iGEM 2021 — 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)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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# Team:Vilnius-Lithuania/Engineering

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# ENGINEERING SUCCESS

![Header](https://static.igem.org/mediawiki/2021/3/34/T--Vilnius-Lithuania--Engineering.jpg)

### Overview

In the process of our project development, we performed many engineering cycles.
We decided to describe five of them, that had the most distinguished stages.

Since engineering stages are four (design, build, test, and learn), **we imagined engineering process as a unit circle, which can also be represented in the form of sinusoid.**  One sinusoid wave is equal to one engineering cycle.

Below you can see the general animation describing our vision. It shows the
engineering process that contains three engineering cycles.

### Protein production - PPDK

### Cycle 1

#### Design

At first, we looked for a protein marker that could be used to identify
*Entamoeba histolytica.* We found a few promising candidates and, in the end, we chose cysteine proteinase 5 (CP5) and **pyruvate phosphate dikinase (PPDK)** as our analytes because they are unique markers of the *E. histolytica*. One of them has even been already considered as a marker for a test in previous papers [1,2]. We decided to put it in the **pET28a(+)
plasmid** as in that way we could manipulate the insert in various ways and get the construct in both
N-His and C-His forms.

#### Build

The cloning of PPDK was unsuccessful - the sequencing showed that there were **a few mutations in our gene of interest that made it impossible to use the protein.** We tried to clone it again.
After four failed attempts we were running out of our ordered construct, so we decided to
**clone it into a pUC19 vector** with simple blunt-end cloning. Since that worked at the first time,
we decided that there was probably something wrong with our plasmid backbone. Finally, **we successfully cloned PPDK into the pET28a(+) vector**.

#### Test

We tested a few different *E. coli* strains and growing conditions for the induction. Some had no
visible protein at all, some had a lot, but none of them had a substantial amount in the soluble
fraction. After determining that **the BL21 (DE3) strain had the most protein** in the insoluble
fraction, we tested out different lysis conditions varrying the saccharose,
NP-40 and Triton X-100 concentrations in the lysis buffers.

#### Learn

After all the testing we found out that **the best conditions for induction are in the *E. coli*
strain BL21 (DE3) with a 1 mM IPTG concentration for 3 hours at 37°C in the TB medium [2].**
The most protein was found in the soluble fraction when we used a lysis buffer with 50 mM
NaH2PO4, 500 mM NaCl, 10 mM imidazole, and 0.5 % NP-40. The protein
was not pure enough to start the SELEX and there was not much of it, therefore we needed to find an alternative.

### Cycle 2

#### Design

Since we could not synthesize a significant amount of our target protein, we decided to **perform
our SELEX process on a denatured (and refolded) protein.** We decided to denature the proteins in the
sample blood on our test.

#### Build

At first, we needed to **find out the specific concentration of a needed strong detergent.** Then we
required **to find a buffer solution,** which could keep the protein soluble and would not interfere
with the SELEX process downstream.

#### Test

We tried to dissolve the PPDK containing biomass with a gradient of urea buffers to find out
at what concentration the protein dissolves the best. After determining the denaturing
lysis conditions, we dialyzed the lysate against a soluble lysis buffer containing
**5 different additives** - arginine (0.375 M), trehalose (0.75 M), proline (0.5 M), mannitol
(0.5 M) and CuCl2 (10 mM) [3, 4].

#### Learn

We found out that **PPDK becomes soluble when a lysis buffer supplemented with 6 M urea is used.**
It is possible to transfer the solubilized protein in a phosphate buffer containing 500 mM of
NaCl and 0.375 M arginine. The protein stays soluble in this solution and can be used in downstream applications.

### Protein production - CP5

### Cycle 1

#### Design

At first, we looked for a protein marker that could be used to identify *Entamoeba histolytica*, we
found a few promising candidates. In the end, we chose **cysteine proteinase 5 (CP5)** and pyruvate phosphate
dikinase (PPDK) as our analytes because they are unique markers of *E. histolytica* and one of them
has even been considered as a marker for a test already in previous papers [1,2]. We decided to put it in
 **the pET28a(+) plasmid** because we could manipulate the insert in various ways and get the construct in
both N-His and C-His forms.

#### Build

The cloning of CP5 into the pET28a(+) vector was successful with both N-His and C-His tags, then it
was transformed into the *E. coli* BL21 (DE3) strain.

#### Test

The protein synthesis was successful and we did not need to optimize the growing conditions
besides the IPTG concentration – **we compared the protein expression at 1 mM and 0.6 mM IPTG
and we found that the lower one works better.** We ran into a bunch of problems while trying
to process the insoluble protein because it had to be denatured, renatured, and then activated [5, 6].
 **The protein was dissolved and purified successfully, but we were not able to refold or activate it.**
After a few consultations with relevant experts from the lab of our PI, we tried to increase our
refolding buffer’s glycerol concentration by adding saccharose, changing the refolding conditions
from 4 °C to 37 °C, but none of it worked. The only condition left that
we could change was the ratio of buffer to protein. However, that was not feasible because we did
not have the equipment to concentrate large volumes of the buffer after refolding.
The method we did try was pulling out all the water from the buffer through a dialysis membrane
with carboxymethyl cellulose. It took more than 4 days with constant cellulose clean-up and
change for the buffer to go from 100 ml to about 20-30 mL.

#### Learn

In the end, we could not find out, what was the problem with CP5 processing due to the following reasons.
It is not possible to determine if the protein folded correctly or did not fold at all, and whether the
problem is in the protein activation process - all we could see was the 32 kDa band on
our SDS gels. Since we could not produce the soluble protein, but could purify insoluble protein
relatively easily, we decided to change our approach to the test itself. **We considered doing our SELEX process on the denatured
CP5 protein and then made a test in which we would denature the proteins in the blood sample too.**

### Cycle 2

#### Design

**The design of the test stayed the same,** we just needed a different approach to get the soluble and active CP5 protein.

#### Build

Since we did not manage to produce an active and soluble protein, we consulted with a relevant expert in our PI’s lab and got advised that we maybe could find some fraction of the soluble protein inside of the cells. **Every
time we grew our *E. coli* cells with the CP5 construct we saw a noticeable decrease in the weight of
the biomass compared to other proteins.** Therefore we made a conclusion that there must have been some
fraction of the proteinase that lysed the cells from the inside.

#### Test

We tried **seven different *E. coli* strains:** BL21(DE3), Rosetta-gami (De3), Rosetta (De3), C41, Arctic express,
HMS(17) and KRX. **None of them produced any soluble CP5 protein in any considerable amount,** although the biomass was
smaller in every instance.

