iGEM 2021 — Team Shanghai_Metro

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# Team:Shanghai Metro/Notebook

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Project Notebook

Contact Info.

---

Email: *binghan423@163.com; runshigu@126.com*

Wechat Official Account: IGEM VIPatent

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# Team:Shanghai Metro/Engineering

Shanghai\_Metro

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Engineering

Background

Biological products produced by biotechnology, especially industrial microbial
fermentation technology, have been widely used in food, medicine, animal feed and daily chemical products.
With the development of strains and genetic modification technologies, the application range of industrial
microbial strains will be further expanded, and its market scale will continue to grow. However, in recent
years, intellectual property disputes related to industrial microbial strains have also been increasing year
by year. According to the currently publicly reported cases, the main considerations for determining the
intellectual property rights of industrial microorganisms include microbial morphology, DNA fingerprinting
and similarity of conservative DNA sequences. Due to the wide distribution of microbes, the limited
morphological characteristics that can be identified for individual microbes, and the strong
conservativeness of some microbial genomes, the process of confirming and protecting related intellectual
property rights is very expensive and lengthy. From the perspective of preventing the propagation and
passage of strains, the development of concise and effective patented strain protection technology has
become an urgent problem to be solved.

Design

Type VI protein secretion system (T6SS) is a protein secretion system widely
distributed in Gram-negative bacteria and can act on both eukaryotic cells and bacterial cells. Under
natural conditions, bacterial cells encoding T6SS transfer cytotoxic or antibacterial effector factors
(amidase, glycoside hydrolase, lipase, etc.) to recipient cells through physical contact, thereby inhibiting
the growth of recipient cells. The bacteria that encode T6SS can translate and produce corresponding immune
proteins to counteract the damage caused by toxic effectors.

The effector factors of T6SS were designed to constitutively express in industrial
strains, and the corresponding immune proteins are designed in a controlled and induced mode (for example:
arabinose, galactose, tetracycline, Zn2+). Only by adding specific inducers to the fermentation broth can
the normal growth of industrial microbial strains be ensured. This can effectively prevent patented strains
from being improperly stolen, and have concise and effective indicators for determining rights when disputes
occur, thereby better protecting the intellectual property rights of strains from infringement.

Build

Tke2 and tke4 are key functional factors that inhibit bacterial growth which are
controlled tet promotor (Figure 1). The immunity effectors ike2/4 are under lac promotor. The anti-bacterial
effectors tke2/4 and the immunity effectors ike2/4 were inserted in pUS232 vector (Figure 2).

![](https://static.igem.org/mediawiki/2021/d/d5/T--Shanghai_Metro--Engineering01.jpg)
Figure 1. T6SS effector and immune protein expression box.

![](https://static.igem.org/mediawiki/2021/d/d0/T--Shanghai_Metro--Engineering02.jpg)
Figure 2. Schematic map of T6SS effector and immune expression plasmids.

Test

pUS232-ike2 or pUS232-ike4 plasmids were transferred into bacterial strains
(Escherichia coli DH5α). The expression of immune effectors needs to be induced by tetracycline in the
appropriate concentration.

![](https://static.igem.org/mediawiki/2021/a/a0/T--Shanghai_Metro--Engineering03.jpg)
Figure 3. Escherichia coli DH5α harboring pUS232-ike4 plasmid. Left: bacteria with pUS232 vector;
right: bacteria with pUS232-ike4 plasmid.

As seen in Figure 3, the control DH5α yield blu strains but the experimental group
pUS232-ike4 had no blue strains, which could indicate that our design worked in DH5α. In addition, we also
determined the concentration range of tetracycline for the experimental bacterial species growth by
measuring the growth curves under tetracycline with different concentrations.

In order to determine the appropriate tetracycline induction concentration range
for better growth of our patent strains, we tested the OD600 of the strains under various concentration of
tetracycline and collected the data against hours and built the model. According to the scatter-plot
analysis by MATLAB, the data points have a tendency of oscillation decline, and there is no model of
elementary function conforming to this trend. Therefore, we decided to use interpolation method to study the
numerical transformation and obtained the final curve.

Interpolation method selection: although the curve obtained by spline interpolation
(Spline) is the smoothest, it will produce negative values, which does not conform to the actual situation.
Therefore, Hermite interpolation method (Pchip) is chosen to keep the shape of the original data.

