control of bacterial co-culture composition *via* optogenetic feedback. *Nat. Commun.* **13**, 4808 (2022).
5. Y.-J. Lan *et al.*, Development of *Escherichia coli* Nissle 1917 derivative by CRISPR/Cas9 and application for gamma-aminobutyric acid (GABA) production in antibiotic-free system. *Biochem. Eng. J.* **168**, 107952 (2021).
6. A. Kan, I. Gelfat, S. Emani, P. Praveschotinunt, N. S. Joshi, Plasmid vectors for in vivo selection-free use with the probiotic *E. coli* Nissle 1917. *ACS Synth. Biol.* **10**, 94-106 (2020).
7. Q. Li *et al.*, A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in *Escherichia coli.* *Acta Biochim. Biophys. Sin.* **53**, 620-627 (2021).
8. K. Wang, Y. Wang, Basic properties and research methods of a new CRISPR-Cas system: CasX as an example. *Microbiology China* **48**, 1227-1238 (2021).
9. F. Jiang, J. A. Doudna, CRISPR-Cas9 structures and mechanisms. *Annu. Rev. Biophys.* **46**, 505-529 (2017).
10. F. Ba *et al*., Expanding the toolbox of probiotic *Escherichia coli* Nissle 1917 for synthetic biology. *Biotechnol. J.* **n/a**, 2300327 (2023).

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Xiamen University, Fujian, China
No.422, Siming South Road, Fujian, P.R.China 361005

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[Common Experiment](#section1)

[Anti-icing Group](#section2)

[Solutions of soil drought](#section3)

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# . Experiments .

## Common Experiments

molecular cloning handbook

#### [Handbook](#collapse-c)

[molecular-cloning-handbook-9-0.pdf](https://static.igem.wiki/teams/4907/wiki/pdf/molecular-cloning-handbook-9-0.pdf)

## Anti-icing Group

CspA mRNA Experiment

#### [Requirements](#collapse51)

● LB liquid medium
● Tecan Infinite M200 microplate reader

#### [Protocol](#collapse52)

1. Revive the transformed bacteria by incubating them at 37 °C with shaking at 200
*r.p.m.* for 12 hours.
2. Inoculate the revived bacteria at 2% concentration into 10 mL of LB medium and shake
at 37 °C for around 2 hours until the OD600 values reaches 0.6-0.8.
3. Incubate the above bacteria separately at 4 °C, 15 °C, and 37 °C, each with shaking at
200 r.p.m. .
4. Use Tecan Infinite M200 microplate reader to measure the green fluorescence intensity and
OD600 values at 2 hours, 4 hours, and 6 hours.
*Note: Excitation wavelength: 488 *nm*, Emission wavelength: 530
*nm*.*
5. Calculate the ratio of green fluorescence intensity to OD600 data.

Construction of Low-Temperature
Stress-Tolerant Engineered Bacteria

Part 1: Mn-SOD - Reactive Oxygen Species Resistance
Test

#### [Requirements](#collapse54)

● LB liquid medium
● Tecan Infinite M200 microplate reader
● TwistAmp® Liquid Basic Kit
● Chloramphenicol (34 mg/mL)

#### [Protocol](#collapse55)

1. Revive the transformed bacteria by incubating them at 37 °C with shaking at 200
*r.p.m.* for 12 hours.
2. Inoculate the revived bacteria at 2% concentration into 10 mL of LB medium and shake
at 37 °C with shaking at 200 *r.p.m.* for approximately 2 hours until the
OD600 values reaches
0.6-0.8.
3. Add 100 µL of 20% L-arabinose to the above bacterial culture and induce at 37 °C with
shaking at 200 *r.p.m.* for 10 hours.
4. Add 10 µL of Chloramphenicol and 100 µL of the above bacterial culture to 10 mL of
non-solidified LB agar medium, mix well and pour onto a Petri dish.
**Note: Set up 3 parallel groups for both experimental and control
groups.**
5. After the agar medium has solidified, place 5 Oxford cups on the plate. 100 µL of 100,
75, 50, and 25 mol/L H2O2 were added to four cups around the
perimeter, and 100 µL of
ddH2O was added to the center cup.
6. Incubate the plate overnight at 37 °C in an incubator.
7. Measure the diameter of the bacterial inhibition zones using a ruler.

Part 2: Mn-SOD/cpn60 and cpn10 - Low Temperature
Stress
Resistance Test

#### [Requirements](#collapse541)

● LB liquid medium
● Tecan Infinite M200 microplate reader
● Chloramphenicol (34 mg/mL)

#### [Protocol](#collapse551)

1. Revive the transformed bacteria by incubating them at 37 °C with shaking at 200 *r.p.m.*
for 12 hours.
2. Inoculate the revived bacteria at 2% concentration into 10 mL of LB liquid medium and
shake at 37 °C, 200 *r.p.m.* for approximately 2 hours until the OD600
values
reaches 0.6-0.8.
3. Add 100 μL of 20% *L*-arabinose to the above bacterial culture and induce at 37 °C
with
shaking at 200 *r.p.m.* for 10 hours.
4. Transfer the induced bacterial culture to -4 °C standing for 8 hours and measure the
OD600 values.
5. Transfer the culture to 37 °C with shaking at 200 *r.p.m.* and incubate for 1 hour,
then
measure the OD600 values.
6. Generate bar graphs of the delta OD600 values for the experimental and control
groups.

Anti-freeze Proteins (AFPs)

Part 1: Differential Scanning Calorimetry for TH
Measurement

#### [Requirements](#collapse41)

● Differential scanning calorimeter
● 25 μL aluminum pan

#### [Before Starting](#collapse121)

Prepare 50 μM TmAFP, SfIBP, and BSA solution in advance.

#### [Protocol](#collapse42)

1. Place the sample in the aluminum pan. Then, seal it and place it in the sample chamber.
Place an empty aluminum pan in the reference chamber.
2. Adjust the temperature to 0 °C at a rate of 2 °C/min after that decreae it to -20 °C and
hold for 5 minutes. Then, increase the temperature at a rate of 2 °C/min until the sample is
fully melted. Determine the melting point (Tm) and freezing point (Tf)
based on the heat
capacity curve.
3. Adjust the temperature again to -20 °C at a rate of 2 °C/min and hold for 5 minutes.
Then, increase the temperature at a rate of 2 °C/min until it reach 0.5 ℃ below Tm. Maintain
this temperature for 5 minutes, and record it as Th. Then the temperature is
decreased to
-20 ℃ again.
4. Calculate TH as TH = Th - To

Part 2: Capillary Method for IRI Measurement

#### [Requirements](#collapse411)

● 150 mM NaCl
● 10 mM Tris-HCl
● 10 μg/mL BSA
● 30% CaCl2 solution
● Capillary tubes
● Polarizing filter

#### [Before Starting](#collapse421)

1. Prepare 0.5, 5, 10, 50, 100, 200, 500, and 1000 nM TmAFP and SfIBP solutions in advance.
2. Prepare the buffer containing 150 mM NaCl, 10 mM Tris-HCl, and 10 μg/mL BSA.

#### [Protocol](#collapse422)

1. Use capillary tubes to draw samples and fill them completely.
2. Arrange capillary tubes containing the same protein side by side between two glass
slides, secure both ends with metal clips and quickly place them in a -80 °C freezer for 1
hour.
3. Remove the glass slides and rapidly transfer them to a 30% CaCl2 solution in a
-20 °C
freezer. Take pictures of ice crystals under a microscope.
4. Place the glass slides in the -20 °C freezer and keep them for 16 hours.
5. Remove the glass slides and quickly take pictures of the ice crystals under a microscope.
6. Compare the two ice crystal images, and the lowest concentration at which the ice
crystals remain unchanged is the Ice Recrystallization Inhibition (IRI).

Verification of Secretion Efficiency of
Signal Peptide LMT/Kp-SP

#### [Requirements](#collapse43)

● LB liquid medium
● Tecan Infinite M200 UV-Visible spectrophotometer

#### [Protocol](#collapse44)

1. Resuscitate the transformed bacteria overnight at 37 °C with shaking at 200
*r.p.m.* .
2. In conditions of 37 °C and 200 *r.p.m.*, inoculate the resuscitated bacteria (2%)
into 10 mL LB liquid culture medium and shake for approximately 2 hours until the
OD600
values reaches 0.6-0.8.
3. Add *L*-arabinose to the above bacterial cultures to a final concentration of 0.2%.
Set up
three parallel control groups for the experimental and control groups, and incubate them at
37 °C with shaking at 200 *r.p.m.* for 12 hours.
4. Take 1.5 mL of the above bacterial cultures into 2.0-mL centrifuge tubes, centrifuge at
room temperature at 12,000 *r.p.m.* for 2 minutes.
5. Measure the fluorescence intensity of the original bacterial culture and supernatant
separately. Express the signal peptide secretion efficiency as the ratio of the fluorescence
intensity of the supernatant to the bacterial culture.

