iGEM 2023 — Team sustech-shenzhen

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# April to May 2023

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# June 2023

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# July 2023

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# August to September 2023

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## Contents

* April to May
* June
* July
* August to September

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# Design

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## Purpose

Due to the inherent characteristics of polypeptides, maintaining a high concentration near target cells and enabling their entry into cells to exert their effects becomes a challenge in treatment and experimentation. The Type VI secretion system (T6SS) of Gram-negative bacteria can deliver effector proteins to the bacterial surrounding environment or into cells. Therefore, we hope to explore the capability of T6SS to act as an exogenous polypeptide delivery system, thus developing new drug delivery methods.

## Deletion of Toxic Genes

In this experiment, we use *Vibrio cholerae* and *Pseudomonas aeruginosa* as chassis cells. As bacteria of the BSL-2 group, how to minimize their toxicity to ensure the safety of experimenters is our primary concern. The toxicity of these two strains mainly comes from their metabolic products (toxins), Type III secreted proteins, and Type VI secreted proteins. Fortunately, our PI has researched the Type VI secretion system, so we already have bacteria with reduced toxicity, deleting T6SS effectors. Based on this, the toxicity of *Vibrio cholerae* has been greatly reduced. Therefore, we mainly focused on modifying *Pseudomonas aeruginosa*.

In this experiment, we mainly want to reduce the toxicity brought by the T3SS of *P. aeruginosa* (see safety section for details). Firstly, using the suicide plasmid pEXG2.0 as a template (where *genR* is a resistance gene, used to screen whether the suicide plasmid is successfully imported into the bacteria, and *sacB* expresses levansucrase protein, which can decompose sucrose, and at the same time makes fructose polymerize into levan, mediating bacterial death, used for the second screening of whether the bacteria successfully lose the plasmid), the up and downstream of 800-1000bp of the target knockout gene was cloned and the complete knockout plasmid was constructed through the Gibson assembly method. This plasmid was introduced into PAO1 by mating. After multiple screenings and PCR checks, a monoclonal bacterium that successfully knocked out the target fragment was finally obtained. After knocking out toxicity, we used fluorescent microscopy to observe whether the Type VI of *Pseudomonas aeruginosa*, with some toxicity genes knocked out, can be normally assembled.

![](https://static.igem.wiki/teams/4739/wiki/engineering/t6ss.png)

Figure 1. The basic structure of T6SS [1]

![](https://static.igem.wiki/teams/4739/wiki/engineering/gibson.png)

Figure 2. Principle of homologous recombination

## Construction of Expression Vectors

The Type VI components used in this experiment are Hcp1, Tse6 (*Pseudomonas aeruginosa*), and VgrG3 (*Vibrio cholerae*). The Type VI secretion system is mainly composed of the membrane complex, baseplate, and tail sheath complex, and the two proteins we selected are associated with the spike at the baseplate place. Tse6 contains a PAAR domain, which can be assembled with Type VI component proteins, and also has an effector domain, which performs functions like eliminating enemies in the natural environment. Simply put, in this experiment, what we did can be understood as replacing the effector domain with the target polypeptide, so that the exogenous polypeptide can be assembled with Type VI and secreted. The same is true for VgrG3, but it is assembled through the VgrG domain. For Tse6 and VgrG3, the Type VI secretion system secretes only one protein at a time, and if a large amount of protein needs to be delivered, Hcp1 will be used, as a single activation of T6SS can deliver tens of Hcp1. Usually, Hcp does not contain effector proteins, so we will connect Hcp1 and the target polypeptide through the AAAGGG linker, which is provided by a previous study[2].

![](https://static.igem.wiki/teams/4739/wiki/engineering-fig3.png)

Figure 3. Schematic of three types of fusion protein we designed

For *Vibrio cholerae*, we choose pBAD33 as the expression vector, which contains the arabinose-inducible operon and can express a large amount of protein under the induction of arabinose; the chloramphenicol resistance gene is a selection marker. For *Pseudomonas aeruginosa*, we choose pPSV37 as the expression vector, which contains the lactose-inducible operon and can express a large amount of protein under the induction of lactose; the gentamicin resistance gene is a selection marker. First, we use AlphaFold 2 to predict the structure of the fusion protein to observe whether fusion with the target protein will affect the structure of the Type VI domain or the structure of the Drug. Then through the Gibson assembly method, the cloned target fragment and vector are ligated and introduced into the corresponding bacteria.