![](https://static.igem.org/mediawiki/2021/2/21/T--Shanghai_Metro--chg2.jpg)
Figure 4. Scatter-plot analysis by MATLAB.

As the curve above, the X-coordinate shows the concentration of tetracycline and
the Y-coordinate shows the corresponding OD600 value of the strain D. In order to find the
appropriate range
of the tetracycline concentration, we referred the OD600 value of the blank control strain after
6 hours
without tetracycline:

Table 2. OD600 value of the strain E: Pus232 after 6 hours
![](https://static.igem.org/mediawiki/2021/b/b0/T--Shanghai_Metro--Engineering05.jpg)

Therefore, we drew a horizontal line where OD600 equals 0.3268 and find the cross
points of these two lines where Xs are 17, 26, 78 and 113. As seen from the graph, it indicates that the
growth of our patent strain (strain D) could grow normally even better the normal strain (strain E) when we
add the tetracycline within the range from 17 µg/L to 26 µg/L, or the more recommended 78 µg/L to
113µg/L.Regarding the range from 0 to 17 µg/L where we didn't have samples to verify the accuracy, thus we
didn’t count it as the recommended concentration range.
To sum up, this model and this recommended concentration range could be used for the future application of
our patented-strain protection technology.

Contact Info.

---

Email: *binghan423@163.com; runshigu@126.com*

Wechat Official Account: IGEM VIPatent

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# Team:Shanghai Metro/Experiments

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EXPERIMENTS

Backbone Chapter I Pus232

![](https://static.igem.org/mediawiki/2021/b/bd/T--Shanghai_Metro--Experiment01.jpg)

Segment Chapter I ike2, ike4

![](https://static.igem.org/mediawiki/2021/5/5d/T--Shanghai_Metro--Experiment02.jpg)

Connecting Backbone and Segment I Pus232-ike2, Pus232-ike4

![](https://static.igem.org/mediawiki/2021/5/54/T--Shanghai_Metro--Experiment03.jpg)

Backbone Chapter II Pus232-ike2, Pus232-ike4

![](https://static.igem.org/mediawiki/2021/0/00/T--Shanghai_Metro--Experiment04.jpg)

Segment Chapter II tke2, tke4

![](https://static.igem.org/mediawiki/2021/8/8a/T--Shanghai_Metro--Experiment05.jpg)

Connecting Backbone and Segment II

![](https://static.igem.org/mediawiki/2021/6/64/T--Shanghai_Metro--Experiment06.jpg)

Experimental Summary

![](https://static.igem.org/mediawiki/2021/3/39/T--Shanghai_Metro--Experiment07.jpg)

Experiment theory

![](https://static.igem.org/mediawiki/2021/f/f3/T--Shanghai_Metro--Experiment08.jpg)

Contact Info.

---

Email: *binghan423@163.com; runshigu@126.com*

Wechat Official Account: IGEM VIPatent

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# Team:Shanghai Metro/Results

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RESULTS

Overview:

We extracted plasmid vector Pus232
and ike/tke components from Pseudomonas putida
respectively and used restriction endonucleases to digest them for connection. At last, we constructed two
new plasmids: Pus232-ike2-tke2 and Pus232-ike4-tke4. Both plasmids have minor genetic differences, thus we
also proceeded with further tests to identify which one is more efficient at growing when the same optimal
amount of tetracycline is added.

**1. Pus232 Electrophoresis**

![](https://static.igem.org/mediawiki/2021/5/52/T--Shanghai_Metro--results01.jpg)

Channel 1~14: Purified plasmid Pus232 from 1~14 E. coli DH5α cultures.

This step is used to test if the plasmids Pus232 extracted from the E. coli DH5α
are successful and could be used to do double enzyme digestion later in the process.

Based on the three types of structures plasmid may have, we collect the supercoiled
bands. Channel 2, 5, 6, and 7 are the best bands and are selected for making double enzyme digestion.
Channel 3, which has a high concentration of plasmid, was once selected but failed.Therefore, channel 2, 5,
6, and 7 plasmids are selected to do double enzyme digestion of BamHI and XbaI for 2 hours.