Verification of Secretion Efficiency of
Surface Display

#### [Requirements](#collapse45)

● LB liquid medium
● Tecan Infinite M200 UV-Visible spectrophotometer
● Chloramphenicol (34 mg/mL)
● PBS buffer
● FITC labelled anti-his tag Antibody
● 5% skim milk
● 1.5-mL centrifuge tubes

#### [Protocol](#collapse46)

1. Resuscitate the transformed bacteria overnight at 37 °C with shaking at 200
*r.p.m.*.
2. In conditions of 37 °C and 200 *r.p.m.*, inoculate the resuscitated bacteria (2%)
into 10
mL LB liquid culture medium and shake for approximately 2 hours until the OD600
values
reaches 0.6-0.8.
3. Add L-arabinose to the above bacterial cultures to a final concentration of 0.2%. Keep
shaking at 37 °C and 200 *r.p.m.* for an additional 6 hours.
4. Take six clean 1.5-mL centrifuge tubes and add 1 mL of bacterial culture to each tube.
Centrifuge at 6500 *r.p.m.* for 3 minutes. Then, discard the supernatant to collect the
pellet.
*Note: Perform three parallel experiments for the experimental group and positive
control.*
5. Add 1 mL of 1×PBS buffer to the centrifuge tubes containing the pellets and mix well.
6. Centrifuge at 6500 *r.p.m.* for 3 minutes, discard the supernatant, and repeat this
step
twice.
7. Add 750 μL of 5% skim milk to each centrifuge tube and mix well. Then, incubate at 37 °C
for 0.5 hours.
8. Centrifuge at 6500 *r.p.m.* for 3 minutes and discard the supernatant.
9. Add 600 μL of 1×PBS buffer and mix well. Then, add FITC labelled anti-his Antibody
(1:3000). Incubate at 37 °C for 1 hour (or overnight at 4 °C).
10. Repeat step 8 once, add 1 mL 1×PBS solution into the centrifuge tube, mix well. Then,
centrifuge at 6500 *r.p.m.* for 3min, discard the supernatant, and repeat twice.
11. Pipette 200 μL of the bacterial culture from each tube into a 96-well microplate. Use a
microplate reader to measure the fluorescence intensity and OD600.
*Note: Excitation wavelength: 492 *nm*, Emission wavelength: 518
*nm*.*
12. Calculate the fluorescence intensity/OD600 for both the negative control
group and the
experimental group.
*Note: Steps 8-11 should be performed in the dark, and subtract the value of PBS
blank.*

*Expected Results:*
The fluorescence intensity/OD600 value in the
experimental group should
be higher than that in the negative control group, indicating successful expression of
mv140
and surface display of CBM on the bacterial surface.

CFU

#### [Requirements](#collapse451)

● LB liquid medium
● Chloramphenicol (34 mg/mL)
● 96-well plate
● L-arabinose stock solution (0.2 g/mL)
● D-glucose stock solution (500 g/L)

#### [Protocol](#collapse461)

1. Resuscitate the transformed bacteria overnight at 37 °C with shaking at 200
*r.p.m.* .
2. Inoculate the overnight revived bacterial culture into fresh LB liquid culture medium at
2% ratio.
3. In the experimental group, add L-arabinose stock solution to achieve a final
concentration of 0.2%, while in the control group, add D-glucose stock solution to achieve a
final concentration of 0.5%.
4. After culturing for 6 hours at 37 °C, 200 *r.p.m.*, take 100 μL of the bacterial
culture
and centrifuge it at room temperature at 6,500 *r.p.m.* for 3 minutes. Discard the
supernatant
and resuspend the pellet in 100 μL of fresh LB liquid culture medium with the same
antibiotic concentration. Repeat this process once.
5. Perform a gradient dilution using a 96-well plate. Take 20 μL of the resuspended
bacterial culture and add it to 180 μL of fresh LB liquid culture medium with the same
antibiotic concentration, mixing thoroughly. Repeat this dilution step six times to create a
total of six dilution levels.
6. From each dilution, spot 5 μL of the diluted culture onto a solid agar plate prepared
with the same antibiotic concentration. Allow the spots to air dry and then incubate the
plates upside down in a 37 °C constant temperature incubator for 12 hours. After incubation,
count the bacterial colonies.
7. Calculate the survival ratio.

*Note:*
1. CFU/mL = Number of colonies × Dilution factor / 0.005 mL
2. Survival Ratio (log10) = log10[(CFU/mL in the experimental
group)/(CFU/mL in the control
group)]

## Anti-drought Group

Determination of *E. coli* Nissle
1917/BL21(DE3) fluorescence growth curve

#### [Requirements](#collapse61)

● Tecan Infinite M200 microplate reader
● LB liquid medium
● Kanamycin antibiotic (50 mg/mL)
● Chloramphenicol antibiotic (34 mg/mL)

#### [Protocol](#collapse62)

1. Revive the transformed bacteria by incubating them at 37 °C with shaking at 200
*r.p.m.* for 12 hours.
2. Inoculate the revived bacteria at 2% concentration into 10 mL of LB medium and shake at
37 °C.
3. Every hour record the OD600 values and fluorescence intensity until the curve
tend to be stable.

Characterization and quantification of HA
production in *E. coli* BL21(DE3)

#### [Requirements](#collapse64)

● Recombinant *E. coli* BL21(DE3) strain
● LB liquid medium
● Ampicillin antibiotic (100 mg/mL)
● 500 g/L glucose solution
● 37 °C shaking incubator
● 0.1% sodium dodecyl sulfate (SDS)
● 0.2 M acetate buffer (pH=6)
● 2.5 g/L CTAB solution containing 2% NaOH
● Tecan Infinite M200 microplate reader
● 96-well plate

A. Determination of HA standard curve

#### [Protocol](#collapse65)

1. Prepare the HA standard solutions with concentrations of 0, 20, 40, 60, and 80 mg/L.
Perform five replicates for each concentration.
2. Add 150 μL of HA standard solution and 350 μL of 0.2 M acetate buffer in a 2-mL
centrifuge tube. Then mix them well.
3. Add 1 mL CTAB solution to the above mixture.
4. After incubation for five minutes, transfer 200 μL to a 96-well plate and measure the
OD400 values.
5. Plot a standard curve with absorbance values to HA concentrations.

B. Measurement of HA samples

#### [Protocol](#collapse67)

1. Take 500 μL of the overnight culture and add it to a 1.5-mL centrifuge tube, add 500 μL
of 0.1% SDS to each sample.
2. Incubate the mixture at 37 °C for 15 minutes.
3. Centrifuge at 4,600 *r.p.m.* for 10 minutes, and pipette out 150 μL of the
supernatant into a new 2-mL centrifuge tube.
4. Add 350 μL of 0.2 M acetate buffer and mix well.
5. Add 1 mL of CTAB solution.
6. After five minutes of incubation, transfer 200 μL to a 96-well plate and measure the
OD400 values.
*Note: Be careful to ensure that the final data falls within the standard curve range.*

Production and test of cross-linked
water-retention material

#### [Requirements](#collapse73)

● Recombinant *E. coli* Nissle 1917 (EcNP) strain
● LB liquid medium
● Ampicillin (100 mg/mL)
● 500 g/L glucose solution
● 37 °C shaking incubator
● Ampicillin (100 mg/mL)
● Kanamycin (50 mg/mL)
● Chloramphenicol (34 mg/mL)

A. Preparation of the water-retention material produced by bacteria

#### [Protocol](#collapse74)

1. Inoculate the recombinant BL21(DE3) strains in 2% ratio into 10 mL of LB medium
containing corresponding antibiotics and glucose (20 g/L) at 37 °C and 200 *r.p.m.* for
20 h.
*Note: Monitor the concentration of glucose in case of product consumption.*
2. Centrifugate the bacteria above to obtain the supernatant containing HA.
3. Inoculate the recombinant EcNP strains in 2% ratio into 10 mL of LB medium containing
corresponding antibiotics and glucose (30 g/L) at 37 °C and 200 *r.p.m.*.
4. Add IPTG at final concentrations of 0.5 mM and the supernatant containing HA to it when
the OD600 reaches 0.8.
5. Shake the cultures at 37 °C with shaking at 200 *r.p.m.* for several hours.
6. Observe the morphology of product and test the water-retention ability.

B. Test of water-retention ability about the cross-linked product

#### [Protocol](#collapse75)

1. Take same amount of soil in bottle caps. Add the water (V1), co-culture
solution (V2) and the mixture of them (V3=V1+V2)
into the soil.
*Note: Make sure the water is evenly mixed with soil and volume will not exceed the
rim.*
2. Place the mixture in 37 ℃ environment for 0.5 hours.
3. Calculate Δweight carefully. Δweight experiment group = the Δweight of soil
with co-culture solution and pure water minus the Δweight of soil with co-culture
solution.

Characterization of the BC/HA complex

A. Verification of HA Entry into the BC Network

#### [Requirements](#collapse76)

●Fourier Transform Infrared Spectroscopy (FTIR)

#### [Protocol](#collapse77)

1. Prepare the samples.
2. In adsorption mode, acquire 32 scans at a resolution of 4 cm⁻¹ within the wave
number range of 4000 to 400 cm⁻¹ using a VERTEX 70 V spectrometer.