![](https://static.igem.wiki/teams/4739/wiki/engineering-fig4.png)

Figure 4. Plasmids we utilized in our project, Cm: Chloramphenicol, Gen: Gentamicin

## Experimental Verification

The Type VI Secretion System of normal *Pseudomonas aeruginosa* is membrane perturbation mediated. The *Pseudomonas aeruginosa* strains used in this experiment have knocked out the *retS* gene so that Type VI can be constitutively activated. Before performing secretion experiments, we first compare whether *retS*-mediated Type VI activation is normal and also want to explore whether extracellular DNA can enhance the activation of Type VI. Firstly, this is explored through the killing assay. After figuring out better secretion conditions, we will first enrich the bacteria, induce secretion under corresponding conditions, and finally check through Western blot, to observe whether secreted proteins appear in the supernatant.

# Engineering

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## Knock out cycle 0:

The first round involved the knockout of *Pseudomonas aeruginosa* type III regulatory gene *vfr*
and type III secretion proteins *exoS* and *exoT*, as well as the second step synthesis enzyme
*phzS* for pyocyanin. The first three genes were successfully knocked out. However, no bacteria
survived the second selection step when knocking out *phzS*, possibly due to toxicity from
accumulated intermediates. Considering that the first-step synthesis enzyme may have other
functions, we decided to abandon the knockout of this gene.

## Expression plasmids construction cycle 0:

In the first cycle, all target peptides were constructed after codon optimization. Some were
fused directly by building overlap on the primers, while others were synthesized by companies,
cloned via PCR to get the fragment, and then assembled through Gibson assembly. Apart from
segments like Lunasin, Adlyxin, Exenatide, Teriparatide, and Teduglutide, all other plasmids
were successfully constructed and sequenced normally. The failure may be due to the difficulty
in recovering short gene fragments during product recovery and the inefficiency of Gibson
assembly for short fragments. In the next cycle, we plan to amplify the PCR cloning system and
extend the adsorption time during product purification.

## Expression plasmids construction cycle 1:

On the one hand, we repeated the Etelcalcetide experiment and confirmed that the peptide is
toxic to bacteria. On the other hand, we re-conducted experiments for unsuccessful fragments,
according to cycle 0's optimization results. Besides the plasmid successfully constructed in
the former round, Only the Tse6-Teduglutide fusion protein was successfully built, proving
that short fragments are indeed difficult to construct. We considered using direct overlap
building on primers but chose to validate only the successfully constructed proteins due to
time constraints and the long synthesis cycle.

# Test

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## Secretion test cycle 0:

At this point, we only tested strains Tse6-KLA and Hcp-minihepcidin. However, Western blot
results showed that only Hcp-minihepcidin was secreted intracellularly, and was unable to
be detected extracellularly. We hypothesize that prolonged induction may reduce T6SS activity,
and intracellular protein might be degraded. Therefore, the induction time needs to be controlled.
Also, since Tse6 is scarce and hard to detect even if secreted, we need to enrich the supernatant
protein with TCA precipitation. It also suggests that T6SS might not be functional, so a control
group with Hcp1 is needed to observe whether the T6SS of this strain is functional. We also tested
the secretion of VgrG3 fusion proteins. Firstly, the result was strange because there were some
bands indicating it existed smaller proteins with 3V5 tags. It might suggest that VrgG3 was easily
degraded in cells. We also discovered that the level of tag in the supernatant of the negative
control group was higher than we expected. We conjecture that this is because of cell lysis, but
the signal level of RpoB is undetectable in the supernatant, and the level of RpoB is low in cells.
Therefore, we will change our RpoB antigen and test for another round.

## Secretion test cycle 1:

At this point, we only tested strains Tse6-KLA and Hcp-minihepcidin. However, Western blot results
showed that only Hcp-minihepcidin was secreted intracellularly, and was unable to be detected
extracellularly. We hypothesize that prolonged induction may reduce T6SS activity, and intracellular
protein might be degraded. Therefore, the induction time needs to be controlled. Also, since Tse6 is
scarce and hard to detect even if secreted, we need to enrich the supernatant protein with TCA
precipitation. It also suggests that T6SS might not be functional, so a control group with Hcp1 is
needed to observe whether the T6SS of this strain is functional. We also tested the secretion of
VgrG3 fusion proteins. Firstly, the result was strange because there were some bands indicating it
existed smaller proteins with 3V5 tags. It might suggest that VrgG3 was easily degraded in cells.
We also discovered that the level of tag in the supernatant of the negative control group was higher
than we expected. We conjecture that this is because of cell lysis, but the signal level of RpoB is
undetectable in the supernatant, and the level of RpoB is low in cells. Therefore, we will change
our RpoB antigen and test for another round.