#### Learn

The **protein was obviously activated and folded correctly in some cells** because every could only grow about half
as much biomass compared to other proteins with identical growth conditions. Since a foreign proteinase was activated inside
*E. coli* cell, the cells were lysed and the active protein was probably denatured and
lost in the growth medium.

### Cycle 3

#### Design

As we could not produce any active protein for the SELEX process, we tried to **produce some soluble
denatured protein in the pro-form** and perform the SELEX process with it and then process the blood
sample in such way that the proteins in it would be similar to our denatured analytes in the lab.

#### Build

At first, we **lysed the cells in denaturing guanidine-hydrochloride and urea buffers to solubilized inclusion bodies.**
Then we decided to just dialyze the whole cell lysate against the soluble lysis buffer with various
different additives that might help to stabilize the protein in solution. Then we centrifuged the insoluble cell
debris and considered to use this solution for downstream SELEX application as the lysate.

#### Test

The additives we tried out were arginine (0.375 M), trehalose (0.75 M), proline (0.5 M), mannitol (0.5 M)
and CuCl2 (10 mM) [5,6]. Some of them were clear as water after the centrifugation - this result showed that nothing
from our lysate was soluble in them, but **a few were cloudy to some degree which was promising.**

#### Learn

We found out that the protein was stable in a soluble lysis buffer containing 10mM CuCl2,
but the amount of the stable protein was very small and not enough for further applications.
In the end, **we decided not to use CP5 as a biomarker for *Entamoeba histolytica.***

### Fusion protein modeling

### Cycle 1

#### Design

Since one of the parts of our project was to create a fusion protein system for the bottleneck
reaction of naringenin synthesis, we decided to model it *in silico*. The model’s primary purpose is
 **to check whether the shorter distance between active sites leads to the higher production of naringenin.**
In the process, we found out that even the modeling part required to cover the main steps of engineering:
design, build, test, and learn. Therefore, we began with the already existing modeling workflow described
for a fusion construct with a malaria pre-erythrocytic vaccine candidate [7].
The described modeling approach was initially designed as follows:

1. Choosing the protein candidates
2. Selecting linkers
3. Primary structural analysis of the fusion system
4. Protein modeling
5. Scoring modeled complexes to choose the model for visualization
6. Plotting Ramachandran graphs before structure refinement
7. Structure refinement
8. Plotting Ramachandran graphs after structure refinement

#### Build

The building stage of the modeling flow consisted of performing the initial design flow.
In this cycle we have raised a hypothesis that **homology-based protein modeling approaches
should not be the most suitable method for our case,** since there are few fusion models existing.

1. The protein candidates were chosen to be 4-coumarate-CoA ligase 2 from *Glycine max* and chalcone synthase from *Arabidopsis thaliana*
2. **Seven linkers were chosen for our fusion system.** Four of them were flexible glycine and serine linkers (GSG, (GGGGS)n, where n is equal to 1, 2, and 3) and the other three were rigid ((EAAAK)n, where n is equal to 1, 2, and 3)
3. For a primary structural analysis we studied PDB: **3TSY structure - an experimentally determined structure of the fusion system that has 4CL protein in it.** We took this structure as a starting point of what we can expect from our models
4. Protein modeling was initially attempted to perform using **homology-based modeling program SWISS-MODEL** [8]
5. The modeled structures in the first cycle were evaluated using VoroMQA [9], QMEAN [10], and QMEANDisCo [11] scores
6. The plot was not drawn in this cycle
7. The structure refinement was not performed in this cycle
8. The plot was not drawn in this cycle

#### Test

The output of SWISS-MODEL showed that **sequence identity between the templates found and the sequence
that is modeled is 66.42 - 69.36%.** According to the specialists in protein modeling,
homology-based methods are suitable, when the sequence identity between the template
and the modeled sequence is more than 25%, belonging to the “daylight" zone [13], thus, **in theory,
the method could be applied.**

The **modeling flow was tested with PyMOL** [12] visualization program using commands
`cealign`, `set seq\_view`. We were looking for an accurate representation of the
linked proteins in the fusion system by comparing them to their distinct versions.
**The main focus on the system was the linker region**, which in the homology-based modeling
case was composed of the uncoiled domain of 4CL that made up a highly disordered massive linker region.
We stopped the flow after the visualization because we decided that **structure refinement using molecular
dynamics will not introduce any significant changes to the structure.**

#### Learn

Nonetheless in theory the homology-based modeling method could be applied,
these modeling results proved that this approach could not be applied to our fusion protein system.
Therefore, **we decided to try running *ab initio* protein modeling programs.**

### Cycle 2

#### Design

The design (main steps) of the modeling flow stayed the same as it was in the first cycle,
yet also we included one more step - energy minimization with Yasara [16] -
that was inserted after the protein modeling step. The main changes in the second
cycle were the choice of protein modeling programs. In this engineering cycle we took *ab initio*
modeling programs trRosetta [14] and RoseTTAFold [15] to model our proteins.

#### Build

1. **Protein candidates stayed the same** as in the first cycle
2. **Linkers stayed the same** as in the first cycle
3. **Primary structure analysis was not required** to be redone in the second cycle
4. Protein modeling was performed **using trRosetta and RoseTTAFold** simultaneously
5. The modeled structures were evaluated **using VoroMQA score**
6. **Energy minimization with Yasara**
7. The Ramachandran plots were drawn before molecular dynamics (MD) simulations
8. We performed **short MD simulations for the trivial linker (GSG)** case
9. The Ramachandran plots were drawn before molecular dynamics (MD) simulations

#### Test

The modeled structures visually were not satisfactory - **the disordered domain of 4CL
protein was still present in the structure.** The energy minimization step with Yasara
and MD simulations did not introduce any significant changes to the structure.

#### Learn

After this step we decided to **consult the bioinformatician** who works with protein modeling.
He suggested we **include multiple sequence alignment files** into our modeling flow. Additionally,
we got advice to use **more scoring functions in the evaluation step.**
Yet also we consulted the protein molecular dynamics specialist to get a professional
insight into how we should run the MD for our system to get a more significant output.
After the consultation **we adjusted the box size of the system and the duration of the simulations.**

### Cycle 3

#### Design

In the third engineering cycle, we included **our own generated multiple sequence alignment (MSA)**
files as input to the protein modeling programs of our choice.
In addition, we took into account the advice from the specialists and **included
QMEAN and QMEANDisCo scores** into the evaluation of the structures. The structure
refinement was not performed in this cycle due to the lack of computational resources at the time.