B. Structural Characterization of BC/HA

#### [Requirements](#collapse78)

● Field Emission Scanning Electron Microscope (FESEM)

#### [Requirements](#collapse79)

1. Prepare freeze-dried samples.
2. Apply a brief (~5 nm) iridium sputter coating for 30 seconds to all freeze-dried samples
before imaging.
3. During imaging, employ an acceleration voltage of 5.0 kV.

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

[1 Overview](#section1)

[2 Anti-icing](#section2)

[2.1 Highlight](#childSection1)

[2.2 Introduction](#childSection2)

[2.3 Result](#childSection3)

[2.4 Conclusion](#childSection4)

[2.5 Future
Prospects](#childSection5)

[3 Anti-Drought](#section3)

[3.1 Highlight](#childSection6)

[3.2 Introduction](#childSection7)

[3.3 Result](#childSection8)

[3.4 Conclusion](#childSection9)

[3.5 Future
Prospects](#childSection10)

[4 Biosafety](#section4)

[4.1 Kill switch](#childSection11)

[4.2 Preventing
HGT](#childSection12)

[4.3 Cytotoxicity of
*ccdB*](#childSection13)

![](https://static.igem.wiki/teams/4907/wiki/background/sidebar1.png)

# . Results .

## **Overview**

Our solution for **freeze** and **drought** can be divided into two sections: produce
anti-icing
proteins to protect crops from freeze, produce water-retention material to prevent crops from
drought. We
organized our experimental results in the order of Anti-icing and Anti-drought. Besides, we
demonstrated the
feasibility of the kill switch, which is also an important part for the sake of biosafety and
biocontainment
of NAIADS. The fundamental aspects of our projects are composed of all the results we obtained.

## **Anti-icing**

### **Highlight**

* Confirm the secretion function of the signal peptide KpSP and LMT
* Improve the growth ability and protein expression ability of bacteria at low temperature
* Use MV140 to display the CBM on the surface of *E. coli* to adsorb cellulose

### **Introduction**

We try to construct engineered bacteria to express antifreeze protein based on synthetic biology
which can
lower the freezing point of water and inhibit the growth of small ice crystals, preventing
further damage to
roots from ice crystals. Since antifreeze protein needs to bind to the surface of ice directly,
it is
necessary that AFPs should be secreted into the soil from our engineered bacteria. First, we
verified the
secretion function of the two signal peptides KpSP and LMT. Second, we combine a logic AND gate
with the
CspA CRE to alleviate oxidative stress in low temperatures. At
last, we use
MV140 to display the CBM on the surface of engineered *E. coli* to adsorb
cellulose, which prevents the
bacteria from rain wash.

### **Results**

#### **Singal peptides KpSP and LMT secret target proteins into extracellular circumstances**

##### **KpSP can secret target proteins into the extracellular circumstances**

To verify whether KpSP can direct target protein to the extracellular circumstance or not, we
tried to fuse
it directly with GFP to obtain KpSP-GFP. But we failed. To optimize it, we added a linker
between KpSP and
GFP to construct fused protein (please see [Engineering Success](https://2023.igem.wiki/xmu-china/engineering) for
details).

The promoter ([BBa\_I0500](http://parts.igem.org/Part%3ABBa_I0500)), RBS ([BBa\_B0034](http://parts.igem.org/Part%3ABBa_B0034)),
KpSP-GFP
coding sequence ([BBa\_K4907002](http://parts.igem.org/Part%3ABBa_K4907002)),
and terminator ([BBa\_B0015](http://parts.igem.org/Part%3ABBa_B0015))
were used to construct a composite part [BBa\_K4907101](http://parts.igem.org/Part%3ABBa_K4907101), which
was then
assembled into the expression vector pSB1C3 by standard assembly. The constructed plasmid was
transformed
into *E. coli* DH10β, then the positive transformants were selected by chloramphenicol and
confirmed by
colony PCR (Fig. 1) and sequencing. Target bands (2557 bp) can be observed at the position
between 2000 bp
and 3000 bp.

![](https://static.igem.wiki/teams/4907/wiki/result/result/fig-1-1.png)

**Fig. 1 Constructing KpSP-GFP fused protein.
a** the
expression gene circuits for KpSP secreting GFP. **b** DNA gel electrophoresis of the
colony PCR
products of BBa\_K4907101\_pSB1C3 in *E. coli* DH10β.

After being cultivated and induced by 0.2% *L*-arabinose at 37 ℃ 12 hours, 1 mL bacterial
liquid was
centrifuged, and the fluorescence intensity of the supernatant and the bacterial liquid was
measured. *E.
coli* DH10β harboring BBa\_K4907100\_pSB1C3 (for expressing GFP intracellularly) was set as the
control
group which has no signal peptide coding sequence. As shown in Fig. 2, the fluorescence
intensity of
supernatant from *E. coli* DH10β harboring BBa\_K4907101\_pSB1C3 was significantly higher
than that of
the control group. It demonstrated that KpSP can transfer target proteins into the extracellular
environment.

![](https://static.igem.wiki/teams/4907/wiki/result/result/fig-4.png)

**Fig. 2 Comparison of the fluorescence intensity
ratio
(supernatant to bacterial culture) between I0500 set as the control group.**

##### **LMT can secret target proteins into the extracellular circumstance**

In order to test whether LMT can secret target proteins to the extracellular circumstance or
not, we add a
linker between LMT and GFP to get the fused protein LMT-GFP ([BBa\_K3739010](http://parts.igem.org/Part%3ABBa_K3739010)).
Then this part was inserted on the expression vector pET-28a(+). The constructed plasmids are
transformed
into *E. coli* BL21(DE3), then the positive transformants were selected by kanamycin and
confirmed by
colony PCR and sequencing (Fig. 3).

![](https://static.igem.wiki/teams/4907/wiki/result/result/fig-3.png)

**Fig. 3 DNA gel electrophoresis of the colony
PCR products
of BBa\_K3739010\_pET-28a(+) in *E. coli* BL21(DE3).**

*E. coli* BL21(DE3) harboring GFP\_pET-28a(+) (for expressing GFP intracellularly) was set
as the control group which has no signal peptide's coding sequence. After being cultivated and
induced by 1 mM IPTG at 37 ℃ for 12 hours, 1 mL bacterial liquid was centrifuged, and the
fluorescence intensity of the supernatant and the bacterial liquid was measured. As shown in
Fig. 4, the fluorescence intensity of supernatant from *E. coli* BL21(DE3) harboring
BBa\_K3739010\_ pET-28a(+) was significantly higher than that of the control group. It
demonstrated that LMT can transfer target proteins into the extracellular environment.

![](https://static.igem.wiki/teams/4907/wiki/result/result/fig-2.png)

**Fig. 4 Comparison of the fluorescence intensity
ratio
(supernatant to bacterial culture), pET-28a(+) set as the control group.**

#### **Response performance of strain with cspA CRE**

To test whether the cspA cold-responsive elements (CRE) own response function at low
temperatures. The cspA
CRE (BBa\_K4907118\_pSB1C3, CspA CRE-*gfp*) was respectively characterized at 37 ℃ and 15 ℃,
controlled
by BBa\_K4907146\_pSB1C3 (as a positive control group) and BBa\_K4907118\_pSB1C3 (as a negative
control group).
As shown in Fig. 5a, the normalized fluorescence intensity of the experimental group decreased
over time at
37 ℃. However, the normalized fluorescence intensity of cspA CRE increased over time when the
temperature
was 15 ℃ (Fig. 5b), indicating that cspA CRE can respond to the low temperature.

![](https://static.igem.wiki/teams/4907/wiki/result/result/5.png)

**Fig. 5 The comparison of normalized
fluorescence intensity
of CspA CREC and BBa\_J23100 at different temperatures. a** 37 ℃. **b** 15 ℃.

We also found that the normalized fluorescence intensity of the experimental group was
comparable to that of
the positive control group at 37 ℃, indicating that the cspA CRE exhibited significant leakage
at 37 ℃ (Fig.
6). The antifreeze proteins will be transported across the membrane constantly, which greatly
increases the
burden on the bacteria. Leakage expression of antifreeze protein at non-target temperature will
undoubtedly
increase the pressure of chassis cells. So, reducing the leakage of cspA CRE under
high-temperature
conditions is very necessary.

![](https://static.igem.wiki/teams/4907/wiki/result/result/6.png)

**Fig. 6 The comparison of normalized
fluorescence intensity
between CspA CREC and BBa\_J23100 at 37 ℃ for 6 h.**

#### **Improve the growth ability and protein expression ability of bacteria at 4 ℃**

##### **Mn-SOD**

Because the environment we put the fertilizer is in cold environments which can cause oxidative
stress in *E. coli* ,
accumulating reactive oxygen species (ROS) in the cell. High levels of ROS can damage
biomolecules and disrupt their functions.
In order to allow our bacteria to survive and maintain activity under low temperature
environment, we use Mn-SOD to improve the stress resistance of the bacteria.
Therefore, to improve the survival of the engineered bacteria at low temperature, we decide to
overexpress Mn-SOD to clear ROS and increase resistance of bacteria to cold stress.
This constructed circuit was transformed into *E. coli* DH10β, followed by positive
transformant selection using kanamycin and confirmation through colony PCR (Fig. 7) and
sequencing.

