iGEM 2020 — Team Vilnius-Lithuania

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# Team:Vilnius-Lithuania/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

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

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Vilnius-Lithuania/Engineering

![](https://static.igem.org/mediawiki/2020/e/e6/T--Vilnius-Lithuania--loader.gif)

1. # project
2. # wet lab
3. # dry lab
4. # human practices
5. # team

Engineering

RESEARCHIMAGINEBUILDTESTLEARNIMPROVEDESIGNENGINEERINGCYCLE

DETECTIONPREVENTIONTREATMENTCLICK TOEXPLORE

## overview

As a team, participating in a synthetic biology competition, we heavily relied on the **good engineering and scientific practices**. However, since synthetic biology is not as well established a field as civil or aerospace engineering
is, not only did we employ a so-called **“middle-out” approach**, but we also **tailored** the **engineering design cycle** to our own **unique** needs. Throughout the project we practiced **accurate** **measurement** of our progress wherever possible - from promoter activity to material integrity - carefully **documenting** our results.

expand all

click on iteration to see more

# Detection

# 1st iteration

# HDA

# 1st iteration

### research & imagine

1

After we discovered the need to empower farmers with the ability to swiftly detect an exact pathogen species, we set a **goal to create a rapid and specific detection kit**. Having these two properties in mind, we decided
to base our tool on isothermal DNA amplification - **HDA** and lateral flow assay - **LFA** to visualize the results.

HDA is an easy to use method meant to amplify short DNA sequences, while LFA, based on DNA-DNA hybridization, is a fast way to identify amplified sequences specific to a single pathogen species[1](#cit1).
We chose **HDA** because this amplification does not require expensive lab equipment or scientific background and due to this can be performed in rural farms.

The HDA reaction imitates a **natural replication process** where helicase is used to unwind DNA and allows amplification to occur at a stable temperature without the need for expensive thermocyclers. This method is
also easy to design since it requires only two primers compared to other isothermal amplification methods such as LAMP[2](#cit2).

### design

2

Our team designed this amplification in a way that the reaction is **asymmetric**, meaning that one primer is used in **an excess concentration**. This type of amplification generates ssDNA fragments which can be
immediately used for our lateral flow assay tests to identify which species of *Flavobacterium* are present.

### build & test

3

We performed HDA reaction according to the manufacturer's protocol. But we were not satisfied with the results since most of the time **no or very little amplicon** was seen in an electrophoresis gel. In order to have
a good working reaction mix, we needed to **optimize reaction conditions**.

### learn & improve

4

A fragment from ***F. psychrophilum*** genomic DNA was amplified via HDA without any modifications. However, ***F. columnare*** proved to be challenging to amplify, so we decided to try a few optimizations
approaches:

* Different pairs of primers;
* Optimization of reaction temperature;
* The quantity of gDNA/primers/MgSO4/NaCl/enzyme;
* Two-step HDA reaction;
* Genomic DNA restriction;
* Additives (DMSO, PEG4000, sorbitol, dextran, ficoll, betaine, formamide, pyrophosphatase).

# 2nd iteration

# 2nd iteration

### build & test

1

We performed amplification reactions using our chosen optimization methods. Based on the results seen in agarose gel, the most efficient additives were **DMSO and sorbitol**. The reason why these additives improved HDA
assay, is their ability to minimise secondary structures of DNA which improves primer annealing[3](#cit3). However, some of the results did not repeat through a series of experiments, so
further optimization should be performed to find the best HDA reaction conditions.

### learn & improve

2

Since no optimization was efficient enough to enhance the reaction, we concluded that primers used for *F. columnare* gene fragment amplification were designed using wrong parameters. To combat this issue, we decided
to **redesign primers**. Newly created primers were longer and had a **greater GC% content**, thus increasing their **melting temperature**. All parameters matched perfectly with the suggested parameters for
HDA primers design. After testing those primers, we saw that the reaction efficiency was still too low. Eventually, we concluded that a more sensitive method then electrophoresis should be used to determine the concentration
of amplicons such as qubit fluorometer or electrophoresis with a more sensitive dye than ethidium bromide.

# 1st iteration

# LFA

# 1st iteration

### research & imagine

1

After researching ways to create a point-of-use test, we decided to base our detection tool on lateral flow assay method[4](#cit4). The main reasons for this decision were that it requires **no prior training or scientific knowledge** to use and is rapid as well as robust. Thus we imagined that it would be the perfect tool for on-site detection. Keeping this in mind, our detection tool will help to **monitor bacterial infections** in aquaculture
farms as well as serve as a foundation for creating a **quantitative LFA** test to be used not only in farms but in the wild environment as well.

### design

2

LFA test is a universal method and with the **utilization of ssDNA** probes it can be designed to detect all kinds of DNA amplicons. It must not be forgotten, however, that a **high level of specificity** has to be
achieved for the test to function properly and to get as little false-positive or false-negative results, as possible.

To assure specificity we found **marker genes** for *F. columnare* and *F. psychrophilum*, which were 16S ribosomal RNA genes, and created detection ([BBa\_K3416101](http://parts.igem.org/Part%3ABBa_K3416101),
[BBa\_K3416107](http://parts.igem.org/Part%3ABBa_K3416107)), capture ([BBa\_K3416102](http://parts.igem.org/Part%3ABBa_K3416102), [BBa\_K3416108](http://parts.igem.org/Part%3ABBa_K3416108)) and
control ([BBa\_K3416103](http://parts.igem.org/Part%3ABBa_K3416103), [BBa\_K3416109](http://parts.igem.org/Part%3ABBa_K3416109)) probes for fragments of those marker genes[5](#cit5).

### build & test

3

Our team analyzed research papers and wrote all protocols as well as designed experiments necessary to create and test a lateral flow assay detection tool. For LFA to work, gold nanoparticles conjugated to the ssDNA detection
probes are crucial. To achieve this conjugation, functionalization reaction must be performed. But we ran into a problem and were **unable to functionalize gold nanoparticles** with ssDNA detection probes using NaCl
“aging” method[6](#cit6). After functionalization reaction gold nanoparticles turned deep blue or colorless. These color changes identify that nanoparticles aggregated during the process.

### learn & improve

4

By trying to make functionalization successful, we put a lot of efforts into **improving our protocol** like using a greater concentration of ssDNA detection probes. Nonetheless, it did not work, and no functionalization
occurred. After these unsuccessful experiments, we talked with **prof. A. Ramanavičienė** and learned about different methodologies for functionalization.

### build & test

5

One of the most promising methods, the professor suggested to us, is based on a **low pH environment** and is suitable for probes which have a poly-A sequence[7](#cit7). In pH 3 adenine becomes
protonated and two poly-A sequences can make parallel duplexes[8](#cit8). Such structures conjugate to gold nanoparticles more efficiently. Since our team created probes with poly-A sequences
we thought this method was worth trying. Besides that, we also decided to omit the sulfhydryl group activation and DTT extraction steps, because they can cause unwanted gold nanoparticles aggregation.

### learn & improve

6

After testing, we saw that the functionalization reaction using a **low pH method was [successful](https://2020.igem.org/Team%3AVilnius-Lithuania/Results)** because gold nanoparticles remained red and they did
not aggregate during NaCl test so we carried on with our experiments. Shortly after we ran into a second problem and saw that the test created for *F. psychrophilum* was **non-specific** and became positive
with *F. columnare* as well as *E. coli* DNA samples.

# 2nd iteration

# 2nd iteration

### research & imagine

1

If the test is non-specific then it can lead to false positive results which would make our test not suitable for *F. psychrophilum* identification. We needed to find a solution to improve specificity. Our team read various research papers and came to a conclusion that running buffers can affect the **performance of the test**[9](#cit9). However, if different optimizations with running buffers would not improve specificity a new marker gene for *F. psychrophilum* identification was found - ***rpoC* gene** (JX657167)[10](#cit10).

### design

2

Two strategies were designed. **First of all**, we wanted to see if buffers with SDS, SSC or formamide which are often used in lateral flow assay tests based on nucleic acid hybridization could improve specificity. **Second of all**,
new ssDNA probes were created for the chosen *rpoC* gene ([BBa\_K3416110](http://parts.igem.org/Part%3ABBa_K3416110), [BBa\_K3416111](http://parts.igem.org/Part%3ABBa_K3416111), [BBa\_K3416112](http://parts.igem.org/Part%3ABBa_K3416112)).
This time ssDNA probes had a greater GC% content and were longer.

### build & test

3

To check the first strategy we tested our lateral flow assay test strips using ultrapure water as well as phosphate buffer. Also two running buffers with different **SDS and SSC concentrations** and one buffer containing
**formamide** were chosen and tested. In addition, new LFA test strips using ssDNA probes created for *rpoC* gene were developed and tested with DNA amplicons from different bacteria species.

### learn & improve

4

Results showed that the overall performance of the test **was improved** by using Running buffer II which contained formamide but the specificity of the test **remained unchanged**, meaning that this strategy was unsuccessful.

However, after testing LFA test created using *rpoC* gene we saw that the **specificity improved**. The test was able to positively identify only *F. psychrophilum* DNA samples. Even though we had a properly working test for *F. psychrophilum* we ran into a third problem - the test created for *F. columnare* was also **not specific enough**.

# 3rd iteration

# 3rd iteration

### research & imagine

1

As mentioned, the test created to identify *F. columnare* 16S rRNA gene was non-specific. It correctly differentiated between *F. psychrophilum* and *F. columnare* but was unable to differentiate between *Flavobacterium* genus and *E. coli*. To combat this issue we researched more and found out that cslA gene can be used for *F. columnare* identification as well
[11](#cit11). We imagine that the LFA test created using cslA gene should be of greater specificity.

### design

2

We created ssDNA probes for hypothetical protein B0A56\_04620 (*Flavobacterium columnare*, NBRC 100251 = ATCC 23463) because we found it to be homologous to *cslA* protein sequence. These probes were also designed to have a **greater GC%** and **length** to improve hybridization reaction ([BBa\_K3416113](http://parts.igem.org/Part%3ABBa_K3416113), [BBa\_K3416114](http://parts.igem.org/Part%3ABBa_K3416114),
[BBa\_K3416115](http://parts.igem.org/Part%3ABBa_K3416115)).

### build & test

3

However, ssDNA probes were created only **in silico**. Due to the pandemic that affected the whole world our team was unable to test the specificity of the newly designed probes in the laboratory. For this reason we encourage **future iGEM teams** to test and if needed improve these lateral flow assay probes for *F. columnare*.

Since LFA requires a lot of optimization our software tool - [OnFlow](https://2020.igem.org/Team%3AVilnius-Lithuania/Software), based on a [mathematical model](https://2020.igem.org/Team%3AVilnius-Lithuania/Model), will be of immense help for future teams in deciding how to build and optimize their LFA tests. OnFlow will determine where to spray capture and control probes and the exact amount of analyte needed to obtain the most visible results on the strip. This software will help to save time and reagents.

### learn & improve

4

What we learned from developing lateral flow assay tests was that a small fragment of 16S rRNA gene is not enough for **accurate Flavobacterium species identification**. Even though it is routinely used for phylogenetic analysis[12](#cit12). Also, during the LFA development process, we gained new knowledge and skills that will be used in a further scientific career.

# 1st iteration

# Helimerase

# 1st iteration

### research & imagine

1

After we decided to base our test on nucleic acid hybridization via LFA method we started to search for a suitable isothermal ssDNA amplification method, which could be as sensitive and accurate as possible. When we came
upon **isothermal helicase dependent amplification (HDA)** method, we started to precisely plan our experiments. However, during the planning process, our team realized that the usage of commercial HDA kits rises
the price of the test.

During literature analysis we have found a significantly cheaper alternative for the HDA assay. The solution is a bifunctional protein complex called helimerase. Helimerase, according to literature, showed the ability of
amplifying significantly longer fragments during HDA reaction[13](#cit13).

Since helimerase complex could be formed in vivo and in vitro, it became crucial for us to find the best conditions of these proteins synthesis and purification. Firstly, based on the research1, we were decided to clone
TteUvrD and BstPol into different plasmids. However, after integrated meeting with dr. Giedrė Tamulaitienė, we decided to clone both genes into pETDuet-1 vector. This change of direction should help to minimise possible
failure risks as much as possible.

### design

2

The cloning design of the helimerase complex was based on pETDuet-1 vector, which is created for the co-expression of two target genes due to its multiple MCS.