#### Build

1. Protein candidates **stayed the same** as in the previous cycle
2. Linkers **stayed the same** as in the previous cycle
3. Primary structure analysis was not required to be redone
4. Protein modeling was performed **using RoseTTAFold**
5. The modeled structures were evaluated **using QMEAN, QMEANDisCo, and VoroMQA scores**
6. The plot was not drawn in this cycle
7. The structure refinement was not performed in this cycle
8. The plot was not drawn in this cycle
9. The distances between active sites in fusion protein systems with rigid linkers were calculated

#### Test

The modeled structures were visually satisfactory -
the **disordered domain of 4CL protein was less disordered** in the modeled structure.

#### Learn

The multiple sequence alignment (MSA) files have a significant impact on the modeling output.

### Cycle 4

#### Design

According to the output of the third cycle, **the fourth engineering cycle was not required.**
However, after the latter cycle was finished, **AlphaFold2 [17] became available for public use.**
Therefore, we decided to **apply this highly evaluated tool** in our modeling workflow.
The structure refinement was not performed in this cycle due to the insignificant impact of
MD for structures recorded in the studies [18].

#### Build

1. Protein candidates **stayed the same** as in the previous cycle
2. Linkers **stayed the same** as in the previous cycle
3. Primary structure analysis was not required to be redone
4. Protein modeling was performed **using AlphaFold2**
5. The modeled structures were evaluated **using QMEAN, QMEANDisCo, ProQ2D, ProQRosCenD, ProQRosFAD, ProQ3D scores**
6. The plots were drawn in this cycle
7. The structure refinement was not performed in this cycle
8. **The distances between active sites** in fusion protein systems with rigid linkers were calculated

#### Test

The output of the fourth cycle had the **less disordered domain** just as the output of the third cycle.

#### Learn

We were **visually satisfied** with the modeled complex.
Nonetheless, **AlphaFold2 models do not require the usage of molecular
dynamics** for structure refinement, we considered applying them to get a
better insight of the system with flexible linkers.

### SELEX

### Cycle 1

#### Design

Before beginning SELEX with purified soluble proteins we chose a few SELEX protocols available from the internet. One in the industry called XELEX [19] and another from an article specifically intended for fool-proof aptamer discovery[20].

#### Build

We tried the **initial SELEX runs with the default parameters** but for some reason, it kept failing, so we chose as many parameters as we could in order to optimize the process. We settled on **the bead and protein amount** in the SELEX mixture, **the heating of aptamers** before the incubation length, **the storage buffer** of the aptamers post-cycle, and the **polymerase** itself.

#### Test

We **lowered the amount of protein and beads** because we suspected that it might interfere with both the SELEX process and the downstream PCR reaction. We also **lowered the heating** of the aptamers before the selection round from 10 to 5 minutes based on another source [21]. We also **substituted the TE exchange buffer with distilled water** because the EDTA in the TE buffer interferes with the PCR reaction polymerase. Finally, we **tested the efficiency of the PCR** reactions with both DreamTaq and Phusion polymerases.

#### Learn

In the end, **we did manage to optimize the SELEX process** enough for it to produce aptamers. We determined that more protein and beads result in obstruction of SELEX. Half of the initial quantity should be applicable for second and further rounds. we also found out that **we should heat the aptamer pool for 5 minutes instead of 10 minutes** after the first round of SELEX. Heating for too long results in degradation or other forms of loss of our dsDNA from the previous round. The aptamer pool post-selection round should be stored in water, not in TE buffer as the EDTA interferes with the functions of polymerase, **Phusion polymerase seems to not be affected as much as DreamTaq,** and works better in both water and TE buffer.

### Cycle 2

#### Design

For the second cycle, we researched that **less bead and protein complex should have better effect in regenerating aptamers.** The first SELEX round should have **higher quantity of beads** for making sure that all possible aptamers bind. However, second and further rounds should have **half of the initial volume.** It also increases stringency parallel to other parameters.

#### Build

In order to check which quantity of complex suits us best, we developed a plan to try changes on separate initial and move on to further rounds.

#### Test

We tested unchanged bead and protein complex quantities for a few rounds and tried various complex volumes for second and further rounds side by side. Comparing volumes reduced by ⅓, ½ and ⅔, we found that **change to half of the initial volume suited our SELEX protocol the best.**

#### Learn

We found that **first round should have larger volume of bead complex and lower in later rounds.** More protein and beads result in obstruction of SELEX. Half of the initial quantity should be applicable for second and further rounds.

### Genome editing

### Cycle 1

#### Design

One of our project’s parts is **genetically modified probiotics** that are capable of performing *de novo* synthesis of natural flavonoid naringenin. This flavonoid has reached our attention as it remarkably reduces *Entamoeba histolytica* viability [22] and does not cause any harm to humans. Conversely, it has been found that naringenin has **antioxidant, antitumor, antiviral, antibacterial, anti-inflammatory, anti-adipogenic, and cardioprotective features [23].** To create an efficiently working naringenin production system, we have been searching for various tools - the synthetic protein quality control (ProQC) system, the most efficient metabolic pathway enzymes, promoters of particular strength, etc. However, it is not only important to construct a fully functional system but also to ensure its stability in the chosen chassis. For this reason, we chose to insert naringenin synthesis metabolic pathway into our probiotic strains: *E. coli* Nissle 1917 and *Lactobacillus casei* BL23. Insertion into genomic DNA has several advantages. Firstly, it **provides stability to the system.** If the new metabolic pathway does not cause too much burden to the chassis, it should be maintained in genome active (otherwise there might be some mutations suppressing its functionality, reduced cell growth, enhanced cell sensitivity to nutrient deficiency [24]). Surprisingly the integration of the genetic circuit of the metabolic pathway into the chassis genome contributes to the burden reduction as it serves as DNA copy number tuning. Secondly, by inserting the desired sequences in the genome we **eliminate the need for antibiotics** or any other environmental pressure usage to maintain needed genetic circuits in the chosen chassis. This is very important when the designed naringenin-producing bacteria will live in the human intestine.

In order to reach this goal, we did a huge research in the genome editing techniques field. With the help of our advisor Inga Songailienė, who is focusing on CRISPR-Cas systems in her Ph.D. thesis, we have **chosen to use the pCas-pTarget plasmids system** which enables the usage of CRISPR-Cas9 and Lambda Red recombination combinment into the one system to acquire the efficient *E. coli* Nissle 1917 genome editing [25]. For *L. casei* BL23 we have chosen **a single plasmid pLCNICK based system** relying on CRISPR-Cas9 D10A Nickase-Assisted genome editing [26]. For the genomic insertion site in *L. casei* BL23 we decided to follow the pLCNICK already provided sequences required for genome editing. We followed this approach because we have run out of resources for new DNA sequences, and for this chassis, there is a need for longer homology arms. Meanwhile, we turned our all focus on E. coli Nissle 1917 to design the functional and robust metabolic pathway. In the future, we would adapt it to the *L. casei* BL23, too.