![](https://static.igem.wiki/teams/4907/wiki/result/fig-7-1.png)

**Fig 7. DNA gel electrophoresis of the colony
PCR products
of BBa\_K4907132\_pSB1C3.**

###### **Antioxidant activity assay by the Oxford cup method**

We used Oxford cup antibacterial experiments to observe and compare the antibacterial circle
diameter of control strains and experimental
strains containing [BBa\_K4907132](http://parts.igem.org/Part%3A%20BBa_K4907132) at hydrogen peroxide
concentrations of 0, 25, 50, 75, 100 mM respectively. The significant difference (Fig. 8)
suggested that the experimental group formed a
smaller antibacterial circle at 50 mM hydrogen peroxide.
[BBa\_K4907132](http://parts.igem.org/Part%3A%20BBa_K4907132) improves the bacterial resistance to
peroxides.

![](https://static.igem.wiki/teams/4907/wiki/result/result/result/fig-9.png)

**Fig. 8 Quantitative diameters of the inhibited
zones in
histograms.**

###### **Survival level of the Mn-SOD-overexpressed DH10β under cold stress at -4 °C**

We subjected both the control and experimental groups to shaking cultivation at -4 °C for 8
hours.
We measured the OD600 values immediately upon removal and after 1 hour of recovery on
a shaking incubator at 37 °C.
It can be observed that the over-expressing Mn-SOD strain exhibited significantly enhanced
colony growth after one hour of recovery (Fig. 9).
[BBa\_K4907132](http://parts.igem.org/Part%3A%20BBa_K4907132) demonstrates a protective effect against
low-temperature stress on the strains.

![](https://static.igem.wiki/teams/4907/wiki/result/result/result/figr9-1.png)

**Fig. 9 The status of Escherichia coli changed
with or without Mn-SOD.**

###### **Survival ability and protein expression ability of the Mn-SOD-overexpressed BL21(DE3) under 4 °C**

Further, we transferred [BBa\_K4907132](http://parts.igem.org/Part%3ABBa_K4907132)\_pSB3K3 (Mn-SOD) and
BBa\_K4907119\_pSB1C3 (CspA CRE-gfp) into *E.
coli* BL21(DE3) at the same time, screened the correct strains with double antibodies, and
characterized
growth ability at 4 ℃. As shown in Fig. 10, the strain carrying Mn-SOD showed better growth
ability and
produced stronger fluorescence intensity under a low-temperature environment. This means that
Mn-SOD can
achieve our original design intention.

![](https://static.igem.wiki/teams/4907/wiki/result/result/9.png)

**Fig. 10 The fluorescence intensity and OD600
with or without Mn-SOD
was at 4 ℃.**

##### **CspA CRE still has a low-temperature induction effect at 4 ℃**

In order to further characterize CspA CRE at lower temperatures, we introduced Mn-SOD
(BBa\_K4907132\_pSB3K3) to enhance the stress resistance of the bacteria. This Mn-SOD-expressing
plasmid and BBa\_K4907118\_pSB1C3 were co-transformed into *E. coli* BL21(DE3) and the
correct dual-plasmid transformants were selected by chloramphenicol and kanamycin. The same
characterization was performed at 4 ℃. It can be seen from Fig. 11 that CspA CREC still has much
stronger expression strength under the condition of 4 ℃, compared to that of promoter J23100.
Such results also showed the excellent performance of CspA CRE as a low-temperature response
expression system when accompanied by the expression of Mn-SOD.

![](https://static.igem.wiki/teams/4907/wiki/result/result/10.png)

**Fig. 11 The comparison of normalized
fluorescence intensity
under CspA CREC and BBa\_J23100 at 4 ℃.**

#### ***hrp* AND Gate**

To verify the function of the *hrp* AND Gate, the promoter ([BBa\_I0500](http://parts.igem.org/Part%3A%20BBa_I0500))
and araBAD
promoter ([BBa\_K4907020](http://parts.igem.org/Part%3ABBa_K4907020))
were used to construct the regulation system of composite part [BBa\_K4907126](http://parts.igem.org/Part%3A%20BBa_K4907126) (Fig.
12), which was then assembled into the expression vector pSB1C3. We also used BBa\_I0500 to
construct the
composite part BBa\_K4907124 and BBa\_K4907125, which was then assembled into the expression
vector pSB1C3. It
was used to prove HrpR or HrpS protein will not activate the *hrpL* promoter alone. We used
the *hrpL* promoter
and GFP (BBa\_K4907036) to construct the reporting system and obtained the composite part
BBa\_K4907123, which
was assembled into the expression vector pSB3K3.

![](https://static.igem.wiki/teams/4907/wiki/result/result/fig-12.png)

**Fig. 12 Gene circuits of composite part
BBa\_K4907126.**

We used a dual-plasmid system to prove the hrp AND gate. As shown in Fig. 13, one control and
three
experimental groups were set up. For the R+S group, Plasmid BBa\_K4907123\_pSB3K3 and plasmid
BBa\_K4907126\_pSB1C3 were transformed into *E. coli* DH10β which can express HrpR and HrpS.
For the
remaining two experimental groups, HrpR ([BBa\_K4907021](http://parts.igem.org/Part%3ABBa_K4907021)) and
HrpS ([BBa\_K4907022](http://parts.igem.org/Part%3ABBa_K4907022)) were
expressed respectively. As for the control, Plasmid BBa\_K4907123\_pSB3K3 and plasmid
BBa\_I0500\_pSB1C3 were
transformed into *E. coli* DH10β. The positive transformants were selected by kanamycin and
chloramphenicol. Colonies harboring the correct plasmid were cultivated and induced. We added
the 2%
*L*-arabinose solution to induce the expression of the *hrp* AND Gate system.

The fluorescence intensity (Fluorescence/OD600) value was measured by the microplate
reader (Fig.
13). The results of fluorescence intensity showed that the pHrpL will be activated when HrpR and
HrpS are
both expressed, while it will not be activated by HrpR or HrpS alone.

![](https://static.igem.wiki/teams/4907/wiki/result/result/13.png)

**Fig. 13 The results of fluorescence intensity
to verify the
function of *hrp* AND Gate.**

#### **CBM's adsorption and CBM receptors can be anchored onto the bacterial surface**

##### **CBM can bind to cellulose effectively**

The plasmid BBa\_K4907027 was transformed into *E. coli* BL21(DE3), then the positive
transformants were selected by kanamycin and confirmed by colony PCR and gene sequencing. The
plasmid verified by sequencing was successfully transformed into *E. coli* BL21(DE3).

![](https://static.igem.wiki/teams/4907/wiki/result/27.png)

**Fig. 14 DNA gel electrophoresis of the colony
PCR
products of BBa\_K4907027\_pET-28a(+) in E. coli BL21(DE3).** Target bands (506 bp) can be
observed at the position around 500 bp.

After being cultivated and induced by 0.75 mM IPTG, the GE AKTA Prime Plus FPLC System was
employed
to collect purified protein from the lysate supernatant. CBM was verified by sodium dodecyl
sulfate
(SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining (Fig.
15).

![](https://static.igem.wiki/teams/4907/wiki/result/result/28.png)

**Fig. 15 SDS-PAGE analysis of CBM-his
protein.**
Target bands (11.9 kDa) can be observed at the position around 10 kDa.

After purification, we got CBM-his successfully, although it was mixed with other proteins. Then
the
CBM-his was diluted to 10 M, and 10 M BSA was set as the negative control. 4 mL of 10 μM CBM and
BSA
was filtered three times by using cellulose filter paper and it was washed three times with
Phosphate Buffered Saline (1×PBS). The eventual concentration of CBM and BSA was tested by the
Bradford method after being diluted to the same volume. The result showed that CBM’s absorption
at
OD595 is higher than BSA’s, illustrating that CBM can bind to cellulose effectively.

![](https://static.igem.wiki/teams/4907/wiki/parts/ctt/tt-cbmbsa.png)

**Fig. 16 The concentration of CBM and BSA
filtered
by cellulose filter paper.**

##### **mv140-linker-cbm-his and mv140-linker-his**

To verify whether MV140 can display heterologous proteins on the surface of the
engineered bacteria or not, a His-tag (6×His) was fused to the C-terminal of MV140.
We
used both [BBa\_I0500](http://parts.igem.org/Part%3ABBa_I0500) (araC/pBAD) and [BBa\_B0034](http://parts.igem.org/Part%3ABBa_B0034) to
construct the expression system and obtained the composite parts [BBa\_K4907136](http://parts.igem.org/Part%3ABBa_K4907136)
and [BBa\_K4907137](http://parts.igem.org/Part%3ABBa_K4907137) (Fig. 17) which are respectively assembled
into the vector pSB1C3 by standard BioBrick assembly. The constructed plasmid was transformed
into
*E. coli* DH10β, then the positive transformants were selected by chloramphenicol and
confirmed
by colony PCR and sequencing (Fig. 18).