## Secretion test cycle 2:

In this round, we test the expression and secretion of Hcp1-Minihepcidin, VgrG3-Dulaglutide, VgrG3-Parkin,
VgrG3-KLA, and VgrG3-Tenecteplase. For *P. aeruginosa*, its expression remains normal in the cell, but we cannot
detect any signal in the supernatant. For *V. cholerae*, this is the second round of our test, and we are
surprised to discover that our plasmid can be expressed stably. We discovered that the level of RpoB was
too low in test cycle 0, so we replaced our RpoB antigen with another product with a stronger level of
signal. As a result, we witnessed a high level of RpoB in our supernatant in this cycle, proving that the
high level of the signal in the negative control group was due to cell lysis. In this group, as we change
some induce conditions, we see that no RpoB was detected in some groups, so we may draw a result that our
induction condition is optimized.

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Results

# Learn

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For *P. aeruginosa* its T6SS seems to be hard to activate, as we observe a low level of Hcp1 fusion
protein in the supernatant, which also suggests that under our new induce condition, the activity
of T6SS is weak, or maybe the downfall was due to our choice of Western blot tag, which makes us
unable to detect the signal of the fusion protein. As a result, we think the engineering of its
T6SS is very difficult. We will try to optimize our experiment by replacing our tag and trying to
find a better induction condition. For *V. cholerae*, we think it is more likely to act as a protein
carrier, we can attempt to further optimize the condition and prove its ability to secrete after wiki freeze.

## References

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References

1. 1.Cherrak, Y.; Flaugnatti, N.; Durand, E.; Journet, L.; Cascales, E. Structure and Activity of the
Type vi Secretion System. Microbiology Spectrum 2019, 7, doi:https://doi.org/10.1128/microbiolspec.psib-0031-2019.

2. 2.Jana, B.; Keppel, K.; Salomon, D. Engineering a Customizable Antibacterial T6SS‐Based Platform in Vibrio Natriegens.
EMBO reports 2021, doi:https://doi.org/10.15252/embr.202153681.

3. 3.Wilton, M.; Wong, M.J.Q.; Tang, L.; Liang, X.; Moore, R.; Parkins, M.D.; Lewenza, S.; Dong, T.G. Chelation
of Membrane-Bound Cations by Extracellular DNA Activates the Type vi Secretion System in Pseudomonas Aeruginosa.
Infection and Immunity 2016, 84, 2355–2361, doi:https://doi.org/10.1128/IAI.00233-16.

## Contents

* Design
* Engineering
* Test
* Learn
* References

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The following are some of the protocols used by our team in this experiment. Comprehensive and detailed protocols can not only help our team obtain reliable and repeatable experimental results but also provide a reference for the experiments of subsequent iGEM teams. In this experiment, we mainly utilized homologous recombination to knock out certain genes on the bacterial genome and used different methods to introduce expression vectors.

Finally, we judged the experimental results based on Western Blot. We have tried our best to present the protocols, optimized according to the experimental results, in a comprehensive and clear manner. We sincerely hope that they can assist subsequent iGEM teams.

# Protocol

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Click here to show or hide Protocol

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# Plasmid Design & Construction

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Click here to show or hide Plasmids

We designed these plasmids to engineer our target bacteria. Including 3 plasmids for knockout *vfr*,
*exoS*, and *exoT*
to ensure the safety, pBAD33-VgrG3 plasmids for engineering *V. cholerae*,
and
pPSV37-PA0085(Hcp1), pPSV37-PA0093(Tse6) plasmids for engineering *P. aeruginasa*.

# PCR and generation homologous arms of *vfr*, *exoS*, *exoT*

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![](https://static.igem.wiki/teams/4739/wiki/result/1-3.jpg)

After getting the primers from a company, we used the DNA of PAO1 as a template and did PCR to get the
homologous arms we needed. Then, we used agarose gel electrophoresis to test the size of the product,
which should be ~1kb, to ensure that the product is correct. After that, we use the Nucleic Acid
Extraction and Purification kit to purify the DNA and test the concentration of the DNA.