### build & test

3

For the development of such plasmid we performed two steps cloning. During the first cloning, in the MCS-1, WZA2-L1-TteUvrD was fused with 10xHisTag and Maltose Binding Protein (MBP) in its N terminal. During the second
cloning WZB1-L1-BstPol was fused with StrepII tag. All these cloning steps were successful, however, during co-expression in E. coli BL21 (DE3) strain, only soluble TteUvrD protein was successfully synthesized.

### learn & improve

4

After result analysis we concluded that the reason why there was no BstPol synthesis could be related with the differences on its copy numbers. Even if pETDuet is low copy number plasmid, 15-20 copies per cell could be
too much to translate both recombinant proteins properly.

In order to tackle this issue we have decided to clone the BstPol gene into another plasmid, thus rejecting the idea to perform co-expression from one plasmid.

### redesign

5

As synthesis of TteUvrD was successful, we have decided to use the same construct for further experiments.

However, after we concluded to change the cloning strategy of BstPol, firstly we needed to find the most suitable plasmid for co-expression. For this purpose we had two main criteria – plasmid copy number must be lower
than 15-20 and it should have a compatible ori site with pETDuet. One such plasmid is pACYC, which copy number reaches only 10 copies, and its ori site is compatible with pETDuet[14](#cit14).

### build & test

6

After BstPol was cloned successful into pACYC plasmid, we performed its synthesis in E. coli BL21 (DE3) strain. Regarding to TteUvrD and BstPol high-solubility, we were able to purify high amounts of both proteins, so no further optimization for protein synthesis was needed. Nonetheless, protein co-expression and fusion in vivo by using these two compatible plasmids was still unsuccessful.

### learn & improve

7

Despite many attempts, further investigation on the recombinant proteins co-expression is needed. Cloning strategy could be improved by swapping TteuvrD and BstPol places in pETDuet or pACYC MCS. Also, different types of plasmids could be examined in order to find the best options for helimerase formation in vivo.

# Treatment

# 1st iteration

# AI-2 Synthesis

# 1st iteration

### research & imagine

1

By analyzing data, we came up that *Flavobacterium* form the biofilm, and as well as *E. coli* use the same **autoinducer 2 (AI-2)** molecule for quorum sensing. It is a universal „language“ for interspecies
communication in bacteria [15-17](#cit15). This AI-2 molecule or its precursor, 4,5-dihydroxy-2,3-pentanedione (DPD), can be used for manipulating genetic circuits[18](#cit18),
but pure AI-2 is costly ( 1mg for 175$). The research goal was to find how we can **synthesize it *in vitro*** and use this AI-2 molecule for the following experiments[19-20](#cit19).
To reach this goal, we decided to make AI-2 ourselves by using its precursor S-adenosyl-homocysteine (SAH) and two recombinant proteins - LuxS and Pfs (Fig. 1).

![](https://static.igem.org/mediawiki/2020/a/ab/T--Vilnius-Lithuania--eng-treat1.png)

**Figure 1.**  AI-2 synthesis from the precursor S-adenosylhomocysteine (SAH)

### design

2

**LuxS** and **Pfs genes** were cloned into pET28a(+) vector and synthesized in *E. coli* BL21 (DE3) strain. Both proteins were purified successfully by using a Ni-NTA column (GE Healthcare Bio-Sciences, Upsala,
Sweden).

### build & test

3

The purified proteins were used for the reaction mixture according to Wattanavanitchakorn et. al. 2014[19](#cit19). The synthesized DPD, a precursor of AI-2, was identified with **Ellman’s reagent** (5,5'-dithiobis-(2-nitrobenzoic acid). This chemical compound is used to quantify the concentration of thiol groups in sample[21](#cit21) (Fig. 2).

![](https://static.igem.org/mediawiki/2020/a/a9/T--Vilnius-Lithuania--eng-treat3.png)

**Figure 2.**  The general scheme of Ellman’s reagent reaction with compounds that have a thiol group. The thiol group cleaves the disulfide bond and the cleaved 2-nitro-5-thiobenzoate (TNB−) ionizes to the TNB2−. This
ion gives a yellow color to a solution.

### learn & improve

4

However, due to the small amount of the DPD we were not able to use this reagent in further experiments. These results led us to think that **SAH quantity was too low** for this reaction. To increase DPD concentration,
we decided to change proteins’ concentrations and the amount of SAH.

# 2nd iteration

# 2nd iteration

### research & imagine

1

After the first trial, we decided to check what else can be done. We have found out that proteins into the mixture should be added one by one. This makes the measurement of the kinetic parameters of each protein much more
**precise**.

### design

2

The Pfs and LuxS protein concentrations’ values and SAH mass were changed.

### build & test

3

The first reaction mixture consisted of everything despite the LuxS. One hour later, LuxS was added. This testing helped to **improve the concentration of DPD** (Fig. 3), an AI-2 precursor, and gather more information
about the enzymatic activity of proteins.

![](https://static.igem.org/mediawiki/2020/0/07/T--Vilnius-Lithuania--eng-treat5.png)

**Figure 3.**  The spectrum after the reaction with Ellman’s reagent. The peak at 412nm identifies dianion TNB2−. The following calculations for determining the AI-2 concentration were done as a [protocol](https://static.igem.org/mediawiki/2020/f/f7/T--Vilnius-Lithuania--LabProtocolsSafety.pdf) requires.

### learn & improve

4

Our experience with Pfs and LuxS proteins showed that every iGEM team that is willing to link their project with a quorum sensing type II system, can easily and cheaply synthesize AI-2 by themselves.

# 1st iteration

# Exolysins

# 1st iteration

### research & imagine

1

**Bacteriophages** (phages) are bacterial viruses that can only infect bacteria and are the most numerous biological entities on our planet[22](#cit22). Bacteriophages use **exolytic proteins** to penetrate the bacteria capsule[23](#cit23). The scientific literature showed that we can use only phage proteins[24](#cit24). We decided to use only phage’s
exolytic protein because it still fulfills its function. Thus, the main goal was to find *Flavobacterium* phage proteins - **exolysins**. This exolysin would be a part of the genetic circuit that will be induced
by AI-2.

### design

2

When the design phase started, we found out that marine phages (e.g. *Synechococcus* phage) are not as well characterized or do not have representative genomes as mostly known non-marine phages([EBI phage database](https://www.ebi.ac.uk/genomes/phage.html))
[25](#cit25). This lack of information would lead to problematic experiments.

### research & imagine

3

The project seeks to solve the problems that are caused by *Flavobacterium* spp. and we had been considering using phages for getting rid of pathogenic bacteria. The bacteriophages have a broad range of specific bacteria
lysing proteins, called exolysins. We have determined to find those proteins but the research has shown that there were no homologous ***Flavobacterium* exolysins** when the ORFs were compared. Then we were
looking for alternatives and consulting with our PI prof. Rolandas Meškys. Then, it was decided to choose the *Klebsiella pneumoniae* KV-3 phages due to easy accessibility.

### design

4

The exolysin is the main part of the whole gene circuit construction (Fig. 4), which is initiated by AI-2 induced *lsrACDBFG*([BBa\_K3416000](http://parts.igem.org/Part%3ABBa_K3416000)) promoter. We had a variety
of *Klebsiella pneumoniae* KV-3 phage depolymerases to choose from. We could have settled to construct all genetic circuits with these depolymerases, but to save time we needed to examine the enzymatic activity
of these proteins.

![](https://static.igem.org/mediawiki/2020/6/6c/T--Vilnius-Lithuania--eng-treat2.png)

**Figure 4.**  The general genetic circuit of exolysin and endolysin is based on quorum sensing Type II molecule AI-2 inducibility

### build & test

5

After enzymatic analysis, the exolysins called gp529([BBa\_K3416029](http://parts.igem.org/Part%3ABBa_K3416029)) and gp531([BBa\_K3416031](http://parts.igem.org/Part%3ABBa_K3416031)) have been chosen for
further treatment strategy development. After proteins’ purification, the enzymatic activity was checked with
*Klebsiella pneumoniae* KV-3 biofilms with the spot test. However, the gp529 showed no depolymerase activity, whereas gp531 was successfully purified as an active form.

### learn & improve

6

To successfully destroy pathogenic bacteria biofilm we need an exolysin with good enzymatic activity. The gp531, due to the most efficient enzymatic activity, was chosen to be used for the following experiments.

# 2nd iteration

# 2nd iteration

### research & imagine

1

The following task was to make the genes from a genetic circuit compatible that could be used for other iGEM projects. The sequence should be compatible with the most-used assemblies in the iGEM library. After deeper analysis,
it was seen that the gp531 native sequence has many repetitive restriction sites. This would have aggravated the construction’s tasks. Thankfully, gp533 exolysin has depolymerase activity and no repetitive restriction
sites.

### design

2

The gp533 exolysin was used for a genetic circuit, that is enhanced by AI-2, construction.

### build & test

3

The genetic circuit construction took many attempts to accomplish. Due to the pandemic, it took a lot of time to obtain the exolysins genes and the selection of the most suitable depolymerase took too long to engineer the
genetic circuit for our project.

### learn & improve

4

Despite these problems, the exolytic protein showed that it is capable of lysing the pathogenic bacteria and could be used for other teams focusing on alternative strategies of pathogenic disease treatment.

# 1st iteration

# toxin-antitoxin

# 1st iteration

### research & imagine

1

The treatment section development arose on how we can ensure that genetically modified bacteria could initiate its lysis. The research has shown that there are commonly used so-called **kill-switches** that ensure that
genetically modified organisms won’t be able to transfer their genetic material. To add more, iGEM the main priority is to guarantee that GMOs are safe for usage, so this was an encouragement for us to create another
treatment system that includes a **classic kill-switch** **- MazF and MazE**[26,27](#cit26).

The iGEM parts library contains MazE and MazF protein-coding sequences but previous teams had uploaded *mazF* and *mazE* genes of *Bacillus subtilis*. After checking these sequences with BLAST, it showed
that these sequences code the same genes but of *E. coli*, not for *B. subtilis*. Moreover, we decided to compare both treatment genetic circuits and found out the differences between these systems. This might
help to establish a better view of how efficient the system is.

![](https://static.igem.org/mediawiki/2020/0/0a/T--Vilnius-Lithuania--eng-treat4.png)

**Figure 5.**  The genetic circuit of exolysin and mazEF system

### design

2

Same as before, exolysin is the main part of this construction. Under AI-2 inducible promoter, *mazF* is expressed. The difference is that genetic circuit accompany Anderson promoter and *mazE* gene. Before picking
the Anderson promoter of the right strength, we needed to **compare these promoters** by measuring the fluorescence intensity and OD600.

### build & test

3

As mentioned in the Design Page we are measuring AI-2 promoters of different strengths. We used Anderson promoters as a standard for our inducible promoter measurement. For the measurements these circuits were constructed:
*lsrACDBF-sfGFP*, EP14r-*sfGFP*, EP01r-*sfGFP*, *J23117-sfGFP*, *J23110-sfGFP, J23104-sfGFP*.

To gain extensive insights about what concentration of AI-2 is best for inducing gene expression in vitro synthesized AI-2 was used for inducing the genetic circuit. The goal is to **determine the strengths of AI-2 inducible promoters**and the concentration threshold of AI-2.

### learn & improve

4

The first trials gave a clearer view of what Anderson promoter we should use. The results indicate that *srACDBFG*([BBa\_K3416000](http://parts.igem.org/Part%3ABBa_K3416000)) and its mutants EP01r([BBa\_K3416001](http://parts.igem.org/Part%3ABBa_K3416001)),
EP14r(
[BBa\_K3416014](http://parts.igem.org/Part%3ABBa_K3416014)) are quite weak promoters. Furthermore, as in results are described, the AI-2 concentrations were determined. We decided to **narrow the window of concentrations’ range**.
The following experiments took place effortlessly.

# 2nd iteration

# 2nd iteration

### build & test

1

After the first iteration, we set a goal for constructing an inducible kill-switch. Everything was working according to plan. After successfully constructing the genetic circuit the measurements were left to be done. We
had difficulties with the accessibility of devices, so it made it pretty difficult for obtaining the right conditions and repetitively of experiments. All in all, the results say everything that you need to know about
this **toxin-antitoxin system** and how it works with the quorum sensing Type II system.