![](https://static.igem.wiki/teams/4907/wiki/result/result/30.png)

**Fig. 17 Graphic description of the expression
gene
circuits for the protein display system. (a** BBa\_K4907136 **b** BBa\_K4907137)

![](https://static.igem.wiki/teams/4907/wiki/result/result/31-1.png)

**Fig. 18 DNA gel electrophoresis of the colony
PCR
products. a** BBa\_K4907136 pSB1C3 in *E. coli* DH10β. Target bands (2188 bp) can be
observed at the position between 3000 bp and 2000 bp. **b** BBa\_K4907137\_pSB1C3 in *E.
coli* DH10β. Target bands (2515 bp) can be observed at the position between 3000 bp
and
2000 bp.

###### **Immunofluorescence (IF)**

2% *L*-arabinose solution was added to induce the expression of the surface-display system.
Then, we use the FITC-labeled anti-His-Tag antibody to target the fused His-tag (6×His)
displayed
*via* MV140, followed by measuring the fluorescence intensity and
OD600
of the culture.

![](https://static.igem.wiki/teams/4907/wiki/result/result/32.png)

**Fig. 19 The results of immunofluorescence to
characterize the function of the two surface display systems.** Fluorescence
intensity/OD600
of *E. coli* DH10β whether express MV140 or not. The left is for
BBa\_K4907136
(*p* = 0.004608) and the right is for BBa\_K4907137 (*p* = 0.003578). *p*-value:
no significance (ns), 0.0332 (\*), 0.0021 (\*\*), 0.0002 (\*\*\*), <0.0001 (\*\*\*\*).

The results showed that the ratio of fluorescence intensity (fluorescence value/OD600)
of
positive control (bacteria harboring surface display system) is higher than that of negative
control
(bacteria without surface display system) (Fig. 19), which indicates that MV140 can
be
successfully located on the surface of engineered bacteria.

When MV140 was localized to the bacterial surface, it would carry CBM and display on
the
bacterial surface. CBM can bind to the cellulose of plant roots, allowing bacteria to secrete
antifreeze proteins near the plant roots. Enriching bacteria in plant roots can not only improve
the
efficiency of antifreeze proteins but also prevent engineered bacteria from escaping with water
after heavy rain.

### Conclusion

In the anti-icing part, we focused on two major themes. **One was the cold-inducible gene
expression
and the support for growth at low-temperatures, the other was the functional module related to
EFE
(extracellular functional element)** (please see  [BBa\_K4195130](http://parts.igem.org/Part%3ABBa_K4195130)
for more details about EFE). For the first theme, we confirmed that the Mn-SOD could improve the
growth
ability of bacteria at 4 °C, characterized the CspA CREC system and constructed AND-logic gate
based
on
*hrp* to reduce the leakage of CspA CREC system. And for the second
theme,
we demonstrated the function both of signal peptide Kp-SP and LMT to direct GFP to the
extracellular
circumstance and surface display system MV140 to anchor the CBM for adsorbing
cellulose.
We
then turned to characterize the anti-freezing proteins (AFPs), which hold the main tasks for
anti-icing
program (please see our [Proof of Concept](https://2023.igem.wiki/xmu-china/proof-of-concept%20)  for details).

## Future Prospects

Combining the CspA CREC system with *hrp* to construct the AND gate closer to the real
conditions need to be further characterized. Even though the secretion of GFP with the help
of
KpSP
and LMT was proved, however, the efficiency of secreting AFPs should be determined in the
future.

## Anti-Drought

### **Highlight**

* Construct two engineered bacteria to produce bacterial cellulose and hyaluronic acid
* Developed the microflora proportional regulation method
* Produce crosslinked products through the co-culture fermentation technique
* Verified the excellent water-retention performance of the crosslinked product in the soil

### **Introduction**

Crosslinked product from bacterial cellulose and hyaluronic acid shows an excellent performance
in
water
retention (please see [Description](https://2023.igem.wiki/xmu-china/description%20)
for
details),
which can be used as a water retention material to against drought. Thus, we construct two
engineered
bacteria to produce bacterial cellulose and hyaluronic acid in *E. coli* Nissle 1917 (EcNP
type,
ΔpMUT1ΔpMUT2) and *E. coli* BL21(DE3) (please see [Design](https://2023.igem.wiki/xmu-china/design)
for details). The microflora proportional regulation gene circuit, co-culture technique, and
hardware
(please see [Hardware](https://2023.igem.wiki/xmu-china/hardware%20) for details) were
developed
to produce the crosslinked product continuously.

### **Results**

#### **Engineered *E. coli* to produce Hyaluronic acid and Bacterial cellulose**

Hyaluronic acid (HA) is a polysaccharide with a highly hydrating effect, and bacterial cellulose
(BC) is
a
biological macromolecule with excellent biocompatibility and water retention. The crosslinked
product of
bacterial cellulose and hyaluronic acid, which exhibits excellent water retention performance,
was
selected
as water-retentive material. Thus, we construct two engineered *E. coli* to produce
hyaluronic
acid
and
bacterial cellulose. Finally, the co-culture technique was developed to produce crosslinked
products.

##### **Engineered *E. coli* BL21(DE3) to produce hyaluronic acid**

The promoter ([BBa\_J23100](http://parts.igem.org/Part%3ABBa_J23100)),
RBS ([BBa\_B0034](http://parts.igem.org/Part%3ABBa_B0034)),
hasA
coding sequence ([BBa\_K4907035](http://parts.igem.org/Part%3ABBa_K4907035))
and terminator ([BBa\_B0015](http://parts.igem.org/Part%3ABBa_B0015))
were used to construct a composite part [BBa\_K4907145](http://parts.igem.org/Part%3ABBa_K4907145) (Fig.
20a),
which
was then assembled into the vector pSB1A2 by standard BioBrick assembly. The constructed plasmid
was
transformed into *E. coli* BL21(DE3) successfully, then the positive transformants were
selected by
ampicillin and confirmed by colony PCR and sequencing (Fig. 20b).

![](https://static.igem.wiki/teams/4907/wiki/result/result/drought/33-hasa-circuit-colonypcr-1.png)

**Fig. 20 Graphic description of the genetic
circuit
for
HA
production. a** DNA gel electrophoresis of the colony PCR products. (BBa\_K4907145\_pSB1A2
in
*E.
coli* BL21(DE3). **b** Target bands (1690 bp) can be observed at the position
between
2000 bp
and
1500 bp).

CTAB (cetyltrimethylammonium bromide) method was employed to measure optical density value at
λ400 (OD400), which can calculate the concentration of hyaluronic acid according to
the
standard
curve in Fig. 21 (please see [**Experiment**](https://2023.igem.wiki/xmu-china/experiments%20)
for details). After that, we measure the concentration of hyaluronic acid in the fermentation
broth
of
engineered strain (*E. coli* BL21(DE3) with BBa\_K4907144\_pSB1A2), controlling by that of
the
*E.
coli* BL21(DE3) with pTet-B0034-*ecfp*-B0015\_pSB1A2.

![](https://static.igem.wiki/teams/4907/wiki/result/result/drought/34-ha-standard-curve.png)

**Fig. 21 Standard curve of HA(R2=0.9490).**

![](https://static.igem.wiki/teams/4907/wiki/result/result/drought/35-ha-positivevsnegative.png)

**Fig. 22 The results of HA concentration
measurement
via
the
CTAB method.**

As shown in Fig. 22, the concentration of hyaluronic acid produced by our engineered strain was
higher
than
that of the control group. It demonstrated that the engineered strain was successfully
constructed
to
produce hyaluronic acid with 80 mg/L, which is consistent with the results in references.

##### **Engineered *E. coli* Nissle 1917 (EcNP) to produce bacterial cellulose**

The promoter ([BBa\_J23109](http://parts.igem.org/Part%3ABBa_J23109)),
RBS ([BBa\_B0034](http://parts.igem.org/Part%3ABBa_B0034)),
*bcsA*
coding sequence ([BBa\_K4907033](http://parts.igem.org/BBa_K4907033)),
and terminator ([BBa\_B0015](http://parts.igem.org/Part%3ABBa_B0015))
were
used
to
construct a composite part ([BBa\_K4907143](http://parts.igem.org/BBa_K4907143)),
which was then assembled into the vector
[BBa\_K4907147](http://parts.igem.org/BBa_K4907147)\_pET-28a(+)
by
standard BioBrick assembly (Fig.23). Besides, the promoter ([BBa\_K731500](http://parts.igem.org/BBa_K731500)),
RBS ([BBa\_B0034](http://parts.igem.org/Part%3ABBa_B0034)),
*bcsB*
coding sequence ([BBa\_K4907034](http://parts.igem.org/BBa_K4907034)), and terminator ([BBa\_B0015](http://parts.igem.org/Part%3ABBa_B0015))
were
used
to
construct another composite [BBa\_K4907144](http://parts.igem.org/BBa_K4907144),
which was assembled into the vector pSB4A5 by standard BioBrick (Fig. 23). The constructed two
plasmids
were
transformed into *E. coli* Nissle 1917 (EcNP) successfully, then the positive transformants
were
selected by ampicillin and kanamycin and confirmed by colony PCR (Fig. 23) and sequencing.