Channel 3 might have undergone too long of a P2 cracking phase, which leads to the
fracture within the DNA. The DNA secreted contains groups of open circular DNA , showing as the bright white
color in channel 3, but less circular plasmid DNA that we are targeting to collect.

**2. ike2/4 Electrophoresis**

![](https://static.igem.org/mediawiki/2021/d/dc/T--Shanghai_Metro--results02.jpg)
Figure 2. Gel electrophoresis of ike2 and ike4 PCR products.

Channel 1: ike2 electrophoresis failed maybe because bacteria added is too much.

Channel 2: ike2 succeeded

Channel 3: ike2 electrophoresis failed maybe because bacteria added is too much.

Channel 4: ike4 succeeded

Channel 5: ike4 succeeded

Channel 6: ike4 succeeded

Channel 7: ike4 succeeded

This step is used to check if the ike2 and ike4 extracted from Pseudomonas putidas
are successful and could be used to do double enzyme digestion later in the process.
PCR clean-up ike2 and ike4 DNA fragments to do double enzyme digestion of BamHI and XbaI overnight.

Channel 1 and 3 failed the test. One possible explanation could be that the
bacteria added into the PCR solution is too much.

**3. Pus232 Double Enzyme Digestion**

![](https://static.igem.org/mediawiki/2021/6/6a/T--Shanghai_Metro--results03.jpg)
Figure 3. Gel electrophoresis of Pus232 double enzyme digestion products.

Channel 1&2: Pus232 control group

Channel 3~6: Products of Pus232-BamHI+XbaI Double Enzyme Digestion

Channel 7~10: Products of Pus232-BamHI+XbaI Double Enzyme Digestion

Channel 3~10 have shorter bands after the double enzyme digestion, and all of them
are successful. Gel clean-up the product of Pus232-BamHI+XbaI double enzyme digestion to obtain
Pus232-backbone. Clean-up the product of ike2 and ike4-BamHI+XbaI overnight double enzyme digestion to
obtain ike2-fragment and ike4-fragment.

T4 DNA ligase is used to connect Pus232-backbone with ike2-fragment and
ike4-fragment overnight separately.

**4. Pus232-ike2/4 sequencing analysis**

![](https://static.igem.org/mediawiki/2021/d/da/T--Shanghai_Metro--results04.jpg)
Figure 4. Blast DNA sequences with theoretical sequences and actual sanger sequencing documents of
Pus232-ike2.

![](https://static.igem.org/mediawiki/2021/c/c1/T--Shanghai_Metro--results05.jpg)
Figure 5. Blast DNA sequences with theoretical sequences and actual sanger sequencing documents of
Pus232-ike4.

The sequencing results show that both Pus232-ike2 and Pus232-ike4 are constructed
successfully.

**5. Electrophoresis of Pus232-ike2/4 Enzyme Digestion**

![](https://static.igem.org/mediawiki/2021/7/74/T--Shanghai_Metro--results06.jpg)
Figure 6. Gel electrophoresis of Pus232-ike2, Pus232-ike4 enzyme digestion products.

Channel 1~4: Products of Pus232-ike2-SacII single enzyme digestion, succeed, cut
the target bands for gel clean-up.

Channel 5~8: Products of Pus232-ike4-SacII single enzyme digestion, succeed, cut
the target bands for gel clean-up.

Channel 9~12: Products of tke4 PCR, contains some unneeded bands, cut the 4k+ bands
for gel clean-up.

**6. Electrophoresis of tke2/4**

![](https://static.igem.org/mediawiki/2021/a/a0/T--Shanghai_Metro--results07.jpg)
Figure 7. Gel electrophoresis of tke2 and tke4 PCR products.

Channel 1~3: tke2 KOD, succeed, bands size are correct and unitary, cut for gel
clean-up.

Channel 4~6: tke4 KOD+DMSO, succeed, contains some unneeded bands, cut the 4k+
bands for gel clean-up.

**7. Recombination of the Linearized Pus232-ike vectors and tke inserts**

The tke insert and the linearized Pus232-ike vector, with overlapped sequences of
15 bp on both 5’- and 3’-end, respectively, are mixed and incubated with recombinase Exnase II at 37°C for
30 min. Pipet 8 μl of the recombination products to 80 μl of the E.coli DH5α competent cells for
transformation. Pipet 4 μl of the control group Pus232 and Pus232-ike2 to 40 μl of the E.coli DH5α competent
cells for transformation.