### learn & improve

2

We hope that genetic circuits that contain AI-2 inducible promoters or having a better understanding of the toxin-antitoxin system will help other iGEM teams to develop even more sophisticated models for manipulating gene
expression.

# Prevention

# 1st iteration

# VHSV

# 1st iteration

### research & imagine

1

To induce an immune response against **Viral Hemorrhagic Septicaemia Virus (VHSV)** in fish by using a subunit vaccine, we had to select an appropriate protein. **Glycoprotein G** from VHSV was shown to be recognized
by fish immune cells[28](#cit28). However, it was also shown that if the protein is expressed in *E. coli*, it does not demonstrate any immunogenic properties, because the proper folding
of a glycoprotein is not guaranteed due to absence of glycosylation processes[29](#cit29). Due to this reason, the protein should be synthesised in yeast recombinant protein expression systems,
where the glycosylation processes occur. After deeper research, we decided to use one of the most common yeast systems, ***S. cerevisiae***.

### design

2

VHSV glycoprotein G coding sequence was fused with a 6xHis purification tag sequence from the N terminus into a **pfX7 vector**, as it is usually used for protein expression in recombinant protein synthesis systems
[30](#cit30). As usual, firstly we decided to perform cloning in *E. coli* DH10B strain. Ligate was first cloned into and purified from *E. coli* DH10B strain. Then transformed
and expressed in *S. cerevisiae* AH22-214 strain.

### build & test

3

After a lot of attempts of cloning there were **no colonies of DH10B cells with the transformed pfX7 VHSV 6His N construct** in a plate. We started to troubleshoot these results.

### learn & improve

4

After numerous unsuccessful attempts to clone the VHSV gene into pfX7 plasmid we started to search for reasons of these failures. Since we obtained this plasmid from VU BTI EGI department, we decided to have a consultation
with Juta Rainytė, who works with this system. After consulting with [Juta Rainytė](https://2020.igem.org/Team%3AVilnius-Lithuania/Human_Practices) we got acquainted that one of the main traits this plasmid
has is its **leaky promoter**. Due to this, there is a possibility that
*E. coli* lyses itself due to the toxicity of the expressed proteins. The ligate could be transformed into a bacterial strain, that would be tailored for toxic protein expression.

# 2nd iteration

# 2nd iteration

### research & imagine

1

After research we found out that *E. coli* **ArcticExpress** strain (DE3), is used to improve bacterial protein expression and solubility[31](#cit31). This strain co-expresses chaperonins
Cpn10 and Cpn60, that optimizes the protein folding process. In addition, it naturally lacks the Lon protease, which can degrade recombinant proteins. This strain could potentially handle the protein that might be toxic
to other strains and help multiply our plasmid for further experiments. Due to this we decided to perform cloning in this strain.

### design

2

To check if the cloning procedure was unsuccessful due to the leakyness of promoter and VHSV toxicity, the next engineering step was to transform VHSV gene containing plasmid into *E. coli* pfX7 plasmid and transforming
it into *E. coli* **ArcticExpress** (DE3) strain. Because this strain possesses an additional plasmid for chaperonin co-expression, it needed to be separated from our plasmid of interest.

### build & test

3

After we made all the required experiments we finally saw that the VHSV cloning step was successful.

### learn & improve

4

Successful cloning procedures in ArcticExpress (DE3) strain led us to the conclusion that the expressed protein due to the leaky promoter **was indeed toxic to DH10B strain**. Now the resulting plasmid could be cloned
into *S. cerevisiae*.

# 3rd iteration

# 3rd iteration

### research & imagine

1

Even though it was shown that some *E. coli* strains do perform **glycosylation**, the process is not well studied nor guaranteed[29](#cit29). Yeast, on the other hand, are more complex
and are able to synthesize properly folded glycoproteins[32](#cit32). Therefore we can use them for VHSV Glycoprotein G production.

### design

2

The protein can be synthesised in different *S. cerevisiae* strains and purified via Ni-NTA column (GE Healthcare Bio-Sciences, Upsala, Sweden), which is used for purification of recombinant proteins with 6xHis
tag. The pH and contents of buffers for purification can be adjusted according to purification results.

### build & test

3

The results of SDS-PAGE electrophoresis indicate that the protein of interest was not synthesised, yet an unknown protein half the size was purified.

### learn & improve

4

The protein synthesis and purification conditions can be improved by:

* Constructing a plasmid with a 6xHis tag on the C terminus of the coding sequence. It might help if 6xHis tag on N terminus is hindered and inaccessible when it is being purified.
* Fusing the glycoprotein G coding sequence with Maltose-Binding protein (MBP) and Tobacco Etch Virus (TEV) Protease coding sequences. MBP tag should help with solubility of the protein and TEV protease site is used
  to remove MBP in that case if this tag is hindering or inhibiting immune response
* Choosing other *S. cerevisiae* strains
* Trying ***Pichia pastoris*** for protein expression. This idea was proposed by Dr. Rasa Burneikiene-Petraityte, gene engineering expert, by stating that *Pichia pastoris* is glycosylating less and with
  more accuracy than *S. cerevisiae*. However, this strategy would require an extensive amount of time to implement, which we unfortunately did not have in our disposal. It could be used in the further developments
  of the project.
* Changing the pH of the buffers used for the lysis of yeast and protein purification. By trying and comparing different buffers such as HEPES, Tris-HCl, PBS and changing pH we can determine the most optimal conditions
  for the protein environment where it would be most soluble and stable.
* Balancing the ionic strength of the protein purification buffers. This might help to achieve higher purity of the specific protein by interacting with the proteins. Only the ones with high affinity to the column
  can bind to the resin, whereas, protein with less affinity to the column will be washed away. Also, ionic strength might help with the solubility when ultrasound lysis occurs by interacting with protein’s amino
  acids and hiding hydrophobic regions.

# 1st iteration

# Gldj

# 1st iteration

### research & imagine

1

Since our project is mainly focused on *Flavobacteria* caused diseases, we paid a lot of attention for the creation of an oral subunit vaccine **against columnariosis** disease, caused by *Flavobacterium columnare*.
We found that this bacteria contains **Gliding motility protein (GldJ)**, which is located on the surface of *F. columnare*[33](#cit33). Previous research showed that this protein
could induce an immune response in fish since GldJ has well-exposed epitopes and is recognized by fish immune cells[34](#cit34).

### design

2

We decided to clone the GldJ coding sequence into **pET28a(+) vector**, as it is commonly used for recombinant protein high-level expression. Due to huge availability to *E. coli* strains we have chosen to optimize
induction conditions. We decided to try three different strains for their intrinsic properties of protein expression:

* BL21 (DE3) strain allows high-efficiency protein expression.
* Rosetta (DE3) contains rare tRNAs that might be needed for our target protein.
* ArcticExpress (DE3) has two heat shock proteins which should help with protein folding.

### build & test

3

After SDS-page gel analysis we saw that protein was induced but there were 2 protein bands that were **not the size** we expected. One of the induced protein sizes was bigger by 5-10 kDa, while the other was smaller
by 5-10 kDa. We concluded that most of the protein was in the insoluble fraction, that is why there was no protein detected after purification with a Ni-NTA column (GE Healthcare Bio-Sciences, Upsala, Sweden). Induction
worked best in ArcticExpress strain but the **protein was mostly in the precipitant**.

### learn & improve

4

When we got unexpected protein bands, it was decided to check whether the gene has any mutations. Sanger **sequencing** revealed that there were none. We thought that it would be great to purify at least a little bit
of the desired protein if protein solubility would increase.

# 2nd iteration

# 2nd iteration

### research & imagine

1

After carrying out literature analysis we found that **trehalose** can enhance the protein solubility by stabilizing it. Trehalose serves as an osmolyte and prevents protein aggregation. It was hypothesized that it might
contribute as a chemical chaperone, that is shown after inspecting change protein folding rates[35](#cit35).

### design

2

It was decided to add **trehalose** into a resuspension buffer which is used for biomass dispersion before ultrasound lysis.

### build & test

3

SDS-page showed that there are more proteins in soluble fraction when trehalose was added. Unfortunately, when we performed Western blot analysis after protein purification, there were **no detectable proteins** in our
elution samples.

### learn & improve

4

Next steps could be:

* Utilizing sonification (resuspension) buffer.
* Trying **other *E. coli* strains**. For example: Hms174(DE3) - an alternative to BL21(DE3); Origami, which employs chaperones; Nova Blue (DE3) intended for toxic genes (we speculate, that maybe the correctly
  folded protein is toxic for bacteria).
* Cloning the gene into other vectors. Changing the vector with a weaker promoter (not T7), could improve protein folding, since in pET28a(+) it could happen too fast.
* Changing and evaluating results with various inductor (IPTG) concentrations.
* Different tag such as MBP should solve the solubility problem.
* Purifying protein under denaturing conditions (with urea or guanidine hydrochloride) might also help.

# 1st iteration

# alginate beads

# 1st iteration

### research & imagine

1

If the immunogenic protein is administered orally, it has to reach the gut of the fish to induce an **effective immune response**[36](#cit36). In order to achieve this, the proteins have to
pass the stomach without any degradation. Scientists have demonstrated that calcium alginate was not digested by stomach enzymes and could only be **degraded with alginate lyase** produced by specific bacteria, for
example *Klebsiella pneumoniae* or *Pseudomonas maltophilia*[37](#cit36). Such kind of bacteria could be found in the midgut of the fish, thus making a perfect place for alginate
degradation. This feature of alginate degradation could be exploited by enveloping the immunogenic proteins in it.

### design

2

The immunogenic protein GLDJ was mixed with alginate and dropped into a calcium chloride bath to form a bead[38](#cit37).

### build & test

3

Expressed proteins were purified by using a Ni-NTA column (GE Healthcare Bio-Sciences, Upsala, Sweden), which was used for recombinant proteins with 6xHis tag purification, and enveloped in calcium alginate. In order to
see whether proteins stay stable after the encapsulation process, the beads were examined in vitro. Therefore, a droplet of digestive enzyme (**trypsin/pepsin**) or a buffer of an appropriate **pH** was applied
directly onto the bead and incubated for up to 2 hours. The resulting changes in the properties of color and solidity of the bead were observed.

### learn & improve

4

Alginate can effectively **protect from the stomach acidic environment** and should protect from alkaline pH. It is important to know if the immunogenic proteins (GLDJ) are spread throughout all the beads in a uniform
manner. This ensures **equal distribution** of the immunogens in every bead. However, it was hard to determine that from the assay. Moreover, the changes after treatment with enzymes and buffers are too minimal to
draw substantial conclusions about the integrity of the beads. In order to improve the tests of protein distribution and digestion, we decided to first optimise it with a visually observable protein. After these steps,
beads production method can be applied to the actual immunogenic proteins.

# 2nd iteration

# 2nd iteration

### research & imagine

1

Green fluorescent protein (**GFP**) is a commonly used protein for visualization of processes that are hardly visible. GFP could be encapsulated in calcium alginate and observed to determine the uniformity of the protein
throughout the bead and its changes after the application of enzymes or buffers. The conditions of the encapsulation process could then be adjusted with the control beads and applied when making vaccines with immunogenic
proteins.

### design

2

GFP coding sequence cloned into a pET28a(+) vector was expressed in *E. coli* BL21(DE3) strain. After protein expression and purification, the **protein was mixed with alginate** and dropped into a calcium chloride
bath to form a bead.

### build & test

3

Expressed GFP protein was purified by using a Ni-NTA column (GE Healthcare Bio-Sciences, Upsala, Sweden) and enveloped in calcium alginate. The uniformity of **protein distribution** in different beads can be easily
assessed. GFP beads can be used to easily determine if an alginate bead would be degraded by digestive enzymes over time. The changes can be seen in mere seconds with the naked eye.

### learn & improve

4

The results of the tests had shown that the beads **did not degrade** in the presence of **digestive enzymes**. Yet, it was crucial to determine if they would release the protein in the midgut of the fish.

# 3rd iteration

# 3rd iteration

### research & imagine

1

In order to determine this, alginate lyase should be present in the environment of the bead. Bacteria species that have this enzyme are *Bacillus circulans, Klebsiella aerogenes, K. pneumoniae, Pseudomonas maltophilia, P. putida* and *P. aeruginosa*. We wanted to use *Pseudomonas sp*., since it is able to synthesize alginate lyase and it lives in the midgut of rainbow trout[37](#cit37). However, due to the
pandemic and this bacteria pathogenicity for humans, we could not acquire it for the testing. Fortunately we found that ***Klebsiella pneumonia* KV-3**, which was used in other parts of our [project](https://2020.igem.org/Team%3AVilnius-Lithuania/Design),
is also able to synthesise the lyase
[39](#cit39). Hence, it could be used to validate if the alginate beads do, in fact, degrade in the presence of this enzyme.