![](https://static.igem.wiki/teams/4907/wiki/result/result/drought/36-bcgenetic-circuit-1.png)

**Fig. 23 Graphic description of the genetic
circuit
for
BC
production.**

To qualitatively determine whether our engineered bacteria can produce bacterial cellulose
normally,
we
coating bacteria broth of EcN (wild type), recombinant EcNP, and *E. coli* BL21(DE3) on an
agar
plate with
Congo red. As shown in Fig. 24, recombinant EcNP exhibited the macroscopic red color because the
Congo
red
bind to the bacterial cellulose produce by recombinant EcNP. It also demonstrated that
recombinant
EcNP
could produce bacterial cellulose.

![](https://static.igem.wiki/teams/4907/wiki/result/result/drought/37-lb-cr.jpg)

**Fig. 24 Image of LB plate with CR.**

The Congo red staining method was employed to measure the value of OD490, which can
calculate
the
concentration of bacterial cellulose according to the standard curve in Fig. 25 (please see [Experiment](https://2023.igem.wiki/xmu-china/experiments%20) for details).

![](https://static.igem.wiki/teams/4907/wiki/result/result/drought/38-bc-standard-curve-1.png)

**Fig. 25 Standard curve of BC
(R2=0.9592).**

![](https://static.igem.wiki/teams/4907/wiki/result/result/drought/39-bc.png)

**Fig. 26 The results of BC concentration
measurement
via
Congo Red method.**

As shown in Fig. 26, the concentration of bacterial cellulose by our engineered strain was
higher
than
that
of the control group. It demonstrated that the engineered strain was successfully constructed to
bacterial
cellulose with 91.34 mg/L at 23th hour.

##### **The fermentation production characteristic for hyaluronic acid**

###### **Produce hyaluronic acid in shake flask**

To pay the way for co-culture, we investigated the growth and product synthesis rule of these
two
engineered
bacteria in a shake flask with 150 mL broth. As shown in Fig. 27, compared with the control
group,
the
expression of hyaluronic acid doesn't have a negative effect on the growth of bacteria.
Hyaluronic
acid
is a
secondary metabolite produced after 16 hours, of which the max concentration is 180 mg/L at the
20th hour (Fig. 27a).
However, we also observed a decrease in the hyaluronic acid concentration after
the cultivation for 20 hours (Fig. 27). We presume that the hyaluronic acid
would be used as a carbon source by bacteria when the glucose was exhausted
after fermentation for 20 hours. Thus, further supplementing the glucose
at the 40th hour, we observe the increase of hyaluronic acid 3 hours later.
It demonstrated that the hyaluronic acid would be used as a carbon source by bacteria when
the glucose was exhausted. So, the fed-batch is the best fermentation way to produce
hyaluronic acid in a bioreactor.

![](https://static.igem.wiki/teams/4907/wiki/result/result/drought/40-ha-flask-od400-600-1.png)

**Fig. 27 The result of the shack flask
fermentation.
a** The OD600 versus time produced by recombinant *E. coli*
BL21(DE3) in shake flask culture. **b**The HA concentration versus time
produced by recombinant *E. coli* BL21(DE3) in shake flask culture.
The HA concentration (mg/L) is represented by OD400. HA: hyaluronic acid.

###### **Produce hyaluronic acid in hardware**

The water retention material will be produced and applied in
the orchard in the future
(please see [Implementation](https://2023.igem.wiki/xmu-china/proposed-implementation)
for
details).
So, we customized hardware for water retention
material production (please see [Hardware](https://2023.igem.wiki/xmu-china/hardware)
for details). The hyaluronic acid fermentation produced by the engineered strain
was carried out in the hardware with a 600 mL working volume. As shown in Fig. 28a,
the max value of biomass (OD600) and hyaluronic acid concentration reached
16.91 and 782 mg/L (Fig. 28a), respectively. The biomass is 9.4-fold higher than that
in the shake flask, while hyaluronic acid concentration is 4.3-fold higher than that
in the shake flask. At the same time, the substrate (glucose) can be exhausted at last.
It demonstrated that the hardware is suitable for both bacteria growth and hyaluronic
acid production.

![](https://static.igem.wiki/teams/4907/wiki/result/result/drought/41.png)

**Fig. 28 Hyaluronic acid production
versus time in hardware. a** The concentration of HA (mg/L) and
biomass (OD600). **b** The glucose concentration,
NaOH (mL), and Feeding (g). HA: hyaluronic acid.

##### **The fermentation production characteristic for bacterial cellulose**

###### **Produce bacterial cellulose in shake flask**

As shown in Fig. 29, the expression of bacterial cellulose doesn't
have a negative effect on the growth of bacteria, in which the highest
OD is 1.78. Bacterial cellulose is a secondary metabolite produced after 16 hours,
of which the max concentration is 95.9 g/L at the 63th hour.

![](https://static.igem.wiki/teams/4907/wiki/result/result/drought/42-bc-flask-product-od600.png)

**Fig. 29 The result of the shack flask
fermentation.
a** The OD600 versus time produced by recombinant
*E. coli* BL21(DE3) in shake flask culture. **b** The BC concentration
versus time produced by recombinant *E. coli* BL21(DE3) in shake flask culture.
The BC concentration (mg/L) is represented by OD400. BC: bacterial cellulose.

###### **Produce bacterial cellulose in hardware**

The bacterial cellulose fermentation produced by the engineered strain was carried out in the
hardware with a 600 mL working volume. As shown in Fig. 30a, the max value
of biomass (OD600) and hyaluronic acid concentration reached 16.93
and 603 mg/L (Fig. 30a), respectively. The biomass is 9.5-fold higher than that
in the shake flask, while hyaluronic acid concentration is 6.28-fold higher than
that in the shake flask. The total produce time reduced from 63 to 38 hours.
It demonstrated that the hardware is suitable for both bacteria growth and bacterial
cellulose production.

![](https://static.igem.wiki/teams/4907/wiki/result/result/drought/43-hardwareha.png)

**Fig. 30 Bacterial cellulose production
versus time in hardware. a** The concentration of BC (mg/L) and biomass
(OD600). **b** The glucose concentration, NaOH (mL), and Feeding (g).
BC: bacterial cellulose.

#### **Constructed the gene circuit to regulate bacterial population density**

##### **Expressed RFP in EcNP**

The promoter ([BBa\_J23100](http://parts.igem.org/BBa_J23100)),
RBS ([BBa\_B0034](http://parts.igem.org/BBa_B0034)),
and *rfp* coding sequence ([BBa\_K4907037](http://parts.igem.org/BBa_K4907037))
were used to construct a composite part [BBa\_K4907147](http://parts.igem.org/BBa_K4907147),
which was then assembled into the vector pET-28a(+) by standard BioBrick assembly (Fig. 31).
The constructed plasmid was transformed into *E. coli* Nissle 1917 (EcNP) successfully,
then the positive transformants were selected by kanamycin and confirmed by
colony PCR and sequencing.

![](https://static.igem.wiki/teams/4907/wiki/result/result/drought/44-rfp-pet-28a.png)

**Fig. 31 Graphic description of the
*rfp* genetic circuit**

The bacterial culture was cultivated overnight,
and then the measurement of OD600 and fluorescence
strength were carried out in triplicate. The pictures of OD600
versus time and fluorescent versus time were shown in Fig 32, which indicated
the growth of EcNP. At the same time, the fluorescence value showed a linear
relationship with the OD600 value (R2=0.9727) when OD600>2,
which was consistent with the results in the references (2). This means that when the
OD600 of bacteria is greater than 2, we can calculate the bacterial population
density of EcNP in the current mixed bacterial solution through the measured RFP
fluorescence intensity, which provides strong support for our subsequent co-culture
regulation. The determination of OD600-t and fluorescence-t showed the
growth of EcNP. At the same time, we observed that when OD600>2, the
fluorescence of EcNP expression showed a linear relationship with its OD600
value (R2=0.9727) (Fig 33), which was consistent with the results in the
references (2). This means that when the OD600 value of bacteria is higher
than 2, we can calculate the bacterial population density of EcNP in the mixed bacterial
broth through the fluorescence intensity measured, which provides strong support for the
co-culture regulation.

![](https://static.igem.wiki/teams/4907/wiki/result/result/drought/45-ecnp-rfp-kan.png)

**Fig. 32 a** Growth curve of EcNP .
**b** RFP fluorescence intensity versus time of EcNP.

![](https://static.igem.wiki/teams/4907/wiki/result/result/drought/46-ecnp-rfp-fluovsod.png)

**Fig. 33 Fluorescence
intensity/OD600 of EcNP**

##### **Expressed GFP in *E. coli* BL21(DE3)**

The promoter ([BBa\_J23100](http://parts.igem.org/BBa_J23100)),
RBS ([BBa\_B0034](http://parts.igem.org/BBa_B0034)),
*gfp*
coding
sequence ([BBa\_K4907036](http://parts.igem.org/BBa_K4907036)),
and
terminator
([BBa\_B0015](http://parts.igem.org/BBa_B0015))
were used to construct the composite part [BBa\_K4907146](http://parts.igem.org/BBa_K4907146),
which was then assembled into the vector pSB3C5 by standard BioBrick assembly.
The constructed plasmid was transformed it into *E. coli* BL21(DE3), successfully,
then the positive transformants were selected by kanamycin and confirmed by colony
PCR (Fig. 34) and sequencing.