**8. Pus232-ike4-tke4 Transformation Plates**

Control group E.coli DH5α/Pus232 and E.coli DH5α/Pus232-ike2 show blue strains,
which are desired results because without the tke presence, lacZ protein in Pus232 won’t be replaced, and
X-Gal we added under the catalysis of lacZ protein, blue products will be produced, thus the strain
appearing blue(Fig. 8 left and middle). However, the white single strain may be microbial contamination
(Fig. 8 left).

![](https://static.igem.org/mediawiki/2021/4/48/T--Shanghai_Metro--results08.jpg)
Figure 8. Transformation plates of recombination reaction.

Experimental group Pus232-ike4-tke4 had a white single strain when the picture was
taken (Fig. 8 right). The result could be satisfactory because tke4 replaced lacZ protein in the plasmid,
stopping it from expressing thus the strain won’t demonstrate any blue color.

**9. Functional Test**

We first identified if we have correct strains with naked-eye observation, with
tke4 present, the strain color should be white, blue strains may be undesired organisms or microbial
contamination. Then we took a single strain for OD600 Testing to further determine the range of optimal
tetracycline amount.

![](https://static.igem.org/mediawiki/2021/6/69/T--Shanghai_Metro--results09.jpg)

OD600 Testing results are below:

![](https://static.igem.org/mediawiki/2021/1/1d/T--Shanghai_Metro--results10.jpg)

With the result, we can generally determine the appropriate tetracycline induction
concentration range is
0-0.1μg/mL or more, only less than 1μg/mL. Due to the large concentration range selected in the preliminary
test, it is impossible to determine the effect of the induced concentration between 0.1-1μg/mL. We perform
further tests to keep a smaller range until we find the optimal tetracycline amount.
The result also proved the effect of tetracycline alone, the graphs below show that more tetracycline in
the. control group Pus232 will suppress the growth of strains. As for our modified plasmid strain, the curve
is roughly similar to the parabola concave down, which has a maximum or optimal amount in this case.

![](https://static.igem.org/mediawiki/2021/6/6a/T--Shanghai_Metro--results11.jpg)
Figure 9. Biomass Concentration curve of Pus323 Strain with tetracycline

![](https://static.igem.org/mediawiki/2021/2/2b/T--Shanghai_Metro--results12.jpg)
Figure 10. Biomass Concentration curve of Pus323-tke4-ike4 Strain with tetracycline

After having a general idea of the best concentration of tetracycline, we set up
different time knots to measure the concentration of strains, each with a two hours interval. Similarly, the
OD600 testing will help us to determine the growth of strains. The results of the test are below.

![](https://static.igem.org/mediawiki/2021/9/9b/T--Shanghai_Metro--results13.jpg)

![](https://static.igem.org/mediawiki/2021/f/f2/T--Shanghai_Metro--results14.jpg)

Despite being less effective than 200µg/L concentration amounts at early hour
measurement, eventually, concentrations at the range of 300-500µg/L are more effective. The results also
proved that adding tetracycline to ike4-tke4 strain will boost the growth of strain, while the effect to
Pus232 strain alone is less obvious. This test is more thorough than our first one, and the induced
concentration of 100-1000μg/L is more detailed. Now it determines that the optimal concentration of
tetracycline is 300µg/L to 500µg/L.

![](https://static.igem.org/mediawiki/2021/2/2d/T--Shanghai_Metro--results15.jpg)
Figure 11. Biomass Concentration curve of Pus323-tke4-ike4 Strain with tetracycline against
hours

![](https://static.igem.org/mediawiki/2021/9/9a/T--Shanghai_Metro--results16.jpg)
Figure 12. Biomass Concentration curve of Pus323 Strain with tetracycline against hours

Future approach:

One disadvantage of our technique is that this only applies to the E.coli bacteria
since the modification is done on the E.coli plasmid. This prevents us from cooperating with companies whose
bacteria is not modified on E.coli. Therefore, possible future approaches could be to discover how we can
protect the patent of bacterias other than E.coli. We may transfer our modified plasmid into other bacteria
strains or design a new plasmid for other types of bacteria.

Contact Info.

---

Email: *binghan423@163.com; runshigu@126.com*

Wechat Official Account: IGEM VIPatent