### design

2

An exact number of uniform sized alginate beads with GFP were dropped into a flask with *Klebsiella pneumonia* KV-3 and grown for 3 days. We dropped the same amount of beads into a flask with *E. coli* BL21 (DE3)
strain as a control. Also, we used alginate beads that lack the protein which was also used as described above with GFP alginate beads.

### build & test

3

After 3 days, the experiment showed that our *Klebsiella pneumonia* KV-3 strain really **does have alginate lyase** because all beads (with and without GFP) were **decomposed** while the ones in *E. coli* flask were still intact. The only question was why did it take exactly 3 days?

### learn & improve

4

This might be due to **biofilm** forming after several days when the rpm is low. Also, the medium might have a big role here. This might be due to catabolite repression mechanism where bacteria prioritize metabolising
most nutritious substances (the ones which give the most energy) inside the LB medium and only after they are malnourished then bacteria begin secreting alginate lyase or other proteins into the environment. In order
to prove our hypothesis, further experiments would be:

* Trying **different temperatures**. Not applying optimal conditions might have an influence on protein expression.
* Testing if the bead maintains its integrity while being spun at a higher rate.
* Trying different **medium** from something minimal like M9 to enriched, such as blood medium or chocolate-blood medium:
  + M9 would be beneficial, because it has very low autofluorescence and we could use plate reader assay to determine when the GPF in the beads dissolve.
  + On the other hand, Blood medium is suitable for pathogenic bacteria (our *Klebsiella pneumonia* KV-3 is pathogenic as it was cultivated from veterinary isolate). This is why the protein expression could
    be different than in LB medium.

# 4th iteration

# 4th iteration

### research & imagine

1

The tests confirmed that calcium alginate gets degraded in the presence of alginate lyase. It was proved earlier that the beads do not degrade when trypsin and pepsin separately are present in their optimum environment.
The last challenge that the beads need to **withstand** while traveling through the fish digestive tract is the **physical pressure** from the peristaltic movements[40](#cit40). According
to research
[41](#cit41), the pressure that the bead should withstand to reach the midgut should be between 0,13-0,32MPa. In accordance to this, the beads should be tested to see if they are strong enough.

### design

2

To ensure the reliability of the testing results, beads of a uniform size were selected. They were **compressed** using a universal testing machine (TIRA test 2300) until rupture.

### build & test

3

The results of the tests indicated that the beads could only withstand a pressure a few times lower than recommended. Also it was clear that the presence of the protein in the bead increased the integrity of it around 20%.

### learn & improve

4

These steps could be taken in order to make the alginate beads stronger and more implementable:

* The beads could be kept in CaCl2 solution after production for a few days, because alginate tends to gradually harden overtime when exposed to CaCl2[42](#cit35).
* The size of the bead could be varied between 3mm to 5mm to see which one is the strongest. This should be done keeping in mind the change of protein concentration in a single bead, to ensure the correct dosage of vaccines
  for immunization.
* The alginate solution **concentration** could be **increased**.
* The concentration of the protein in an alginate-protein mixture could be increased, as it tends to increase the integrity of the bead as observed earlier.

#

#

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2. Vincent, M., Xu, Y. & Kong, H. Helicase‐dependent isothermal DNA amplification. EMBO Rep 5, 795–800 (2004).
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11. Mabrok, M. et al. Development of a species-specific polymerase chain reaction for highly sensitive detection of Flavobacterium columnare targeting chondroitin AC lyase gene. Aquaculture 521, 734597 (2020).
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13. Motré, A., Li, Y. & Kong, H. Enhancing helicase-dependent amplification by fusing the helicase with the DNA polymerase. Gene 420, 17–22 (2008).
14. Morgan, K. Plasmids 101: Origin of Replication. addgene blog share science Available at: https://blog.addgene.org/plasmid-101-origin-of-replication. (Accessed: 27th October 2020)
15. Federle, M. J. Autoinducer-2-Based Chemical Communication in Bacteria: Complexities of Interspecies Signaling. in Contributions to Microbiology (eds. Collin, M. & Schuch, R.) vol. 16 18–32 (KARGER, 2009).
16. Pereira, C. S., Thompson, J. A. & Xavier, K. B. AI-2-mediated signalling in bacteria. FEMS Microbiol Rev 37, 156–181 (2013).
17. Sun, J., Daniel, R., Wagner-Döbler, I. & Zeng, A.-P. Is autoinducer-2 a universal signal for interspecies communication: a comparative genomic and phylogenetic analysis of the synthesis and signal transduction pathways. BMC
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18. Stephens, K. & Bentley, W. E. Synthetic Biology for Manipulating Quorum Sensing in Microbial Consortia. Trends in Microbiology 28, 633–643 (2020).
19. Wattanavanitchakorn, S., Prakitchaiwattana, C. & Thamyongkit, P. Rapid and simple colorimetric method for the quantification of AI-2 produced from Salmonella Typhimurium. Journal of Microbiological Methods 99, 15–21 (2014).
20. Ascenso, O. S. et al. An efficient synthesis of the precursor of AI-2, the signalling molecule for inter-species quorum sensing. Bioorganic & Medicinal Chemistry 19, 1236–1241 (2011).
21. Ellman, George L. "Tissue sulfhydryl groups." Archives of biochemistry and biophysics 82.1 (1959): 70-77.
22. Hendrix, R. W. Bacteriophages: Evolution of the Majority. Theoretical Population Biology 61, 471–480 (2002).
23. Latka, A., Maciejewska, B., Majkowska-Skrobek, G., Briers, Y. & Drulis-Kawa, Z. Bacteriophage-encoded virion-associated enzymes to overcome the carbohydrate barriers during the infection process. Appl Microbiol Biotechnol 101,
    3103–3119 (2017).
24. Pires, D. P., Oliveira, H., Melo, L. D. R., Sillankorva, S. & Azeredo, J. Bacteriophage-encoded depolymerases: their diversity and biotechnological applications. Appl Microbiol Biotechnol 100, 2141–2151 (2016).
25. Perez Sepulveda, B. et al. Marine phage genomics: the tip of the iceberg. FEMS Microbiology Letters 363, fnw158 (2016).
26. Cho, J., Carr, A. N., Whitworth, L., Johnson, B. & Wilson, K. S. MazEF toxin-antitoxin proteins alter Escherichia coli cell morphology and infrastructure during persister formation and regrowth. Microbiology 163, 308–321 (2017).
27. Nigam, A., Ziv, T., Oron-Gottesman, A. & Engelberg-Kulka, H. Stress-Induced MazF-Mediated Proteins in Escherichia coli. mBio 10, e00340-19, /mbio/10/2/mBio.00340-19.atom (2019).
28. Shin, C., Kang, Y., Kim, H.-S., Shin, Y. K. & Ko, K. Immune response of heterologous recombinant antigenic protein of viral hemorrhagic septicemia virus (VHSV) in mice. Anim Cells Syst (Seoul) 23, 97–105 (2019)
29. Upreti, R. K., Kumar, M. & Shankar, V. Bacterial glycoproteins: Functions, biosynthesis and applications. PROTEOMICS 3, 363–379 (2003).
30. Sasnauskas, K. et al. Yeast Cells Allow High-Level Expression and Formation of Polyomavirus-Like Particles. Biological Chemistry 380, 381-386 (1999).
31. Agilent Technologies. ArcticExpressTM competent cells. Arct. Express Instr. Man. 1–17 (2010).
32. Upreti, R. K. & Kumar, M. Review Bacterial glycoproteins : Functions, biosynthesis. Proteomics 363–379 (2003).
33. Dumpala, P. R., Gülsoy, N., Lawrence, M. L. & Karsi, A. Proteomic analysis of the fish pathogen Flavobacterium columnare. Proteome Sci 8, 26 (2010).
34. Luo, Z. et al. Immunogenic proteins and their vaccine development potential evaluation in outer membrane proteins (OMPs) of Flavobacterium columnare. Aquaculture and Fisheries 1, 1–8 (2016).
35. Leibly, D. J. et al. Stabilizing Additives Added during Cell Lysis Aid in the Solubilization of Recombinant Proteins. PLoS ONE 7, e52482 (2012).
36. Kwon, K.-C., Lamb, A., Fox, D. & Porphy Jegathese, S. J. An evaluation of microalgae as a recombinant protein oral delivery platform for fish using green fluorescent protein (GFP). Fish & Shellfish Immunology 87, 414–420 (2019).
37. Tavafi, H., Abdi- Ali, A. A., Ghadam, P. & Gharavi, S. Screening of Alginate Lyase-Producing Bacteria and Optimization of Media Compositions for Extracellular Alginate Lyase Production. Iran Biomed J 21, 48–56 (2017).
38. Lee, K. Y. & Mooney, D. J. Alginate: properties and biomedical applications. Prog Polym Sci 37, 106–126 (2012).
39. Egerton, S., Culloty, S., Whooley, J., Stanton, C. & Ross, R. P. The Gut Microbiota of Marine Fish. Front. Microbiol. 9, (2018).
40. Olsson, C. & Holmgren, S. The control of gut motility. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 128, 479-501 (2001).
41. Maurice, S., Nussinovitch, A., Jaffe, N., Shoseyov, O. & Gertler, A. Oral immunization of Carassius auratus with modified recombinant A-layer proteins entrapped in alginate beads. Vaccine 23, 450–459 (2004).
42. Lotfipour, F., Mirzaeei, S. & Maghsoodi, M. Evaluation of the effect of cacl2 and alginate concentrations and hardening time on the characteristics of Lactobacillus acidophilus loaded alginate beads using response surface analysis.
    Advanced Pharmaceutical Bulletin 2, 71–78 (2012).

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## human practices

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# Team:Vilnius-Lithuania/Experiments

1. # project
2. # wet lab
3. # dry lab
4. # human practices
5. # team

Experiments

Experiments

Calendar

Lab Weekly Calendar

Protocols and Safety

PDF

Benchling

Detection

Treatment

Prevention

Weekly Summary

Detection

Treatment

Prevention

March 30 - April 5

Since its inception iGEM has worked to ensure that excellence in synthetic biology goes beyond what happens in the lab. Decisions in science and engineering shape, and are shaped by, the societies we create. Social, political, economic, and ethical aspects of synthetic biology cannot be an afterthought of research and development. Rather, they should be considered from project conception all the way through the innovation process.

Synthetic biology is not just science or engineering. It is a practice of building life and the societies and environments that support that life. It is a practice that is intimately human, with all our flaws and virtues. This approach is embedded in the values we espouse, including: integrity; good sportsmanship; respect; honesty; celebration; cooperation; effort; and excellence.

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## wet lab

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* [engineering](https://2020.igem.org/Team%3AVilnius-Lithuania/Engineering)
* [parts](https://2020.igem.org/Team%3AVilnius-Lithuania/Parts)
* [safety](https://2020.igem.org/Team%3AVilnius-Lithuania/Safety)

## dry lab

* [model](https://2020.igem.org/Team%3AVilnius-Lithuania/Model)
* [software](https://2020.igem.org/Team%3AVilnius-Lithuania/Software)
* [hardware](https://2020.igem.org/Team%3AVilnius-Lithuania/Hardware)
* [measurement](https://2020.igem.org/Team%3AVilnius-Lithuania/Measurement)

## human practices

* [integrated human practices](https://2020.igem.org/Team%3AVilnius-Lithuania/Human_Practices)
* [education & public engagement](https://2020.igem.org/Team%3AVilnius-Lithuania/Education)
* [the 6th SynBio Sense](https://2020.igem.org/Team%3AVilnius-Lithuania/The_6th_SynBio_Sense)

## team

* [members](https://2020.igem.org/Team%3AVilnius-Lithuania/Team)
* [collaborations](https://2020.igem.org/Team%3AVilnius-Lithuania/Collaborations)* [attributions](https://2020.igem.org/Team%3AVilnius-Lithuania/Attributions)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Vilnius-Lithuania/Results

1. # project
2. # wet lab
3. # dry lab
4. # human practices
5. # team

Results

Results

Detection

Overview

**Goal** - To create an LFA test that can be used for F. columnare and F. psychrophilum species specific HDA amplicon identification in the farm. As well as to enhance HDA efficiency by using a binary helimerase complex.