![](https://static.igem.wiki/teams/4907/wiki/result/result/drought/47-gfp-circuit-colonypcr-2.png)

**Fig. 34 a** A graphic description of the
*gfp*
genetic
circuit.
**b** DNA gel electrophoresis of the colony PCR products.
(BBa\_K4907146\_pSB3C5 in *E. coli* BL21(DE3). Target bands (1195 bp)
can be observed at the position between 1000 bp and 1500 bp.)

The bacterial culture was cultivated overnight, and then the measurement of
OD600 and fluorescence strength were carried out in triplicate.
The fluorescence value showed a linear relationship with the OD600
value (R2=0.9590) when OD600>1, which was consistent with the
results in the references (2). This means that when the OD600 of bacteria
is greater than 1, we can calculate the bacterial population density of *E. coli* BL21(DE3)
in
the
current
mixed bacterial solution through the measured GFP fluorescence intensity, which provides strong
support
for
our
subsequent co-culture regulation.

![](https://static.igem.wiki/teams/4907/wiki/result/result/drought/48-bl21-de3-gfp-curve.png)

**Fig. 35 a** Growth curve of
*E. coli* BL21(DE3) with BBa\_K4907146. **b** Fluorescent intensity curve
of *E. coli* BL21(DE3) with BBa\_K4907146.

![](https://static.igem.wiki/teams/4907/wiki/result/result/drought/49-bl21-de3-gfp-fluovsod.png)

**Fig. 36 Fluorescence
intensity/OD600
of *E. coli* BL21(DE3).**

#### **Regulated the bacterial population density by different concentrations of antibiotics**

We attempted to regulate the ratio of these two engineered bacteria through antibiotics, which
was
preferentially carried out in the shake flask. So, we culture the *E. coli* BL21(DE3)
harboring
plasmid of J23100-B0034-*gfp*-B0015\_pSB3C5 and EcNP harboring plasmid of
J23100-B0034-*rfp*-T7t\_pET-28a(+).
Chloramphenicol and kanamycin were added to regulate the ratio of these two engineering
bacteria,
whose
population quantity was characterized by the fluorescence intensity. After that, we could
harvest
the
relationship between the concentration of antibiotics and the bacteria. As shown in Fig. 37
**a** and Fig 37 **b**
(data
of
EcNP and *E. coli* BL21(DE3) respectively), various concentrations of antibiotics also have
different effects on the quantity of bacteria. These results will pave the way for the
regulation
of
bacteria population quantity.

![](https://static.igem.wiki/teams/4907/wiki/result/antibiotic-37.png)

**Fig. 37 The effect of the antibiotic on the
bacteria
growth rate of *E. coli* BL21(DE3). a** The effect of the antibiotic on the bacteria
growth of EcNP (The concentration of antibiotic is 0, 1.36, 34 mg/L respectively). **b**
The effect of the antibiotic on the bacteria growth of *E. coli* BL21(DE3) (The
concentration of antibiotic is 0, 20, 100 mg/L respectively).

#### **Pave the way for co-culture fermentation**

##### **Co-culture fermentation in shake flask**

The two engineered bacteria constructed were cultured in the shake flask to produce the
hyaluronic
acid
and bacterial cellulose, which was mixed to produce water-retention material. As shown in Fig.
38,
we
can observe the flocculent water-retention material both in the mixed culture broth and sediment
after
centrifugation. Thus, we have successfully produced the water-retention material which has been
applied
in characterizing the water-retention efficiency in the soil (please see  [Proof of concept](https://2023.igem.wiki/xmu-china/proof-of-concept%20)  for
details).

![](https://static.igem.wiki/teams/4907/wiki/result/result/drought/fig-co-culture.png)

**Fig. 38 Co-culture results of these two
engineered
bacteria. a** co-culture broth. **b** sediment after centrifugation.

##### **Co-culture fermentation in hardware**

Two cycles of repeated fed-batch were carried out in hardware to ensure the functionality of
hardware. The crosslinked compound of bacterial cellulose and hyaluronic acid produced
successfully (as shown in Fig. 39), and the product was filtered out to be used in further
analysis.

![](https://static.igem.wiki/teams/4907/wiki/result/result/39.jpg)

**Fig. 39 The outlook of the harvested
crosslinked compound from repeated fed-batch fermentation.**

We conducted an Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy
(ATR-FTIR) assay to confirm whether the crosslinking occurred. As shown in Fig. 40, a peak
at 1028 cm-1 can be observed, indicates that the crosslinking between bacterial
cellulose and hyaluronic acid occurred during co-culture fermentation.

![](https://static.igem.wiki/teams/4907/wiki/result/result/40.png)

**Fig. 40 ATR-FITR diagram, comparing the
characteristic of harvested crosslinked product and standard compound.**

Also, a water retention ability test in soil was carried out with the bacterial culture
containing the crosslinked product. As shown in Fig. 41, the bacterial culture has
greater water retention ability than pure water, indicates that our product produced in
the hardware functions well as a water retention material which can maintain the
moisture of soil.

![](https://static.igem.wiki/teams/4907/wiki/result/result/41.png)

**Fig. 41 A diagram showing the result of
water retention ability of the harvested crosslinked compound in soil.**

### **Conclusion**

In the anti-drought part, we develop a water-retention material
produce by the co-culture of two bacteria. We also developed a
customized hardware to produce this material. We also demonstrated that
the co-culture product exhibits excellent performance as a water-retention
material in the soil (please see [Proof
of
Concept](https://2023.igem.wiki/xmu-china/proof-of-concept)
for details).

### **Future Prospects**

We will further optimize the proportion of the two engineered
bacteria and enhance the production of the water-retention material in hardware.

## **Biosafty**

### Kill switch

In our implementation, we plan to mix engineered bacteria or products into the soil. To
avoid
the
leakage of engineered bacteria, MazF is selected as the toxin by cleaving the mRNA of
bacteria
inside.

The kill switch system is mainly composed of an inducible promoter (pBAD/araC) and a
*mazF* gene
(express toxin protein of MazF).

#### Kill switch of anti-icing part

We introduce the inverter to make engineered bacteria survive when arabinose is present
and die
when
it
runs out. So we can mix arabinose and engineered bacteria together and put them into the
soil,
and
when
the arabinose runs out, engineered bacteria expresses toxins and dies, preventing them
from
leaking
out.

We use pBAD/araC ([**BBa\_I0500**](http://parts.igem.org/Part%3ABBa_I0500%20))
, inverter ([**BBa\_Q04510**](http://parts.igem.org/Part%3A%20BBa_Q04510%20)),
RBS ([**BBa\_ B0034**](http://parts.igem.org/Part%3A%20BBa_%20B0034%20)), Terminator ([**BBa\_B0015**](http://parts.igem.org/Part%3ABBa_B0015%20))
, mazF ([**BBa\_ K1096002**](http://parts.igem.org/Part%3ABBa_%20K1096002%20))
to construct the composite part BBa\_K3332081.

![]( https://static.igem.wiki/teams/4907/wiki/result/fig-56.png)

**Fig. 42 CFU assay for characterizing
the
killing
effect
of kill switch in detection system**

CFU assay was carried out to characterize the effect of kill switch. We discovered that
the
number
of
colonies of *E. coli* carrying BBa\_K3332081 (without induction) and pBAD/araC-mazF
(with
induction)
decreased significantly after 8 hours. And there was almost no bacterial growth in 15
hours
(Fig.
42),
which could verify that BBa\_K3332081 has the killing function without L-arabinose. In
conclusion,
This
demonstrated that toxin MazF could be lethal to the bacteria and the kill switch
(BBa\_K3332081)
in
detection system did work to prevent engineered *E. coli* escaping and could
achieve our
purpose of
bio-containment.

#### Kill switch of anti-drought part

As glucose is the production ingredient, we choose to induce expression of MazF directly
by
arabinose.
When glucose exhausts, arabinose will play its role to lead bacteria to death.

We use pBAD/araC ([**BBa\_I0500**](http://parts.igem.org/Part%3ABBa_I0500%20)),
RBS
([**BBa\_B0034**](http://parts.igem.org/Part%3ABBa_B0034%20)), terminator ([**BBa\_B0015**](http://parts.igem.org/Part%3ABBa_B0015%20)), mazF ([**BBa\_ K1096002**](http://parts.igem.org/Part%3ABBa_%20K1096002%20)) to
construct the
composite part BBa\_K3332083, which were assembled on pSB1C3 backbone by standard
assembly.