During the design stage, we first **considered genomic sequences** that would be used for metabolic pathway insertion. From literature, we learned that **16S rRNA genes might be used for this purpose** [27]. However, it would not let us implement a ProQC system due to 16S rRNA transcript being created as polycistronic RNA. For this reason, we targeted **non-essential gene** - *colicin*. We have also considered possible metabolic flux enhancement toward naringenin synthesis. It covers acetate kinase (ackA), phosphate acetyltransferase (pta) knockouts creation to channel carbon flows toward acetyl-CoA [28]. Subsequently, it leads to the enhanced malonyl-CoA amounts needed for bottle-neck reaction in naringenin production [29]. Also, tyrosine-specific transporter (tyrP) knockout mutants are potentially able to produce **10% higher amounts of L-tyrosine** (naringenin synthesis precursor) than that of the original strains [30]. In addition, aldehyde-alcohol dehydrogenase (adhE) knockout disrupts the conversion of acetyl-CoA to ethanol in the cell, therefore more acetyl-CoA can be converted to malonyl-CoA [31] further enhancing naringenin synthesis.

#### Build

To fulfill our experimental plan, we have designed **sgRNA for genomic insertion** and earlier mentioned knockouts generation. For this reason, we used **CRISPOR tool** [32] to assess the probability of sgRNA efficiency and off-targets generation. Furthermore, after selecting sgRNA for each gene we have checked how many other similar DNA sequences exist in the *E. coli* Nissle 1917 genome. For this reason, we have used **NCBI Blast tool** [33] to analyse whether there are any other identical sequences to the seed region of the chosen sgRNA. It is important to be aware of this because it might contribute to possible off-targets [34]. Also, for genome insertion we decided to use **approximately 70-90 bp long homology sequences** flanking the inserts as it should work on this system [35]. By following these considerations we have constructed sgRNAs specific to *ackA* ([BBa\_K3904401](http://parts.igem.org/Part%3ABBa_K3904401)), *pta* ([BBa\_K3904402](http://parts.igem.org/Part%3ABBa_K3904402)), *adhE* ([BBa\_K3904403](http://parts.igem.org/Part%3ABBa_K3904403)), *tyrP* ([BBa\_K3904404](http://parts.igem.org/Part%3ABBa_K3904404)), *colicin* ([BBa\_K3904405](http://parts.igem.org/Part%3ABBa_K3904405)) genes.

#### Test

We have performed genome editing as it is described in the literature [25]. However, genome editing has not been very efficient as very **few cells have survived** and not all of them contained desired modifications.

#### Learn

As we have received these results, we have hypothesized that some of the cells **might lose pCas plasmid** as it has a temperature-sensitive replicon.

### Cycle 2

#### Design

In the second genome editing cycle we have used the same parts but **changed some experimental conditions.** We have contacted our advisor Inga Songailienė, who gave us some insights about this system.

#### Build

Here is experimental conditions which we have decided to change:

1. Lower growth temperature **from 30°C to 27.5°C** because a water filled incubator might show a bit lower temperature than it is in it.
2. Cells have been induced for recombination at **0.5-0.8 OD instead of 0.4-0.6 OD** during preparation for co-electroporation with pTarget and recombination template.
3. Enhanced the L-arabinose concentration till **0.2 % (previously it was 0.15 %).**

#### Test

We get *pta* and *ackA* gene knockouts. However, even after several repeats and recombination template amount increase (600 ng instead of 400 ng), new recombination template preparation, ***adhE* and *tyrP* genes left unmodified.**

#### Learn

By these changes, we learned that the **timing of recombination induction is very important.** However, we still had some unsolved problems with two left genes.

### Cycle 3

#### Design

As we saw there were still some undetected problems. After further literature research, we found some alterations to this system [26]. Also, by this time we had constructed our **kill-switch system,** so we decided to insert it and superfolder green fluorescent protein (sfGFP) into the *colicin* gene. We used sfGFP to check how well the designed system is working and kill-switch VapXD system integration into genomic DNA is important for evaluation of how this system would perform in the final product.

#### Build

In this engineering cycle, we have changed the induction timing - **L-arabinose to final 0.2 % concentration has been added at 0.2-0.3 OD600** and cells harvested by centrifugation then **the OD600 has reached 0.6-0.8.** Recombination template amount for kill-switch constructs, GFP - 400 ng, while for *tyrP* and *adhE* - 800 ng as previously there were many colonies resistant to both antibiotics but **none of them insertion-positive.**

#### Test

Both kill-switch systems and sfGFP constructs have been inserted into genomic DNA with almost 100 % accuracy leading to **7-12 insertion-positive colonies** and just **two survived colonies** in the control plate. Meanwhile, *adhE* and *tyrP* genes knockout mutants still have not been generated. There were from hundreds to thousands of colonies in control plates, of those two mutant creation experiments. A bit smaller numbers occurred in plates where mutants were expected but not found.

#### Learn

The results of kill-switch and sfGFP insertion into the genome proved the system and chosen sgRNA for the *colicin* gene is working. However, just a bit of GFP fluorescence could be seen by the eye after blue light use. This might be because very little GFP is produced from only one DNA copy of this construct. Thus,urther investigation needed to be done.

The amount of colonies raised after several tries to generate *tyrP* and *adhE* genes knockouts lead to two possible explanations. Firstly, there was a huge possibility that chosen **sgRNA is incapable to properly lead Cas9 endonuclease to a particular genomic site** and causing double-strand break. This lead to almost all bacteria surviving even if they have Cas9 and gene-specific sgRNA inside. Second possible explanation would be that those **two genes are too important for bacteria surveillance,** so either it escapes double strand break occurrence, or it dies because of nonfunctional gene creation after successful genomic recombineering.

### Cycle 4

#### Design

After taking earlier mentioned considerations together, we decided to design **new sgRNA for *adhE* and *tyrP* genes** to test whether our previously designed **sgRNA are truly inefficient** or can not be created mutants of *tyrP* and *adhE* genes knockouts. Moreover, as GFP levels from colonies with genomic insertion of its construct **did not generate any clear fluorescence,** we looked deeper into the pattern of genome wide *E. coli* transcriptional map [27]. From the literature and consultation with our advisor Inga Songailienė, we have chosen **another genomic target for metabolic pathway insertion.** *nupG* gene is responsible for broad-specific nucleoside permease synthesis.

#### Build

*nupG* **specific sgRNA** have been selected by CRISPOR [22] tool. Recombination template created by three steps PCR with long overhangs.

#### Test

GFP, linked 4CL-CHS, TAL and CHI genes have been inserted into *nupG* and *colicin* genes. *tyrP, adhE* knockouts have not been obtained.