**Results** - The test created for F. psychrophilum worked as we expected and was able to positively identify only F. psychrophilum species, whereas specificity of F. columnare test was lower and potentially could lead to false positive results. Proteins, synthesized for helimerase showed activity, however they did not form electrostatic interactions in vivo or in vitro.

**Future directions** - To improve overall performance of the test, experiments are needed to intensify the colour of the test and control lines as well as specificity of F. columnare LFA test. This could be achieved by doing sample dilutions or testing a different marker gene. Also, further optimization of TteUvrD and BstPol proteins interaction in vivo and in vitro needs to be done with the aim to enhance HDA amplification efficiency.

Infections caused by Flavobacterium genus spread incredibly fast and are of an immense threat to aquaculture farms. For this reason, it's crucial to detect F. columnare or F. psychrophilum caused infections as soon as possible. However, majority of the time this identification is delayed and causes huge losses for aquaculture farms. To solve this issue, we decided to create a rapid detection tool based on **helicase dependent amplification (HDA) and lateral flow assay test (LFA)**.

Bioinformatic analysis

Since we decided to base our detection system on nucleic acid hybridization, the first crucial step was to find unique marker genes for each Flavobacterium species. **16S ribosomal RNA gene** (AY577821) was selected for F. columnare (ATCC 23463) strain. Whereas ***rpoC* gene** (JX657167.1) was chosen for F. psychrophilum (ATCC 49418) strain[1](#citations).

To make sure that the test is highly specific, we made a **multiple sequence alignment with 16S rRNA genes** from other species within the same genus using [Clustal Omega tool](https://www.ebi.ac.uk/Tools/msa/clustalo/) (1.2.4.). Probe placement for **F. columnare** was selected based on the absence of matching alignments between sequences (Fig. 1). For the *rpoC* gene, meant to identify **F. psychrophilum** we chose 205 - 250 bp region to place detection and capture probes.

![](https://static.igem.org/mediawiki/2020/3/34/T--Vilnius-Lithuania--results-detectionFig1.png)

**Figure 1.** *Flavobacterium* species 16S rRNA partial gene sequences alignment. Black boxes highlight sequence parts chosen for probe placement. Gene sequences: *F. columnare* - AY577821, *F. branchiophilum* - AB680752, *F. psychrophilum* - AY662493.

**ssDNA probes were created for the chosen regions** in marker genes (Table 1). Detection and capture probes were created to be complementary to the negative strand of the DNA. Control probe is complementary to the detection probe. ssDNA probes for F. columnare were created according to parameters proposed in the research paper[2](#citations). When creating probes for F. psychrophilum we decided to make them longer and of greater GC-content in hopes that hybridization reaction in the LFA test would be improved.

| Species | Probe type | Sequence and its modification | Location | Parameters |
| --- | --- | --- | --- | --- |
| *F. columnare* 16S rRNA gene | [Detection probe](http://parts.igem.org/Part%3ABBa_K3416101) | ThioMC6-D-(A)20-TTTCAGATG | 172 - 180 bp | Tm = 15.4°C GC% = 33.3% size = 29 nt |
| [Capture probe](http://parts.igem.org/Part%3ABBa_K3416102) | GCCTCATTTGATT-(A)20-bio | 181 - 193 bp | Tm = 36.5°C GC% = 38.5% size = 33 nt |
| [Control probe](http://parts.igem.org/Part%3ABBa_K3416103) | biosg-(A)20-CATCTGAAA | - | Tm = 15.4°C GC% = 33.3% size = 29 nt |
| *F. psychrophilum rpoC* gene | [Detection probe](http://parts.igem.org/Part%3ABBa_K3416110) | ThioMC6-D-(A)20-ATTCCTTACGGTTCAAGTAT | 205 - 224 bp | Tm = 48.5°C GC% = 35% size = 40 nt |
| [Capture probe](http://parts.igem.org/Part%3ABBa_K3416111) | AAATGACGCTCAAGTTGTAG-(A)20-bio | 231 - 250 bp | Tm = 48.5°C GC% = 35% size = 40 nt |
| [Control probe](http://parts.igem.org/Part%3ABBa_K3416112) | biosg-(A)20-ATACTTGAACCGTAAGGAAT | - | Tm = 48.5°C GC% = 35% size = 40 nt |

**Table 1.** Parameters of ssDNA probes created for nucleic acid lateral flow assay test. Tm and GC% was calculated without poly-A sequence using IDT oligo analyzer tool. (A)20 marks poly-A sequence of 20 adenines. Bio and Biosg means biotin modification and ThioMC6-D - Thiol group modification at appropriate ends of the sequence.

HDA

After exact placement of probes was known, **primers for HDA** were created to make sure that the amplicon will have necessary detection and capture probe hybridization sites (Table 2). Primers were made using IDT primer quest tool. Blast analysis results showed that **amplicons are specific** only to the exact species of bacteria we wanted to identify.

|  | | Primer | | | Amplicon | |
| --- | --- | --- | --- | --- | --- | --- |
| **bp** | **GC%** | **Tm, °C** | **bp** | **Tm, °C** |
| **Recommended parameter ranges for HDA** | | **20-35** | **30-60** | **60-80** | **70-120** | **68-77** |
| CAGGGGGATAGCCCAGAGAAATTTGG | F\_Col | 26 | 53.8 | 73.9 | 122 | 75 |
| ACCACACCAACTAGCTAATGGGACGC | R\_Col | 26 | 53.8 | 72.2 |
| ACGGGTATTCTTCTTGCTACAAATA | F\_Psy | 25 | 36.0 | 62.9 | 104 | 70 |
| GGATCCCATTTACAAATAACATCTCC | R\_Psy | 26 | 38.5 | 65.3 |

**Table 2.** Used primers and amplicons parameters. Calculations were made using the [Thermo Fisher multiple primer analyzer tool](https://www.thermofisher.com/lt/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/multiple-primer-analyzer.html). All parameters are in the ranges suggested by HDA amplification kit manufacturers.

Symmetric and asymmetric HDA as well as PCR were performed with the created primers (Fig. 2). PCR amplification using F. psychrophilum gDNA as a template and F\_Psy, R\_Psy primers proved to be specific to species (Fig. 2, C). In contrast, PCR amplification of F. columnare gDNA fragment with F\_Col and R\_Col primers showed less specificity than we imagined (Fig. 2, A). Nonetheless both **asymmetric amplifications were successful** and the average concentration of ssDNA was 2.53-2.96 μM as measured by qubit fluorometer.

Also, HDA amplification with F. psychrophilum gDNA was successful and the product could be seen in a gel (Fig. 2, B). On the other hand HDA using F. columnare gDNA as a template proved to be difficult to perform and improve. Even though the product could not be seen in a gel after electrophoresis, by using qubit fluorometer we determined that the **average concentration of amplicon** after symmetric and asymmetric HDA was around **0.7 μM** for both species. This concentration is lesser than we expected but still suitable to be detected by the LFA test.

![](https://static.igem.org/mediawiki/2020/0/0a/T--Vilnius-Lithuania--results-detectionFig2.png)

**Figure 2.** Left to right: A. **L** - gene ruler 50 bp ladder (SM0371), **1** - F. columnare symmetric PCR with F\_Col and R\_Col primers, **2** - F. psychrophilum symmetric PCR with F\_Col and R\_Col primers, **3** - *E. coli* symmetric PCR with F\_Col and R\_Col primers, **4** - F. columnare asymmetric PCR with F\_Col and R\_Col primers (1:15), B. **L** - gene ruler 50 bp ladder (SM0371), **5** - F. columnare symmetric HDA, **6** - F. psychrophilum symmetric HDA, C. **7**- F. psychrophilum symmetric PCR with F\_Psy and R\_Psy primers , **8** - F. columnare symmetric PCR with F\_Psy and R\_Psy primers, **9** - *E. coli* symmetric PCR with F\_Psy and R\_Psy primers, **10** - F. psychrophilum asymmetric PCR with F\_Psy and R\_Psy primers (1:15), **L** - gene ruler 50 bp ladder (SM0371). After amplification with F\_Col and R\_Col primers fragment size should be 122 bp and for F\_Psy, R\_Psy - 104 bp.

Gold nanoparticles synthesis

Created detection probes (Table 1) need to be immobilized on the gold nanoparticles. We decided to chemically synthesize these **13 nm gold nanoparticles** using a modified Turkevich method. Synthesized AuNP were characterized by using NanoSight NS300 instrument. The results showed that the mode of size for gold nanoparticles was 25.4 nm. The size is greater than expected but some slight variation can be present using such characterization method.

To get the most accurate results, analysis using dynamic light scattering and transmission or scanning electron microscopy should be performed as well. We concluded that synthesized gold nanoparticles were of **suitable parameters** for further functionalization reactions.

During the **functionalization reaction**, ssDNA detection probes are conjugated to the gold nanoparticle itself. Since created probes had a **poly-A sequence**, a **low pH assisted method** was used. No change in colour of the gold nanoparticles solution during NaCl test indicated a lack of aggregation and successful functionalization reaction. However, a more appropriate evaluation method was required.

For this reason **absorption spectrum** of nanoparticles before and after functionalization was determined (Fig. 3). Before functionalization gold nanoparticles had an absorption peak at 522 nm whereas after conjugation to F. columnare or F. psychrophilum detection probes, **absorption peak shifted** to 527 and 529 nm respectively. This shift is expected and indicates a successful functionalization reaction[2](#citations).

![](https://static.igem.org/mediawiki/2020/2/2c/T--Vilnius-Lithuania--results-detectionFig3.png)

**Figure 3.** Gold nanoparticles (Au-NP) absorption spectra determined with UV-Visible spectrophotometry. Blue line indicates nanoparticles absorption before conjugation to detection probes, yellow line - after functionalization with F. psychrophilum probes whereas orange line shows absorption after functionalization with F. columnare detection probes.

Lateral flow assay

Finally, having all necessary reagents and membranes, the LFA tests were assembled (Fig. 4). Functionalized gold nanoparticles can be found in the **conjugate pad**. Capture and control probes were sprayed on the test line and control line respectively. After this we had **two sets of tests** created to identify F. columnare and F. psychrophilum. Different running buffers were tested and it was determined that running buffer II (10X SSC, 3.5% Triton X-100, 0.25% SDS, 12.5% formamide) was the most suitable since tests resulted in the most **clearly visible red lines**. The next step was to evaluate specificity and accuracy.

![](https://static.igem.org/mediawiki/2020/4/4f/T--Vilnius-Lithuania--results-detectionFig4.png)

**Figure 4.** LFA test dimensions.

At first the LFA test using 16S ribosomal RNA gene was created to identify F. psychrophilum but its specificity was low and could not be improved, meaning that the test became positive using amplicons other than F. psychrophilum. However, a redesigned test using the ***rpoC* gene** proved to be **more specific** and was able to differentiate F. psychrophilum from F. columnare as well as from *E. coli* and F. piscis (Fig. 5). These results meant that **the test can be used for accurate** F. psychrophilum species identification.

![](https://static.igem.org/mediawiki/2020/4/4c/T--Vilnius-Lithuania--results-detectionFig5.png)

**Figure 5.** Specificity experiment of F. psychrophilum identification LFA test. Tests were evaluated using 100 nM of DNA in 100 μL of running buffer II. **1** - F. psychrophilum asymmetric HDA with F\_Psy and R\_Psy primers, **2** - F. psychrophilum symmetric HDA with F\_Psy and R\_Psy primers after denaturation, **3** - F. psychrophilum asymmetric PCR with F\_Psy and R\_Psy primers, **4** - F. psychrophilum symmetric PCR with F\_Psy and R\_Psy primers after denaturation, **5** - F. psychrophilum asymmetric PCR with F\_Col and R\_Col primers, **6** - F. columnare asymmetric PCR with F\_Psy and R\_Psy primers, **7** - F. piscis asymmetric PCR with F\_Psy and R\_Psy primers, **8** - *E. coli* asymmetric PCR with F\_Psy and R\_Psy primers, **9** - no DNA template. **CL** indicates control line, **TL** - test line.