![]( https://static.igem.wiki/teams/4907/wiki/result/fig-57.png)

**Fig. 43 Survival Ratio of different
groups
against
time
(h).**

CFU assay was implemented to characterize the effect of toxin MazF. We observed that the
number
of
colonies on the plate decreased significantly after induction (Fig. 43). This
demonstrated that
toxin
MazF could be lethal to the bacteria and the kill switch ([**BBa\_ K3332083**](http://parts.igem.org/Part%3ABBa_%20K3332083%20)) in
detection
system
did
work to prevent engineered *E. coli* escaping and could achieve our purpose of
bio-containment.

### Preventing HGT

Our genetically modified organisms will be engineered as biofertilizers for release into
the
soil
environment of crops. Given the presence of thousands of microorganisms in soil,
preventing
Horizontal
Gene Transfer (HGT) is of paramount importance. Our kill switch consists of *ccdA*
(encoding
the
*ccdA* antitoxin protein) and *ccdB* (encoding the *ccdB* toxin protein).
We aim
to
ensure that in the soil environment, our expression vector only functions within our
host
bacteria.
In
the event of HGT, the corresponding bacteria will undergo cell death, successfully
blocking HGT.

Before incorporating the constitutive promoter and *ccdA* gene into the genome,
during the
validation phase, we use an inducible promoter.

#### Anti-toxicity of*ccdA*

We use pBAD (BBa\_K206001), RBS (BBa\_B0034), *ccdB* (BBa\_K3512001) to construct the
composite
part
BBa\_K4907139, which were assembled on pSB4K5 backbone by standard assembly. This
constructed
circuit
was
transformed into *E. coli*DB3.1, followed by positive transformant selection using
kanamycin
and
confirmation through colony PCR (Fig. 44) and sequencing.

![]( https://static.igem.wiki/teams/4907/wiki/result/fig-50.png)

**Fig. 44 DNA gel electrophoresis of the
colony
PCR
products of BBa\_K4907139.**

We use pBAD (BBa\_I13453), RBS (BBa\_B0034), *ccdA* (BBa\_K4907032) to construct the
composite
part
BBa\_K4907138, which were assembled on pSB1C3 backbone by standard assembly. This
constructed
circuit
was
transformed into *E. coli*DH10β, followed by positive transformant selection using
chloramphenicol
and confirmation through colony PCR (Fig. 45) and sequencing.

![]( https://static.igem.wiki/teams/4907/wiki/result/fig-51.png)

**Fig. 45 DNA gel electrophoresis of the
colony
PCR
products of BBa\_K4907138.**

To validate the resistance of *ccdA* to *ccdB*, we performed a dual-plasmid
transformation.

| Experiment | Dual-plasmid system | Strain | Result |
| --- | --- | --- | --- |
| Verfication of *ccdA* in plasmid4 | BBa\_K4907139\_pSB4K5   BBa\_K4907138\_pSB1C3 | in DH10β | ![](https://static.igem.wiki/teams/4907/wiki/parts/yxy/139-ban1.jpg) |
| BBa\_K4907139\_pSB4K5   BBa\_I13453\_pSB1C3 | ![](https://static.igem.wiki/teams/4907/wiki/parts/yxy/139-ban2.png) |
| Verfication of *ccdA* in genome ( *ccdB* is controlled by pBAD) | BBa\_K4907139\_pSB4K5   BBa\_K4907138\_pSB1C3 | in DB3.1 | ![](https://static.igem.wiki/teams/4907/wiki/parts/yxy/139-ban3.jpg) |
| in DH10β | ![](https://static.igem.wiki/teams/4907/wiki/parts/yxy/139-ban4.png) |

**Table. 1** Performance of different dual-plasmid systems in *E. coli*
DH10β and
DB3.1

The results showed that *E. coli* DB3.1 transformed with toxin genes grew well. *E.
coli* DH10β transformed with both toxins and antitoxins also exhibited good growth.
However,
*E. coli*DH10β with toxins only did not grow. This confirmed the killing effect of
the
toxin *ccdB* again and the neutralization of antitoxin *ccdA*.

Besides, the *E. coli*DB3.1 transformed with toxin controlled by pBAD promoter
without
antitoxin both grew better compared with *E. coli*DH10β. From these results, we can
draw
the conclusion that whether the *ccdA* is in plasmid or genome can play the role of
neutralisation to *ccdB*.

Therefore, in light of the serious leak of BBa\_K206001, we use pRha(BBa\_K914003), RBS
(BBa\_B0034), *ccdB* (BBa\_K3512001) to construct the composite part BBa\_K4907131,
which were
assembled on pSB4K5 backbone by standard assembly. This constructed circuit was
transformed into*E.
coli*DH10β, followed by positive transformant selection using kanamycin through
colony PCR
(Fig. 46) and sequencing.

![]( https://static.igem.wiki/teams/4907/wiki/result/fig-52.png)

**Fig. 46 DNA gel electrophoresis of the
colony
PCR products of BBa\_K4907131.**

| Experiment | Dual-plasmid system | Strain | Result |
| --- | --- | --- | --- |
| Verfication of *ccdA* in plasmid | BBa\_K4907131\_pSB4K5   BBa\_K4907138\_pSB1C3 | in DH10β | ![](https://static.igem.wiki/teams/4907/wiki/parts/yxy/131-ban1.jpg) |
| BBa\_K4907131\_pSB4K5   BBa\_I13453\_pSB1C3 | ![](https://static.igem.wiki/teams/4907/wiki/parts/yxy/131-ban2.png) |
| Verfication of *ccdA* in genome ( *ccdB* is controlled by pRha) | BBa\_K4907131\_pSB4K5   BBa\_K4907138\_pSB1C3 | in DB3.1 | ![](https://static.igem.wiki/teams/4907/wiki/parts/yxy/131-ban3.jpg) |
| in DH10β | ![](https://static.igem.wiki/teams/4907/wiki/parts/yxy/131-ban4.png) |

**Table. 2** Performance of different dual-plasmid systems in *E. coli*
DH10β and
DB3.1

we also performed a dual-plasmid transformation. The result is the same as
BBa\_K4907139(Table.
2).

### Cytotoxicity of *ccdB*

We use pBAD/araC (BBa\_I0500), RBS (BBa\_B0034), *ccdB* (BBa\_K3512001) to construct
the
composite part BBa\_K4907140, which were assembled on pSB4K5 backbone by standard
assembly. This
constructed circuit was transformed into *E. coli*DH10β, followed by positive
transformant
selection using kanamycin and confirmation through colony PCR (Fig. 47) and sequencing.

![]( https://static.igem.wiki/teams/4907/wiki/result/fig-53.png)

**Fig. 47 DNA gel electrophoresis of the
colony
PCR products (BBa\_K4907140).**

Given that BBa\_K206001 and BBa\_K914003 both have serious leaks, we need to test if
BBa\_I0500
will leak. The *ccdB* circuit was induced by L-arabinose. Bacteria (*E. coli*DH10β
harboring BBa\_K4907140\_pSB4K5 and BBa\_I13453\_pSB1C3) were induced with L-arabinose and
glucose
separately for 6 hours, with 3 replicates set for each. And the changes in
OD600 and
CFU count before and after induction were measured.

![]( https://static.igem.wiki/teams/4907/wiki/result/fig-54.png)

**Fig. 48 The function of BBa\_I0500 was
characterized by survival. a: OD600 values of bacteria upon addition of
L-arabinose (left) and glucose (right). b: CFU values of bacteria upon addition of
glucose
(left) and L-arabinose (right).**

The results indicated that in the arabinose-induced group, the number of viable cells
was
significantly lower compared to the glucose group.

The experimental group (*E. coli* DH10β harboring BBa\_K4907140\_pSB4K5 and
BBa\_I13453\_pSB1C3, and the control group (*E. coli* DH10β harboring
BBa\_K206001\_pSB4K5 and
BBa\_I13453\_pSB1C3) were separately induced with L-arabinose, with 3 replicates set for
each.
Then measure OD600 and CFU count at 0, 2, 4, 6 and 8 hours.

![]( https://static.igem.wiki/teams/4907/wiki/result/fig-55.png)

**Fig. 49 Growth curve and survival
assay for
characterizing the function of *ccdB*. a The value of OD600 against
time (h)
for different groups. b CFUs/mL calculated of different groups are plotted against
time (h).**

We used promoter
BBa\_I0500
to regulate the expression of *ccdB*. After induction by *L*-arabinose,
OD600values were measured every two hours. The bacteria that had no
*ccdB*
expressed grew rapidly, while the one expressing *ccdB* showed a significant growth
defect,
as the optical density (at 600 nm) increased very slightly (Fig. 49a).

At each time, the spot assay was also performed, then the cell viability was measured by
CFU
count (Fig. 49b). Consistent with the trend of OD600 value against time, only
the
absence of *ccdB* allowed the host cells to survive. All these results indicated
that *ccdB*
was toxic enough to the engineered bacteria so that this toxin could be applied to the
cases
when the suicide of genetically engineered microorganisms (GEMs) were strongly needed.

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Xiamen University

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Xiamen University, Fujian, China
No.422, Siming South Road, Fujian, P.R.China 361005

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