# Knockout plasmid construction and verification

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![](https://static.igem.wiki/teams/4739/wiki/result/4-6.jpg)

We generated the plasmid we needed through the method of Gibson Assembly and transformed the plasmids
into T-Fast. Then we used PCR and agarose gel electrophoresis to verify that the plasmid was
successfully constructed (if successful, the size of the product should be ~2kb). The plasmids were then
transformed into WM6026. And later we tested whether the plasmids were successfully transformed using
antibiotic plates.

# Knockout

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![](https://static.igem.wiki/teams/4739/wiki/result/result7.png)

Figure 7 *vfr* knock out test

![](https://static.igem.wiki/teams/4739/wiki/result/result8.png)

Figure 8 *exoT* knock out test

![](https://static.igem.wiki/teams/4739/wiki/result/result9.png)

Figure 9 *exoS* knock out test

After the above process, we used homologous recombination to knock out the *exoS, exoT, vfr* genes in the
PAO1. Here we used agarose gel electrophoresis to test the size of the DNA and checked whether the toxic
genes (*exoS, exoT, vfr*) between the homologous arms were
knocked out. If they are knocked out, the size
should be 2 kb.

# Test of T6SS assembly of the toxic genes knocked out strains

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![](https://static.igem.wiki/teams/4739/wiki/result/result10.png)

Figure 10 toxic genes knocked out strains.

Then we fused Fluorescence molecules sfGFP to the *tssB1* and
used the microscope to test the T6SS activity
of the toxic genes knocked out strains.

# Expression plasmids construction

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![](https://static.igem.wiki/teams/4739/wiki/result/11-14.png)

Even though we intended to construct several expression plasmids, the small DNA fragments were difficult
to fuse with the vectors. Etelcalcetide seemed to have toxicity to the PAO1 since cell death was
observed after being mated with WM6026. At last, KLA, Somatotropin, Dulaglutide, and Dulaglutide genes
could be fused with *tse6*; Minihepcidin genes could be fused
with *hcp1*. For *vgrG3*, Dulaglutide, KLA,
Parkin and Tenecteplase genes can successfully fuse (we only showed the results of Parkin and
Tenecteplase since we directly sequenced others’ sequences).

# Western blot analysis of the expression and secretion of the fused proteins

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![](https://static.igem.wiki/teams/4739/wiki/result/result15.png)

Figure 15 cycle 0 Western blot analysis

![](https://static.igem.wiki/teams/4739/wiki/result/result16.png)

Figure 16 cycle 0 Western blot analysis

The expression and secretion of the fusion protein were detected by Western blot. In the first analysis,
Only Hcp1-Minihepcidin (~19 kDa) was successfully expressed and detected in the engineered strain PAO1
but was not successfully secreted. While for VgrG3 fused proteins (VgrG3-Dulaglutide 106 kDa, VgrG3-KLA
78 kDa, VgrG3-Parkin 128 kDa, VgrG3-Tenecteplase 135 kDa), all of them could be expressed. However, we
detected signals in the supernatant from T6SS negative strains, which suggested the inducing condition
might be inappropriate.

![](https://static.igem.wiki/teams/4739/wiki/result/result17.png)

Figure 17 cycle 1 Western blot analysis

The result indicates the RpoB antibody might be insufficient since the signal inside cells was low. The
detection of the signal in supernatant still suggests that the inducing condition was inappropriate.

![](https://static.igem.wiki/teams/4739/wiki/result/result18.png)

Figure 18 cycle 2 Western blot analysis

Compared with last cycle, the RpoB signal is indeed better. However, the Hcp fused proteins can only be
detected in cells. Even though the *Pseudomonas aeruginosa* lysed (since we detected the RpoB signal in
the supernatant), we still couldn't detect the signal of FLAG. This may suggest that *P. aeruginosa* may
be difficult to secrete fused protein. As for *Vibrio cholerae*, we found that our plasmids can be
expressed stably since we can detect the signals. However, the induce conditions were not good enough to
prevent all from cell lysis in the supernatant.

## Contents

* Plasmid Design & Construction
* PCR and generation homologous arms
* Knockout plasmid construction and verification
* Knockout
* Test of T6SS assembly of the toxic genes knocked
  out strains
* Expression plasmids
  construction
* Western blot analysis

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