However, the LFA test created for F. columnare identification showed less exciting results (Fig. 6). We determined that it was able to differentiate between F. psychrophilum and F. columnare as well as F. piscis but **was unable to distinguish** F. columnare from *E. coli*. This can lead to false positive results. In the future further **optimization experiments should be performed** to see if specificity of the test could be improved.

Also, in silico created ssDNA probes using ***cslA* gene** for F. columnare identification should be tested. Nonetheless, as a control experiment we tested F. columnare amplicon on the LFA test created using **F. branchiophilum** 16S rRNA gene and saw no positive results indicating that in the future LFA test for F. branchiophilum species identification **could be created**.

![](https://static.igem.org/mediawiki/2020/c/ce/T--Vilnius-Lithuania--results-detectionFig6.png)

**Figure 6.** Specificity experiment of F. columnare identification LFA test. Tests were evaluated using 100 nM of DNA in 100 μL of running buffer II. **1** - F. columnare asymmetric HDA with F\_Col and R\_Col primers, **2** - F. columnare symmetric HDA with F\_Col and R\_Col primers after denaturation, **3** - F. columnare asymmetric PCR with F\_Col and R\_Col primers, **4** - F. columnare symmetric PCR with F\_Col and R\_Col primers after denaturation, **5** - F. columnare asymmetric PCR with F\_Psy and R\_Psy primers, **6** - F. psychrophilum asymmetric PCR with F\_Col and R\_Col primers, **7** - F. piscis asymmetric PCR with F\_Col and R\_Col primers, **8** - *E. coli* asymmetric PCR with F\_Col and R\_Col primers, **9** - F. columnare asymmetric PCR with F\_Col and R\_Col primers on the test created for F. branchiophilum identification, **10** - no DNA template. **CL** indicates control line, **TL** - test line.

After testing specificity we aimed to determine the **lowest amount of detectable DNA** by F. psychrophilum identification test (Video 1). To do this we used **serial dilutions**. As seen in a timelapse, the lowest amount of DNA detected was around **15 nM**, because the test line was still visible. F. columnare LFA test showed similar results. This **detection limit is sensitive enough** to identify fragments amplified during HDA even if this amplification is not as efficient as PCR.

[

Your browser does not support the video tag.
](https://static.igem.org/mediawiki/2020/6/66/T--Vilnius-Lithuania--results-LFA66.mp4)

**Video 1.** Timelapse of F. psychrophilum LFA test (lower) and control (higher) lines development using serial dilutions of DNA. The starting amount was 500 nM followed by 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.63 nM, 7.81 nM, 3.91 nM, 1.95 nM, 0.98 nM, 0.45 nM, 0 nM.

Vilnius-Lithuania 2020 iGEM team results show that HDA-LFA based detection tool can be developed to identify specific Flavobacterium genus species. In the future further optimization must be performed to improve this detection tool. Optimizations using our [onFlow software](https://2020.igem.org/Team%3AVilnius-Lithuania/Software) could help in improving test and control lines colour intensity as well as specificity. In addition onFlow will help in the process of creating a **quantitative LFA**. Also, to minimize wastage LFA with **multiple test lines** for different species identification could be created. In hopes that **future iGEM teams** will find our project useful and will try to improve on it. All ssDNA probes can be found as DNA parts in the iGEM repository.

Helimerase

However, according to WHO recommendations on the point-of-care diagnostic systems development, one of the criteria is its low price[3](#citations). To fulfil this criteria we found a significantly cheaper alternative for HDA assay – the helimerase.

Helimerase – is a bifunctional protein complex, made up of two enzymes - BstPol and TteUvrD. These proteins are physically linked to each other through the coiled-coil structure. TteUvrD helicase is fused with one part of this structure, WinZip-A2 (WZA2), through the linker L1 and is positioned in the N-terminal end of the sequence. The research has shown that this type of coiled-coils structures provide predictable tertiary structure and stability[4](#citations).

Firstly gene sequence WZA2-L1-TteUvrD (Fig. 7A.) was cloned into pETDuet vector. This protein was fused with a 10xHis tag and a maltose binding protein (MBP) at N-terminus. Analogously, WZB1-L1-BstPol (Fig. 7B.) gene was cloned into the second MCS of pACYC plasmid and fused with StrepII tag at the N-terminus.

![](https://static.igem.org/mediawiki/2020/8/82/T--Vilnius-Lithuania--results-helimerase.svg)

**Figure 7.** Helimerase structure. **A** - TteUvrD helicase fused with one part of coiled-coil structure WinZip-A2(WZA2) through the linker L1; **B** - Bst polymerase I large fragment (BstPol) fused with one part of coiled-coil structure WinZip-B1(WZB1) through the linker L1.

The optimal expression conditions for WZA2-L1-TteUvrD and WZB1-L1-BstPol was observed in *E. coli* BL21(DE3) strain after 16 hours induction with 1 mM IPTG at 30 °C. Both proteins were purified under native conditions.

WZA2-L1-TteUvrD protein was purified by using Ni-NTA affinity column. The concentration of recombinant protein observed after purification under native conditions from 1 L medium reached 5.5 mg/ml. However, as seen in SDS-PAGE gel, further optimization procedures are needed to remove impurities. Yield of WZB1-L1-BstPol obtained after purification from 1 L medium was 2.2 mg/ml (Fig. 8).

![](https://static.igem.org/mediawiki/2020/2/25/T--Vilnius-Lithuania--results-detDidelis.jpg)

**Figure 8.** SDS-PAGE electrophoresis after WZA2-L1-TteUvrD (136.598 kDa) and WZB1-L1-BstPol (74.225 kDa) purification. L - PageRuler Plus Prestained Protein Ladder (#26620), 1 - WZA2-L1-TteUvrD supernatant, 2 - WZA2-L1-TteUvrD 3 elution fraction, 3 - WZA2-L1-TteUvrD 5 elution fraction, 4 - WZA2-L1-TteUvrD 14 elution fraction, 5 - WZA2-L1-TteUvrD after protein concentration, 6 - WZB1-L1-BstPol supernatant, 7 - WZB1-L1-BstPol 4 elution fraction, 8 - WZB1-L1-BstPol 6 elution fraction, 9 - WZB1-L1-BstPol 7 elution fraction, 10 - WZB1-L1-BstPol after protein concentration.

To verify that WZA2-L1-TteUvrD and WZB1-L1-BstPol proteins are stable at high temperatures we performed fluorescence thermal shift assay by utilizing a thermodynamic model[5](#citations). This measurement is based on protein property to unfold upon increasing temperature of environment and solvatochromic property of fluorescence dye to fluorescent more intensively in the hydrophobic environment[6](#citations). The results (Fig. 9A and Fig. 9B) show that both proteins are stable at high temperatures, thus letting us use them in activity assays. Also, according to obtained melting temperatures (TM) WZB1-L1-BstPol enzyme is more stable and starts to denaturate at 76 °C, while WZA2-L1-TteUvrD protein denaturation starts at 61.1°C.

**A**

![](https://static.igem.org/mediawiki/2020/5/5f/T--Vilnius-Lithuania--results-helicase-stability.svg)

**B**

![](https://static.igem.org/mediawiki/2020/c/c7/T--Vilnius-Lithuania--results-polymerase-stability.svg)

**Figure 9.** Fluorescence thermal shift assay for proteins stability characterization. **A** - simulated dependence of the WZA2-L1-TteUvrD melting temperature; **B** - simulated dependence of the WZB1-L1-BstPol melting temperature.

According to obtained enzyme stability data, kinetic experiments were performed. WZA2-L1-TteUvrD activity was measured based on SYBR GreenI fluorescent dye, which emits fluorescence upon binding with double-stranded DNA. Otherwise, if helicase separates dsDNA, fluorescence then declines proportionally[7](#citations). As shown in Fig. 10A and Fig. 10B the bigger amount of WZA2-L1-TteUvrD protein is in the assay, the more dsDNA is unwinded.

**A**

![](https://static.igem.org/mediawiki/2020/1/1d/T--Vilnius-Lithuania--results-helicase-concentration-raw.svg)

**B**

![](https://static.igem.org/mediawiki/2020/8/84/T--Vilnius-Lithuania--results-helicase-concentration-norm.svg)

**Figure 10.** Time dependent analysis of the helicase activity assay. Three different concentrations were used: 4 nM, 44 nM, 1000 nM. **A** - raw data of helicase activity assay, where fluorescence intensitivity decreases depending on helicase concentration; **B** - normalised data of helicase activity assay, which shows the percentage of substrate unwound depending on the helicase concentration.

WZB1-L1-BstPol activity (Fig. 11A and Fig. 11B) was also measured by observing its fluorescence depending on time. The bigger the polymerase concentration is in the assay, the more amplified fragments are being generated. Linear dependence of polymerase activity on its substrate in solution was also determined by using fluorescence measurements.

**A**

![](https://static.igem.org/mediawiki/2020/c/c5/T--Vilnius-Lithuania--results-polymerase-concentration.svg)

**B**

![](https://static.igem.org/mediawiki/2020/2/2a/T--Vilnius-Lithuania--results-polymerase-substrate-concentration.svg)

**Figure 11.** Time dependent analysis of the BstPol activity assay. **A** - generation of dsDNA depending on BstPol amount in the assay. **B** - polymerase activity assay depending on the substrate concentration.

Based on these enzymes' activity assays we determined that the synthesis of active helimerase proteins was successful. However, after a lot of attempts we were unable to fuse these proteins via coiled-coil amino acid structures in vivo and in vitro. We hypothesized that newly formed affinity tags, as such as tertiary structures, could influence these proteins' interaction with each other, so in order to tackle this problem, new affinity tags and linkers should be tested. Also, with the aim to co-express these proteins in vivo, they could be cloned into other compatible plasmids.

Treatment

Overview

**Goal.** To create genetically engineered bacteria that are able to sense pathogenic bacteria biofilm secreted AI-2 and express a sufficient amount of exolysin and toxin.
**Results.** The inducible kill-switch is created and exolysin depolymerase activity was determined. The AI-2 inducible promoters work as expected.
**Future directions.** To create a more precise AI-2-sensing circuit and improve lysis protein function.

The main goals were to construct two genetic circuits that are capable of expressing a sufficient amount of depolymerase and initiate cell-lysis. The genetic circuit is based on a quorum sensing Type II system that uses autoinducer-2 for bacterial interspecies communication. The treatment circuits have an exolysin gene accompanied by *E. coli* lysing proteins: endolysin lysECD7 or mazEF system.

AI-2 Synthesis

Before starting to construct the whole treatment genetic circuit, we needed to determine the strength of autoinducer-2 inducible promoters. To do that it was decided to compare *lsrACDBFG* and its mutants, EP01r, EP14r, with Anderson’s promoters that is a conventional part collection. The literature shows that these AI-2 inducible promoters are quite weak. Before narrowing the window of possible promoters‘ strength values, there was a need to get AI-2 as a 4,5-dihydroxy-2,3-pentanedione (DPD). Keeping in mind that AI-2 is costly and there are not well-described parts that are the base of AI-2 synthesis, it was decided to synthesize AI-2 in vitro[10,11](#citations). As described in [Engineering](https://2020.igem.org/Team%3AVilnius-Lithuania/Engineering) for the production of AI-2 there is a need for luxS and pfs proteins that are key enzymes for converting S-adenosylhomocysteine (SAH) into AI-2.

The protein-coding genes *pfs* (693bp) and *luxS* (510bp) cloned into pET28a(+) vector (restriction sites: NcoI and XhoI). The plasmid was transformed in *E. coli* BL21(DE3) strain. After the induction with IPTG, the protein purification followed. After performing SDS-PAGE (Fig.1, Fig.2) pfs and luxS proteins were successfully purified and soluble in native conditions. The purified proteins were used for the following experiments.

![](https://static.igem.org/mediawiki/2020/a/a8/T--Vilnius-Lithuania--results-treatF1.png)

**Figure 1.** Purification of pfs enzyme in BL21(DE3) *E. coli* strain. L- PageRuler Plus Prestained Protein Ladder (Thermo Fisher Scientific), 1 - BL21(DE3) pET28a(+)-pfs biomass sample before induction, 2 - BL21(DE3) pET28a(+)-pfs supernatant (soluble) fraction after ultrasound lysis, 3 - purified, concentrated pfs protein(25.5kDa).