#### Learn

From GFP fluorescence measurements we have learned that *nupG* it is transcriptionally more active. Also, we have found out that our designed primers pair for the third PCR round of recombination template creation for insertion into *nupG* gene are not suitable. These primers do not generate any PCR product as they probably do not anneal to the genomic DNA. This might be because of some nucleotides variation in the *nupG* gene in the Nissle genome. In order to evade this problem firstly for genomic editing chosen gene should be sequenced and only after that all over needed DNA constructs should be designed.

## References

1.

Wong, W. K., Tan, Z. N., Othman, N., Lim, B. H., Mohamed, Z., Olivos Garcia, A., & Noordin, R. (2011). Analysis of Entamoeba histolytica Excretory-Secretory Antigen and Identification of a New Potential Diagnostic Marker. Clinical and Vaccine Immunology, 18(11), 1913–1917. [To the article.](https://journals.asm.org/doi/full/10.1128/CVI.05356-11)

2.

Saidin, S., Yunus, H. M., Zakaria, D. N., Razak, A. K., Huat, B. L., Othman, N., & Noordin, R. (2014). Erratum to: Production of recombinant Entamoeba histolytica pyruvate phosphate dikinase and its application in a lateral flow dipstick test for amoebic liver abscess [BMC Infect Dis, 14, (2014), 182, doi: 10.1186/1471-2334-14-182]. BMC Infectious Diseases, 14(1), 1–9. [To the article.](https://doi.org/10.1186/1471-2334-14-533)

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Olechnovič, K., & Venclovas, Č. (2019). VoroMQA web server for assessing three-dimensional structures of proteins and protein complexes. Nucleic acids research, 47(W1), W437-W442. [To the article.](https://academic.oup.com/nar/article/47/W1/W437/5487673?login=true)

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Benkert, P., Tosatto, S. C. E., & Schomburg, D. (2008). QMEAN: A comprehensive scoring function for model quality assessment. Proteins: Structure, Function, and Bioinformatics, 71(1), 261–277. doi:10.1002/prot.21715 [To the article.](https://pubmed.ncbi.nlm.nih.gov/17932912/)

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Studer, G., Rempfer, C., Waterhouse, A. M., Gumienny, R., Haas, J., & Schwede, T. (2020). QMEANDisCo—distance constraints applied on model quality estimation. Bioinformatics, 36(6), 1765-1771. [To the article.](https://doi.org/10.1093/bioinformatics/btz828)

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DeLano, W. L. (2002). Pymol: An open-source molecular graphics tool. CCP4 Newsletter on protein crystallography, 40(1), 82-92. [To the article.](http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.231.5879&rep=rep1&type=pdf#page=44)

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Venclovas, Č. (2011). Methods for sequence–structure alignment. Homology Modeling, 55-82. [To the article.](http://www.bti.vu.lt/dokumentai/skyriu_medziaga/bi/publications/Venclovas_Methods_for_Sequence-Structure_Alignment_2012.pdf)

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Yang, J., Anishchenko, I., Park, H., Peng, Z., Ovchinnikov, S., & Baker, D. (2020). Improved protein structure prediction using predicted interresidue orientations. Proceedings of the National Academy of Sciences, 117(3), 1496-1503. [To the article.](https://www.pnas.org/content/117/3/1496.short)

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Baek, M., DiMaio, F., Anishchenko, I., Dauparas, J., Ovchinnikov, S., Lee, G. R., ... & Baker, D. (2021). Accurate prediction of protein structures and interactions using a 3-track network. bioRxiv. , [To the article.](https://doi.org/10.1101/2021.06.14.448402)

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Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., ... & Hassabis, D. (2021). Highly accurate protein structure prediction with AlphaFold. Nature, 1-11. [To the article.](https://doi.org/10.1038/s41586-021-03819-2)

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Simpkin, A. J., Rodríguez, F. S., Mesdaghi, S., Kryshtafovych, A., & Rigden, D. J. (2021). Evaluation of model refinement in CASP14. Proteins: Structure, Function, and Bioinformatics. [To the article.](https://doi.org/10.1002/prot.26185)

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Roboklon. (2019, May). XELEX DNA Core Kit. [To the article.](https://roboklon.com/pdf/240_det_en.pdf)

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Wang, T., Yin, W., AlShamaileh, H., Zhang, Y., Tran, P., Nguyen, T., … Duan, W. (2019). A detailed protein-SELEX protocol allowing visual assessments of individual steps for high success rate. Human Gene Therapy Methods. [To the article.](doi.org/10.1089/hgtb.2018.237)

21.

Sefah, K., Shangguan, D., Xiong, X., O’Donoghue, M. B., & Tan, W. (2010). Development of DNA aptamers using Cell-SELEX. Nature Protocols, 5(6), 1169–1185. [To the article.](doi.org/10.1038/nprot.2010.66)

22.

Quintanilla-Licea, R., Vargas-Villarreal, J., Verde-Star, M. J., Rivas-Galindo, V. M., & Torres-Hernández, Á. D. (2020). Antiprotozoal activity against Entamoeba histolytica of flavonoids isolated from Lippia graveolens kunth. Molecules, 25(11), 2464. [To the article.](doi.org/10.1038/nprot.2010.66)

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Salehi, B., Fokou, P., Sharifi-Rad, M., Zucca, P., Pezzani, R., Martins, N., & Sharifi-Rad, J. (2019). The Therapeutic Potential of Naringenin: A Review of Clinical Trials. Pharmaceuticals (Basel, Switzerland), 12(1), 11. [To the article.](https://doi.org/10.3390/ph12010011)

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Jiang, Y., Chen, B., Duan, C., Sun, B., Yang, J., & Yang, S. (2015). Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Applied and environmental microbiology, 81(7), 2506-2514. [To the article.](https://doi.org/10.3390/ph12010011)

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Song, X., Huang, H., Xiong, Z., Ai, L., & Yang, S. (2017). CRISPR-Cas9D10A nickase-assisted genome editing in Lactobacillus casei. Applied and environmental microbiology, 83(22), e01259-17. [To the article.](https://doi.org/10.3390/ph12010011)

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Riedel, C. U., Casey, P. G., Mulcahy, H., O'Gara, F., Gahan, C. G., & Hill, C. (2007). Construction of p16Slux, a novel vector for improved bioluminescent labeling of gram-negative bacteria. Applied and environmental microbiology, 73(21), 7092–7095. [To the article.](https://doi.org/10.1128/AEM.01394-07)

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Ku, J. T., Chen, A. Y., & Lan, E. I. (2020). Metabolic engineering design strategies for increasing acetyl-CoA flux. Metabolites, 10(4), 166. [To the article.](https://doi.org/10.1128/AEM.01394-07)

29.