![](https://static.igem.org/mediawiki/2020/3/39/T--Vilnius-Lithuania--results-treatF2.png)

**Figure 2.** LuxS purification experiment in BL21(DE3) strain in pET28a(+) plasmid. 1 – BL21(DE3) pET28a(+)-luxS biomass sample before induction, 2 - BL21(DE3) pET28a(+)-luxS biomass sample after induction, 3 - Supernatant (soluble) fraction after ultrasound lysis, 4 - Precipitant (insoluble) fraction after ultrasound lysis, 5 - purified, concentrated protein (20.6kDa), L- PageRuler Plus Prestained Protein Ladder (Thermo Fisher Scientific).

The purified proteins were used for in vitro reaction of AI-2 synthesis (Fig.3). This reaction product and its concentration was identified with Ellman reagent (5,5'-dithiobis-(2-nitrobenzoic acid). This reagent reacts with compounds that have a thiol group. The reaction product 2-nitro-5-thiobenzoate (TNB−) ionizes to the TNB2− that gives a yellow color to the solution. In that way, the concentration of TNB2−(ion amount is equal to the compound’s amount that has thiol groups) can be measured via measuring solution's absorption at 412nm and calculating the concentration of DPD.

![](https://static.igem.org/mediawiki/2020/e/e9/T--Vilnius-Lithuania--results-treatF3.png)

**Figure 3.** The chemical reaction catalyzed by pfs and luxS from a precursor S-adenosylhomocysteine (SAH) to AI-2 as a linearised form DPD.

The Measurement of Genetic Circuits

The following step after successfully producing AI-2, was to test whether the genetic circuit J23XXX-sfGFP reacts to AI-2, and if so, at what concentrations the signal is increased. Before determining the most suitable promoter for treatment genetic circuit, a few promoters from the Anderson promoter collection compared with AI-2 inducible promoters: *lsrACDBFG*, EP01r, EP14r. The measurements that followed are [described here](https://static.igem.org/mediawiki/2020/f/f7/T--Vilnius-Lithuania--LabProtocolsSafety.pdf). After a few trials, the genetic circuit that has a weak Anderson promoter ([J23117](http://parts.igem.org/Part%3ABBa_J23117)) was chosen. To add more, even for a more efficient system it could have an even weaker promoter but due to the lack of a DNA distribution kit, there was no possibility to get weaker Anderson promoters. The fluorescence intensity and OD600 were measured and the ratio was calculated. All bar plots are indicating a ratio at 6h hours of bacterial growth (exponential growth phase).

![](https://static.igem.org/mediawiki/2020/a/a7/T--Vilnius-Lithuania--results-treat-lsrACDBFG_sfGFP_FI_OD_barplot_35.svg)

**Figure 4.** EP01r promoter strength measurement inducing with AI-2. From left to right: ‘+’- a positive control (J23117-sfGFP), without AI-2, 12μM, 18μM, 24μM of AI-2.

![](https://static.igem.org/mediawiki/2020/d/de/T--Vilnius-Lithuania--results-treatx1.png)

**Figure 5.** *lsrACDBFG* promoter strength measurement inducing with AI-2 after 6h of bacteria growth. From left to right: ‘+’- a positive control (J23117-sfGFP), without AI-2, 12μM, 18μM, 24μM of AI-2.

The results did not reiterate as expected. Only AI-2 inducible promoter *lsrACDBFG*-sfGFP signal showed a correlation with increasing AI-2 concentration(Fig.5 and Fig. 6). The EP01r is expected to be a stronger promoter than *lsrACDBFG*4. To add more, all three AI-2 inducible promoters are leaky: when there is no AI-2 added there can be seen an emission of enhanced sfGFP. There is little known about these promoters. Thus, the conclusions are not valid enough. This might be affected by changing the plate reader devices, non-optimized sequences, or unprocessed experiment conditions. However, after the last experiments construct *lsrACDBFG-mazF-J23117-sfGFP* showed positive results: there can be seen a positive toxin mazF activity. The increase of AI-2 concentration leads to cell-lysis of *E. coli*.

![](https://static.igem.org/mediawiki/2020/7/76/T--Vilnius-Lithuania--results-treat-fig-6.png)

**Figure 6.** lsrACDFBG promoter strength measurement inducing by AI-2 changes over time.
The concentrations varied from 0μM to 24μM.

![](https://static.igem.org/mediawiki/2020/b/b5/T--Vilnius-Lithuania--results-treat-fig-7.png)

**Figure 7.** *lsrACDBFG-mazF-J23117-sfGFP* dependency on AI-2 concentration over time.

However, counting the last days left until wiki freeze, the genetic circuit *lsrACDBFG-mazF-J23117-sfGFP* showed expected results. The mentioned genetic circuit activity was dependent on AI-2 concentration (Fig.7). The increase of AI-2 lead to cell lysis due to increased *lsrACDBFG* activity and mazF expression. Fig. 8 illustrates the difference between positive control or colonies without the induction of AI-2 between those colonies that were induced.

![](https://static.igem.org/mediawiki/2020/0/07/T--Vilnius-Lithuania--results-treatx3.png)

**Figure 8.** *lsrACDBFG-mazF-J23117-sfGFP* dependency on AI-2 concentration.

To verify that our proteins had exolysin activity we had to do bioinformatic analysis. By comparing the sequences of the genes and their gene products, we can predict that they are tail fiber proteins. Also, by using the Basic Local Alignment Search Tool (BLASTx) we have found that these proteins show high similarities(>89%) with K. pneumoniae phage K64-1 tail spike/ structural proteins which have been proven to be responsible for depolymerization of the bacteria polysaccharides[13](#citations) (Table 1). Also, gp533 (BBa\_K3416033) is homologous (85,58%) to the gene which will result in a protein with pectate lyase enzymatic activity (Table 1). After exolysin sequence analysis, we can deduct that our proteins have pectate lyase activity.

**Table 1.** BLAST comparative analysis of Klebsiella phage RAK-2 and K64-1 proteins.

| RAK-2 protein / Accession | K64-1 protein / Accession | E value | Percentage identity |
| --- | --- | --- | --- |
| gp529 YP\_007007684.1 | Putative tail fiber protein YP\_009153199.1 | 0.0 | 99.49% |
| gp531 YP\_007007686.1 | Putative tail fiber protein YP\_009153201.1 | 3e-137 | 91.94% |
| gp533 YP\_007007688.1 | Putative structural protein YP\_009153203.1 | 0.0 | 89.96% |
| gp533 YP\_007007688.1 | S2-6 gene for pectate lyase LC121105.1 | 0.0 | 85.83% |

Pectate lyase is responsible for eliminating cleavage of pectate, yielding oligosaccharides with 4-deoxy-α-D-mann-4-enuronosyl at their non-reducing ends (Fig. 9).

![](https://static.igem.org/mediawiki/2020/6/6f/T--Vilnius-Lithuania--results-treatF4.png)

**Figure 9.** Pectate lyase catalyzed reaction

To test exolysins' activity, we tried to induce and purify three different exolysins (BBa\_K3416029), (BBa\_K3416031), (BBa\_K3416033). Plasmids with the cloned exolysins were kindly provided by prof. Rolandas Meškys.
Plasmid pET28b(+)-gp529 was transformed into Rosetta (DE3) strain and induced with 0.5mM IPTG for 19 hours in 16°C in 200mL LB. Biomass was resuspended into 50mM Tris-HCl pH=8 and ultrasound lysed until the solution became clear. His-tagged protein was purified using Ni-NTA. Dialysed and concentrated. Yielding a 1.2mg of the protein from 200mL of LB medium. But the protein was not in its active form. Thus, more optimization is required.
Plasmid pET16b(+)-gp531 was transformed into Rosetta (DE3) strain and induced with 0.5mM IPTG for 17 hours in 16°C in 200mL LB. Biomass was resuspended into 50mM Tris-HCl pH=8 and ultrasound lysed until the solution became clear. His-tagged protein was purified using Ni-NTA. Dialysed and concentrated Yielding 0.8mg from 200mL of LB medium. Synthesis should be optimized if a larger yield of the protein is desired but the protein amount was sufficient to see a visible activity on the spot test.
Plasmid pET16b(+)-gp533 was transformed into Rosetta (DE3) strain and induced with 0.5mM IPTG for 21 hours in 17°C in 200mL LB. Biomass was resuspended into 50mM Tris-HCl pH=8 and ultrasound lysed until the solution became clear. His-tagged protein was purified using Ni-NTA. Dialysed and concentrated. (Fig. 10) Yielding 0.78mg from 200mL of LB medium. Synthesis should be optimized if a larger yield of the protein is desired but the protein amount was sufficient to see a visible activity on the spot test.

![](https://static.igem.org/mediawiki/2020/6/67/T--Vilnius-Lithuania--results-treatF5.png)

**Figure 10.** SDS-PAGE electrophoresis results after gp529, gp531, gp533 purification. L - PageRulerTM Prestained Protein Ladder, #SM0671 (Fermentas), 1 – gp529 biomass sample before induction, 2 – gp529 biomass sample after induction, 3 -purified and highly concentrated gp529 (62.5 kDa), 4 - gp531 biomass sample before induction, 5 – gp531 biomass sample after induction, 6 - gp531 purified protein (97.8 kDa), 7 - gp533 biomass sample before induction, 8 - gp533 biomass sample after induction, gp533 purified protein (82.2 kDa).

After purification, we tested exolysin activity using the spot test on Klebsiella sp. KV-3 strain.
First, we performed an experiment to determine a minimal amount of protein needed to form a lysis halo (Fig.3). 5μL of 4μM initial (101) starting concentration of all exolysins were used. Dilutions were 101, 10-1, 10-2, 10-3, 10-4, 10-5. After spot test analysis it was seen the exolysin gp529 did not show any depolymerase activity towards this strain, while gp531 had the most visible activity. After dilution experiments, the smallest measured exolysin concentration, which can induce cell lysis is 10-2 – 10-3 of initial concentration. However, even if this part works, after sequencing we saw that this gene sequence is not compatible with iGEM standard construction. Due to this, we decided not to use it in our genetic circuit. However, gp533 showed minimal but detectable depolymerase activity so we decided to construct it under AI-2 induced promoter. For the spot test positive control we have chosen RAK-2 phage, which was kindly provided by doc. Algirdas Noreika. Phage dilution was 101 10-2, 10-4, 10-6, 10-8, 10-10. As the negative control, we used 50 mM Tris– HCl pH=8.

![](https://static.igem.org/mediawiki/2020/3/32/T--Vilnius-Lithuania--results-treatF6.png)

**Figure 11.** A) Spot test with serial dilutions of gp529, gp531, gp533 exolysins. From top to bottom the proteins were gp529, gp531, gp533, RAK-2(positive control), blank(negative control). Tris- HCl was used as a negative control. From left to right the protein dilutions were 101, 10-1, 10-2, 10-3, 10-4, 10-5 and phage dilutions were 101 10-2, 10-4, 10-6, 10-8, 10-10. B) Spot test with gp529, gp531, gp533 exolysins (24μmol) incubated at 42oC, 60oC, 65oC, 70oC . From top to bottom the proteins were gp529, gp531, gp533, RAK-2(positive control), blank(negative control). RAK-2 phage (1.2 \* 1010 PFU/mL) was used as a positive control. Tris- HCl was used as a negative control.

Prevention

Overview

**Goal** - To synthesize and purify immunogenic proteins GldJ and VHSV glycoprotein G and examine how alginate beads withstand pressure and interact with digestive system in vitro.
**Results** - Both GldJ and VHSV glycoprotein G were successfully cloned into appropriate protein expression vectors. However, no immunogenic protein synthesis was achieved. Physical and chemical alginate bead tests provided valuable insight into further developments of orally administered recombinant protein vaccines for fish immunization.
**Future directions** - To purify both recombinant immunogenic proteins as well as to test the beads with immunogenic proteins in vivo, therefore proving their potential to be used as vaccines.

GldJ

GldJ is an immune response inducing protein. We decided to synthesize it using *E. coli* bacteria strains. Thus, our team successfully cloned *gldJ* gene (1731 bp) into pET28a(+) vector. This was confirmed by Sanger sequencing. However, after induction with 1mM IPTG at 16°C for 18 hours in 50mL LB medium. After SDS-page analysis (Fig. 1), we found out that two proteins were expressed, but their sizes indicated that it was not GldJ, which should have been 66 kDa.