Dunstan, M. S., Robinson, C. J., Jervis, A. J., Yan, C., Carbonell, P., Hollywood, K. A., ... & Scrutton, N. S. (2020). Engineering Escherichia coli towards de novo production of gatekeeper (2 S)-flavanones: naringenin, pinocembrin, eriodictyol and homoeriodictyol. Synthetic Biology, 5(1), ysaa012. [To the article.](https://doi.org/10.1128/AEM.01394-07)

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Scholz, S. A., Diao, R., Wolfe, M. B., Fivenson, E. M., Lin, X. N., & Freddolino, P. L. (2019). High-resolution mapping of the Escherichia coli chromosome reveals positions of high and low transcription. Cell systems, 8(3), 212-225. [To the article.](https://doi.org/10.7717/peerj.8881)

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# Team:Vilnius-Lithuania/Experiments

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# EXPERIMENTS

![Header](https://static.igem.org/mediawiki/2021/1/19/T--Vilnius-Lithuania--Experiments.JPG)

[PROTOCOLS](https://static.igem.org/mediawiki/2021/e/e0/T--Vilnius-Lithuania--protocols_and_materials.pdf)
[BENCHLING

 PREVENTION](https://static.igem.org/mediawiki/2021/1/16/T--Vilnius-Lithuania--BENCHLING_PREVENTION.zip)
[BENCHLING

 DETECTION](https://static.igem.org/mediawiki/2021/8/85/T--Vilnius-Lithuania--BENCHLING_DETECTION.zip)

## Prevention

Recovery of *Escherichia coli* Nissle 1917 and *Lactobacillus casei* BL23 cultures.

 Searching for the most suitable and effective *E. coli* Nissle 1917 and *L. casei* BL23 genome editing strategy.

## Detection

pET28a(+)-CP5, pET28a(+)-PPDK plasmid construction.

 Recombinant CP5 production optimization.

## Prevention

Preparation of *E. coli* Nissle 1917 and *L. casei* BL23 competent cells.

 Genome editing strategy development.

## Detection

Recombinant CP5 purification.

 pET28a(+)-N-His, pET28a(+)-N-His-TEV plasmid construction.

## Prevention

pTRKH2+sfGFP and pTRKH2+loop+sfGFP construction for fluorescence measurements.

 sgRNAs, recombination templates, primers construction *in silico*.

## Detection

Recombinant CP5 refolding and activation.

## Prevention

pTRKH2+sfGFP and pTRKH2+loop+sfGFP construction for fluorescence measurements.

 pTarget receiving from our advisor and multiplication in *E. coli* DH5 alpha strain.

## Detection

Recombinant CP5 refolding and activation.

## Prevention

pTRKH2+sfGFP and pTRKH2+loop+sfGFP construction for fluorescence measurements.

## Detection

ePCR preparation.

 Recombinant CP5 refolding and activation.

## Prevention

pTRKH2+sfGFP and pTRKH2+loop+sfGFP construction for fluorescence measurements.

 pLCNICK plasmid construction with sfGFP.

## Detection

ePCR preparation.

 Recombinant CP5 refolding and activation.

 Recombinant PPDK production optimization.

## Prevention

pTRKH2+sfGFP and pTRKH2+loop+sfGFP construction for fluorescence measurements.

 sgRNA and gRNA oligos annealing and ligation with pTarget plasmids.

 Construction of recombination template with *colicin* homology arms and sfGFP insertion, reactions’ conditions optimizations.

 pLCNICK plasmid construction with sfGFP.

## Detection

Recombinant PPDK production optimization.

## Prevention

pTRKH2+sfGFP and pTRKH2+loop+sfGFP construction for fluorescence measurements.

 pTarget plasmid construction.

 pLCNICK plasmid construction with sfGFP.

## Detection

Recombinant PPDK solubilisation and purification.

## Prevention

pTRKH2+sfGFP and pTRKH2+loop+sfGFP fluorescence measurements.

 Terminator insertion to the pTRKH2+loop system.

 pTarget amplification, purification and preparation for sequencing.

 *Colicin, adhE, ackA, pta, tyrP* PCR from genomic DNA optimization.

## Detection

Experimental PDA synthesis.

 Recombinant and denatured PPDK and CP5 stabilization experiments.

## Prevention

pTRKH2+TAL/4CL/CHS/CHI and pTRKH2+loop+TAL/4CL/CHS/CHI construction.

 Analysis of *ackA, pta, adhE, tyrP* genes sequencing results and previously designed sgRNA validation.

## Detection

Anti-HSA aptamer conjugation with PDA.

 Testing of PDA with Anti-HSA aptamer.

 Recombinant PPDK purification.

## Prevention

pTRKH2+TAL+4CL+CHS+CHI and pTRKH2+loop+TAL+4CL+CHS+CHI construction.

 pCas9 plasmid amplification.

 Nissle electroporation with pCas9.

 sfGFP insertion into *L. casei* BL23.

## Detection

Protein purification with magnetic beads.

 SELEX preparation, first round.

## Prevention

pTRKH2+TAL-sfGFP and pTRKH2+loop+TAL-sfGFP construction.

 pTRKH2+TAL+4CL+CHS+CHI and pTRKH2+loop+TAL+4CL+CHS+CHI construction.

 sfGFP insertion into *colicin* gene in *E. coli* Nissle 1917.

 sfGFP insertion into *L. casei* BL23.

## Detection

SELEX rounds 1-8.

## Prevention

VapXD system construction.

 pTRKH2+TAL-sfGFP and pTRKH2+loop+TAL-sfGFP construction.

 pTRKH2+TAL+4CL+CHS+CHI and pTRKH2+loop+TAL+4CL+CHS+CHI construction.

 *AckA, pta, adhE, tyrP* knockouts creation.

## Detection

Aptamer ligation into pUC19 for Sanger sequencing.

 NGS preparation.

## Prevention

VapXD system construction.

 pTRKH2+TAL-sfGFP and pTRKH2+loop+TAL-sfGFP construction.

 pTRKH2+TAL+4CL+CHS+CHI and pTRKH2+loop+TAL+4CL+CHS+CHI construction.

 *AckA, pta, adhE, tyrP* knockouts and their combinations creation.

## Detection

NGS preparation.

## Prevention

VapXD serial dilution spotting.

 pTRKH2+TAL-sfGFP and pTRKH2+loop+TAL-sfGFP fluorescence measurements.

 pTRKH2+TAL+4CL+CHS+CHI and pTRKH2+loop+TAL+4CL+CHS+CHI construction.

 Fusion protein construction.

 *AckA, pta, adhE, tyrP* knockouts and their combinations creation.