![](https://static.igem.org/mediawiki/2020/5/5a/T--Vilnius-Lithuania--results-prev1.png)

**Figure 1.** SDS-PAGE electrophoresis results after GldJ induction in different *E. coli* strains. L- PageRuler Plus Prestained Protein Ladder (#26620), 1BL – *E. coli* BL21(DE3) biomass sample before induction, 1RO – *E. coli* Rosetta(DE3) biomass sample before induction, 1AE - *E. coli* ArcticExpress(DE3) biomass sample before induction, 2BL – *E. coli* BL21(DE3) biomass sample after induction, 2RO - *E. coli* Rosetta(DE3) biomass sample after induction, 2AE - *E. coli* ArcticExpress(DE3) biomass sample after induction, 3BL - *E. coli* BL21(DE3) supernatant sample after ultrasound lysis, 3RO - *E. coli* Rosetta(DE3) supernatant sample after ultrasound lysis, 3AE - *E. coli* ArcticExpress supernatant sample after ultrasound lysis.

The protein of interest was also not detected by Western blot analysis with Anti-His antibodies (Fig. 2).

![](https://static.igem.org/mediawiki/2020/0/08/T--Vilnius-Lithuania--results-prev2.png)

**Figure 2.** Western blot results after GldJ induction in different *E. coli* strains. L- PageRuler Plus Prestained Protein Ladder (#26620), 1 – Control protein, 2 – *E. coli* ArcticExpress supernatant sample after ultrasound lysis, 3 - *E. coli* Rosetta(DE3) supernatant sample after ultrasound lysis, 4 - *E. coli* BL21(DE3) supernatant sample after ultrasound lysis, 5 - *E. coli* ArcticExpress(DE3) biomass sample after induction, 6 - *E. coli* Rosetta(DE3) biomass sample after induction, 7 - *E. coli* BL21(DE3) biomass sample after induction.

After using trehalose to enhance the protein solubility[12](#citations), we did not see any difference in the size of the proteins after SDS-PAGE electrophoresis (Fig. 3). However, Western blot analysis confirmed that the amount of soluble recombinant protein was significantly higher after sample treatment with trehalose (Fig. 4).

![](https://static.igem.org/mediawiki/2020/6/66/T--Vilnius-Lithuania--results-prev3.png)

**Figure 3.** SDS-PAGE electrophoresis results of GldJ with and without trehalose from ArcticExpress *E. coli* strain. L- PageRuler Plus Prestained Protein Ladder (#26620), 1 – supernatant with a 0.7M trehalose after Ni-NTA purification, 2 – Precipitant fraction without trehalose, 3 - Precipitant fraction with trehalose, 4 - Supernatant fraction without trehalose, 5 - Supernatant fraction with trehalose, 6 - *E. coli* ArcticExpress biomass sample after induction, 7 - *E. coli* ArcticExpress biomass sample before induction.

![](https://static.igem.org/mediawiki/2020/a/a3/T--Vilnius-Lithuania--results-prev4.png)

**Figure 4.** SDS-PAGE electrophoresis results of GldJ with and without trehalose purified from ArcticExpress *E. coli* strain. L- PageRuler Plus Prestained Protein Ladder (#26620), 1 – First elution fraction Ni-NTA purification with trehalose in resuspension buffer, 2 – second elution fraction Ni-NTA purification with trehalose in resuspension buffer, 3 - precipitant fraction after ultrasound lysis without trehalose, 4 - precipitant fraction after ultrasound lysis with trehalose, 5 - supernatant fraction after ultrasound lysis without trehalose, 6 - supernatant fraction after ultrasound lysis with trehalose, 7 - *E. coli* ArcticExpress(DE3) biomass sample after induction.

VHSV

*VHSV glycoprotein* G gene (1545 bp) with a 6xHis tag on the N-terminus of the sequence was cloned into a pfX7 vector. Sanger sequencing confirmed that the procedure was successful. After transforming the plasmid into *S. cerevisiae* AH22-214 strain and inducing the cells with 25mL 12% galactose and growing at 30°C for 24 hours with 25 mL 2xYEP medium containing 0.1% formaldehyde, SDS-PAGE analysis was performed. No trace of VHSV glycoprotein G (57kDa) was found (Fig. 5a). Only an unknown protein around half the desired size was purified. After performing identical procedures with AH22-214Δpep *S. cerevisiae* strain (Fig. 5b), similar results were yielded, with no protein of interest and a purified unknown protein. Western blot analysis showed no substantial results.

![](https://static.igem.org/mediawiki/2020/9/9c/T--Vilnius-Lithuania--results-prev6.png)

**Figure 5.** SDS-PAGE electrophoresis results after glycoprotein G synthesis induction in (a) S. cerevisiae AH22-214 strain and (b) AH22-214Δpep strain. L - PageRuler Plus Prestained Protein Ladder (#26620), b- protein sample before induction, a - protein sample after induction, s - supernatant sample, p - precipitate sample, ff - flow through, f1-5 - chromatography elution fractions.

Alginate Beads

GFP and digestive system in vitro

In order to test whether our immunogenic protein would reach the midgut of the fish undigested, we designed an experiment where different conditions were applied onto alginate beads containing GFP. We tried to imitate decomposition of alginate beads by using EDTA. Both of them are chelators which interact and bind to Ca2+ ions therefore disrupt the bead structure (Table 1). All tested beads had a uniform diameter of 5mm and final concentration of GFP in each bead was 0.4mg/mL with 2% alginate used. 1mL of 1mg/mL concentration of either pepsin/ trypsin/0.1M EDTA was used on 2 alginate beads in accordance with experiment conditions provided in the table.

**Table 1.** Alginate beads with GFP affected by digestive enzymes and chelating substances. 1 - alginate beads with pepsin pH=3, 2 - alginate beads without pepsin pH=3, alginate beads with pepsin pH=4, 4 - alginate beads without pepsin pH=4, 5- alginate beads with trypsin pH=9, 6- alginate beads without trypsin pH=9, 7- alginate beads with EDTA.

![](https://static.igem.org/mediawiki/2020/7/7d/T--Vilnius-Lithuania--results-prevTable2.png)

We found out that the buffer of pH 3 affected GFP inside alginate beads as the protein's fluorescence weakened after 2h. EDTA also affected bead structure and ruptured it over time. There were no visible changes in pH 9 with and without trypsin and in pH 4 with and without pepsin. This means that beads were not affected by these conditions, that would be affecting the bead while it would travel through the fish gut.

Alginate Beads

When our team found out that *E. coli* does not have an alginate lyase gene while *Klebsiella pneumonia* KV-3 does, we decided to make an experiment where we incubated beads with bacteria in order to test how long it would take for bacteria to digest the beads[13](#citations). We tested *Klebsiella pneumonia* KV-3 and *E. coli* strain BL21(DE3) difference in alginate degradation and beads inside *Klebsiella pneumonia* KV-3 were completely disintegrated whereas *E. coli* bacteria were not able to decompose alginate beads at all (Table 2).

**Table 2.** Alginate beads with GFP incubated with *E. coli* and *Klebsiella pneumonia* KV-3 in LB medium from left to right - without beads, with beads that do not contain protein and beads with GFP. 1 - *E. coli* BL21(DE3) before adding bacteria to medium, 2 - *E. coli* BL21(DE3) 3 days after adding bacteria to medium, 3 - *Klebsiella pneumonia* KV-3 before adding bacteria to medium, 4 - 3 days after adding *Klebsiella pneumonia* KV-3 to medium.

![](https://static.igem.org/mediawiki/2020/c/c2/T--Vilnius-Lithuania--results-prevTable1.png)

Strength of Alginate Beads

Alginate beads with GFP and beads without a protein were compressed using a universal testing machine TIRA test 2300. By applying force over time at a rate of 10mm/min onto the bead, it was measured at what point alginate starts to rupture (Fig. 6). The parameters at the point of breakage were recalculated according to Maurice et. al.[14](#citations). The stress and strain at failure values were below the recommended values of a 0.13-0.32MPa (Table 3). Which means that the produced beads would not be able to withstand physical challenges of the fish digestive tract. It is important to note that a significant difference in rigidity of the beads was observed between control beads and the beads containing a protein. Which means that the stiffness depends on protein concentration and should be considered when producing vaccines for in vivo experiments.

**Table 3.** Stress and strain at failure of control alginate beads without the protein and beads with GFP. The results are averaged from twenty measured beads.

| Bead type | Stress at failure (MPa) | Strain at failure (-) |
| --- | --- | --- |
| With GFP | 0.0351 ±0.0026 | 0.5522 ±0.0338 |
| Empty control | 0.0273 ±0.0084 | 0.5784 ±0.0354 |

![](https://static.igem.org/mediawiki/2020/f/ff/T--Vilnius-Lithuania--results-prev5.png)

**Figure 6.** Example of force applied on the bead with GFP at a rate of 10mm/min over time. The yellow line indicates the rupture point of the bead.

Future Directions

Effective orally administered vaccines against columnariosis and viral hemorrhagic septicemia diseases could be developed if our current research would be further implemented and improved. This could be achieved by optimising protein purification and testing alginate beads more thoroughly. Also, other types of gliding motility lipoproteins could be used, since most of them have immunogenic properties. For VHSV glycoprotein G, it would be beneficial to try using *Pichia pastoris* and algae for protein expression, as this could help to solve the glycosylation problem. Finally, it is crucial to perform ELISA assay and testing the beads with immunogenic proteins in vivo, to prove their potential to be used as vaccines.

### References

1. Strepparava, N., Wahli, T., Segner, H. *et al.* Detection and quantification of Flavobacterium psychrophilum in water and fish tissue samples by quantitative real time PCR. *BMC Microbiol* **14**, 105 (2014)
2. Kolm, C., Martzy, R., Führer, M. *et al.* Detection of a microbial source tracking marker by isothermal helicase-dependent amplification and a nucleic acid lateral-flow strip test. *Sci Rep* **9**, 393 (2019).
3. Kosack, C. S., Page, A. L. & Klatser, P. R. A guide to aid the selection of diagnostic tests. *Bulletin of the World Health Organization* **95**, 639–645 (2017).
4. Motré, A., Li, Y. & Kong, H. Enhancing helicase-dependent amplification by fusing the helicase with the DNA polymerase. *Gene* **420**, 17–22 (2008).
5. Matulis, D., Kranz, J. K., Salemme, F. R. & Todd, M. J. Thermodynamic Stability of Carbonic Anhydrase: Measurements of Binding Affinity and Stoichiometry Using ThermoFluor. (2005) doi:10.1021/bi048135v.
6. Cimmperman, P. *et al.* A quantitative model of thermal stabilization and destabilization of proteins by ligands. *Biophysical Journal* **95**, 3222–3231 (2008).
7. Siddiqui, S., Khan, I., Zarina, S. & Ali, S. Use of the SYBR Green dye for measuring helicase activity. *Enzyme and Microbial Technology* **52**, 196–198 (2013).
8. Wattanavanitchakorn, S., Prakitchaiwattana, C. & Thamyongkit, P. Rapid and simple colorimetric method for the quantification of AI-2 produced from Salmonella Typhimurium. *Journal of Microbiological Methods* **99**, 15–21 (2014).
9. Ascenso, O. S. *et al.* An efficient synthesis of the precursor of AI-2, the signalling molecule for inter-species quorum sensing. *Bioorganic & Medicinal Chemistry* **19**, 1236–1241 (2011).
10. Ellman, George L. "Tissue sulfhydryl groups." *Archives of biochemistry and biophysics* **82.1** (1959): 70-77.
11. Hauk, P. *et al.* Insightful directed evolution of *Escherichia coli* quorum sensing promoter region of the *lsrACDBFG* operon: a tool for synthetic biology systems and protein expression. *Nucleic Acids Res* gkw981 (2016) doi:10.1093/nar/gkw981.
12. Leibly, D. J. *et al.* Stabilizing Additives Added during Cell Lysis Aid in the Solubilization of Recombinant Proteins. *PLoS ONE* **7**, e52482 (2012).
13. Tavafi, H., Abdi- Ali, A. A., Ghadam, P. & Gharavi, S. Screening of Alginate Lyase-Producing Bacteria and Optimization of Media Compositions for Extracellular Alginate Lyase Production. *Iran Biomed J* **21**, 48–56 (2017).
14. Maurice, S., Nussinovitch, A., Jaffe, N., Shoseyov, O. & Gertler, A. Oral immunization of Carassius auratus with modified recombinant A-layer proteins entrapped in alginate beads. *Vaccine* **23**, 450–459 (2004).

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