## Detection

Sanger sequencing data analysis.

 NGS preparation.

## Prevention

pTRKH2+TAL-sfGFP and pTRKH2+loop+TAL-sfGFP fluorescence measurements.

 VapXD serial dilution spotting.

 pTRKH2+TAL+4CL+CHS+CHI and pTRKH2+loop+TAL+4CL+CHS+CHI construction.

 *AckA, pta, adhE, tyrP* knockouts and their combinations creation.

 VapXD system insertion into *colicin* gene in *E. coli* Nissle 1917.

## Detection

Sanger sequencing data analysis. NGS of aptamers.

## Prevention

Plate reader measurements of VapXD construct in pTRKH2 plasmid.

 pTRKH2+TAL+4CL+CHS+CHI and pTRKH2+loop+TAL+4CL+CHS+CHI construction by Gibson assembly.

 *AckA, pta, adhE, tyrP* knockouts and their combinations creation.

## Detection

NGS data analysis.

## Prevention

Fusion protein construction.

 New crRNA (for *tyrP, adhE, nupG*) annealing with gRNA and ligation into pTarget.

 Recombination template construction for VapXD system insertion into *nupG* gene.

 sfGFP insertion into wild type, new knockouts combinations creation with newly obtained sgRNA.

 Genes of naringenin synthesis enzymes insertion into *colicin* and *nupG* genes into wild type and *ackA-pta* knockout strains.

## Detection

Anti-PPDK aptamer conjugation with PDA.

 pTRKH2+TAL+4CL+CHS+CHI and pTRKH2+loop+TAL+4CL+CHS+CHI construction by Gibson assembly.

## Prevention

Plate reader measurements of VapXD construct in *E. coli* Nissle 1917 genome.

 pTRKH2+TAL+4CL+CHS+CHI and pTRKH2+loop+TAL+4CL+CHS+CHI construction by Gibson assembly.

 Genes of naringenin synthesis enzymes insertion into *colicin* and *nupG* genes into wild type and *ackA-pta* knockout strains.

 Intermediate compounds of naringenin synthesis from *E. coli* Nissle 1917 growth medium and cell lysate identification by MS.

## Detection

Diagnostic test assembly.

 IT aptamer evaluation.

###### JUNE

###### JULY

###### AUGUST

###### SEPTEMBER

###### OCTOBER

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# Team:Vilnius-Lithuania/Results

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# RESULTS

![Header](https://static.igem.org/mediawiki/2021/0/05/T--Vilnius-Lithuania--Results.jpg)

### Prevention

#### Reconstruction of shuttle vector

For the construction of the naringenin synthesis cassette that would work in both *Escherichia coli* Nissle 1917 and *Lactobacillus casei* BL23, we chose to use the **pTRKH2 shuttle** vector. We modified the plasmid’s original multi-cloning site (MCS). This was successfully done by amplifying the pTRKH2 vector with MCS deletion primers in PCR reaction (Fig. 1), and generating the new MCS site with designed oligonucleotides (Fig. 1).

![](https://static.igem.org/mediawiki/2021/d/d8/T--Vilnius-Lithuania--multiple_assembly_gel.png)

**Fig. 1.**  Restriction analysis of pTRKH2 vector with new MCS. 1st sample - restriction control, 2nd - pTRKH2 with old MCS, 3rd-6th - pTRKH2 new old MCS.

#### Promoter characterization

Before the actual experiments, we optimized our measurement condition with an iGEM measurement kit according to the [standard protocol](https://www.protocols.io/view/calibration-protocol-plate-reader-fluorescence-cal-6zrhf56). This optimization allowed choosing the most appropriate gain settings in the plate reader.

To assure the most efficient possible naringenin production pathway, we had to select the most suitable promoters for the expression of naringenin synthesis genes.
Evaluation was done by analysing the results of superfolder green fluorescent protein (**sfGPF**) **expression rates** under the promoters of interest and dividing the intensiveness of the signal by the OD600 during the course of 6 hours (Fig. 2).

![](https://static.igem.org/mediawiki/2021/e/e7/T--Vilnius-Lithuania--promoter-old-1.png)

**Fig. 2.**  Evaluation of promoter strength.

![](https://static.igem.org/mediawiki/2021/1/1b/T--Vilnius-Lithuania--promoter-old-2-fixed.png)

**Fig. 3.**  First graph - comparison of promoters strength by indicating the curves of the two strongest promoters and the generalized curve of the remaining weaker promoters; second graph - comparison
of acceleration versus velocity by indicating the curves of the two strongest promoters.

#### Evaluation of transcription efficiency dependency on *E. coli* Nissle 1917 genomic site

We decided to insert naringenin metabolic pathway encoding genes into ***E. coli* Nissle 1917 genome**. This experimental decision helps to overcome the problem of additional antibiotic usage, reduce the fluctuations gained because of unstable plasmid copy numbers in cells. Furthermore, metabolic pathway genomic insertion helps to overcome horizontal gene transfer problem. This is particularly important as probiotics interact with a huge variety of microorganisms in our intestine.

To measure the transcription activity from two genomic regions, we have inserted **sfGFP under slpA promoter into *colicin*** and ***nupG* genes** (fig. 4, 5) and compared the amount of fluorescence (fig. 6). As we can see in fig. 4, GFP insertion into *colicin* gene has been successful with **89 percent efficiency** and GFP insertion into *nupG* gene a bit lower (fig. 5).

![](https://static.igem.org/mediawiki/2021/6/60/T--Vilnius-Lithuania--GFP-colicin.png)

**Fig. 4.**  GFP insertion into *colicin* gene results. Here are represented with XbaI digested cPCR products from chosen transformant colonies. If insertion is successful, 1 kbp and 148 bp
products are expected. L - GeneRuler 1 bkp Ladder, 1 - negative control (wild type *E. coli* Nissle 1917), 2 - colicin-GFP (1), 3 - colicin-GFP (2), 4 - colicin-GFP (3), 5 - colicin-GFP (4), 6 - colicin-GFP (5), 7 - colicin-GFP
(6), 8 - colicin-GFP (7), 9 - colicin-GFP (8), 10 - colicin-GFP (9).

![](https://static.igem.org/mediawiki/2021/3/3c/T--Vilnius-Lithuania--GFP-nupG.png)

**Fig. 5.**  GFP insertion into *nupG* gene results. L - GeneRuler 1 kb Ladder (Thermo Fisher), 1 - nupG-GFP (1), 2 - nupG-GFP (2), 3 - nupG-GFP (3), 4 - nupG-GFP (4), 5 - nupG-GFP (5), 6
- nupG-GFP (6), 7 - nupG-GFP (7), 8 - nupG-GFP (8), 9 - nupG-GFP (9), 10 - nupG-GFP (10).

The results of fluorescence measurement (fig. 6) showed that **transcription activity** was **higher** and **more stable in *colicin* site**.

![](https://static.igem.org/mediawiki/2021/a/a3/T--Vilnius-Lithuania--positioning.png)

#### GFP expression in *L. paracasei* BL23

After promoters evaluation in *E. coli* DH5 alpha strain and finding out that p-slpA was the strongest promoter, we decided to insert sfGFP construct under p-slpA control into *L. paracasei* BL23 genome. For this reason, we have inserted sfGFP coding gene into pLCNICK plasmid and placed it under transcriptional control of slpA promoter. This new plasmid has been obtained by digestion of pLCNICK with BglI and BshTI restriction endonucleases and ligation with sfGFP construct. GFP insertion into pLCNICK plasmid has been validated by visible fluorescence. Designed system was only suitable for DH5 alpha strain bacteria (fig. 7). In consequence, we decided to further investigate *E. coli* Nissle 1917 as our chassis.

![](https://static.igem.org/mediawiki/2021/5/5c/T--Vilnius-Lithuania--Fluorescence.png)

#### mRNA cyclization system evaluation

During the same set of experiments as for promoter characterization, mRNA cyclization ([BBa\_K3904217](http://parts.igem.org/Part%3ABBa_K3904217)) performance was tested in our system.

At first, the fluorescence of *E. coli* Nissle 1917 bacteria containing pTRKH2+sfGFP and pTRKH2+loop+sfGFP plasmids with different promoters was measured.

Since mRNA cyclization system performance was not as expected, we decided to repeat the experiment while using longer sequence protein TAL fused with sfGFP. What is more, these measurements were conducted in *E. coli* DH5 alpha and at different temperatures of 37 °C and 24 °C, since during the construction of mRNA cyclization system, we noticed that it begins to fluoresce after incubation at room temperature.

Each experiment had three controls: positive – fluorescent *E. coli* DH5 alpha with J23101 Anderson promoter, negative – non-fluorescent bacteria, contamination – media with an antibiotic.

In the 8 figure, one can see that there is no significant difference in the mRNA cyclization performance in 37 °C and 24 °C because the shift of the curves is only affected by the lower OD600 values in 24 °C. The bottom graph illustrates averaged data at 37 °C. Interestingly, it was noticed that mRNA cyclization does not allow the accumulation of the sfGFP protein. After some time, the system reaches equilibrium, and the fluorescence/OD600 ratio stabilizes.

![](https://static.igem.org/mediawiki/2021/c/cf/T--Vilnius-Lithuania--promoter-new-1.png)

**Fig. 8.**  mRNA cyclization system performance.

##### Conclusions

One of our chosen protein synthesis enhancing mechanisms - mRNA cyclization - appeared not to work as expected. In our case, it did not increase protein production. Interestingly, protein synthesis stabilization was noticed as a novel property of this system and may be further tested.

#### Metabolic pathway construction

To construct the pTRKH2 vector containing all four genes of the naringenin metabolic pathway we amplified pTRKH2 vector and all four naringenin synthesis genes using primers that contain specific restriction endonuclease recognition sites. This way we should have been able to digest each sequence with appropriate restriction enzymes and create a library of inserts with sticky ends, that can be ligated into the target vector as the ending part of composite insert or as a part of the whole naringenin synthesis cassette. However, we were only able to construct plasmids containing only TAL and TAL+4CL under slpA promotor (Fig. 9), cassettes that later were found to have been mutated by Sanger sequencing.

![](https://static.igem.org/mediawiki/2021/e/e4/T--Vilnius-Lithuania--multiple_assembly.png)

**Fig. 9.** Restriction analysis (using NdeI restriction endonuclease) of the multiple assembly constructs: 1st-3rd samples contain pTRKH2+TAL construct, 4th sample contains pTRKH2+TAL+4CL construct.

To get the construct containing all four genes we chose the strategy of Gibson assembly. By amplifying the pTRKH2 vector and all naringenin synthesis genes with primers containing flanking regions that form homologous pairs with each other in the manner that a complete naringenin synthesis cassette should be constructed in a single tube reaction. Nevertheless, we were not able to obtain the desired construct. In a parallel, fused proteins were successfully constructed in the pTRKH2 vector and analysed further.

#### Endogenous metabolism modulation toward enhanced naringenin synthesis

To enhance naringenin synthesis in *E. coli* Nissle 1917 we have created *ackA-pta* double knockout. Firstly, we knockouted *ackA* (fig. 10), and *pta* (fig. 11) genes separately with 100 percent and 60 percent efficiency, respectively. Later on, we used *ackA* knockout to generate *ackA-pta* double knockout (fig. 12) with 80 percent efficiency. For further experiments used *ackA* knockout have been verified by *ackA* gene sequencing.

![](https://static.igem.org/mediawiki/2021/9/92/T--Vilnius-Lithuania--ackA-knockout.png)

**Fig. 10.**  Restriction of cPCR product representing ackA knockout generation. Colony PCR product is 300 bp long and restriction by BcuI generates two separate fragments - 131 bp and 108 bp, which
in this gel are seen as one line. L - GeneRuler 1 kbp DNA Ladder, 1 - *ackA* knockout (1), 2 - *ackA* knockout (2), 3 - *ackA* knockout (3), 4 - *ackA* knockout (4), 5 - *ackA* knockout (5), 6 - *ackA* knockout (6), 7 - *ackA* knockout (7), 8 - *ackA* knockout (8), 9 - *ackA* knockout (9), 10 - *ackA* knockout (10), 11 - *ackA* knockout (11), 12 - *ackA* knockout (12), 13 - *ackA* knockout
(13), 14 - *ackA* knockout (14), 15 - wild type *E. coli* Nissle 1917.

![](https://static.igem.org/mediawiki/2021/3/39/T--Vilnius-Lithuania--pta-knockout.png)

**Fig. 11.**  Restriction of cPCR product representing *pta* knockout generation. *pta* gene have been amplified from genomic DNA and restricted by BcuI. 2161 bp fragments represent wild
type genotype, 1797 bp and 364 bp - while knockouts. 1 - wild type (negative control), 2 - *pta* knockout (1), 3 - *pta* knockout (2), 4 - *pta* knockout (3), 5 - *pta* knockout (4), 6 - *pta* knockout
(5), 7 - *pta* knockout (6), 8 - *pta* knockout (7), 9 - *pta* knockout (8), 10 - *pta* knockout (9), 11 - *pta* knockout (10).

![](https://static.igem.org/mediawiki/2021/3/3a/T--Vilnius-Lithuania--acka-pta-knockout.png)

**Fig. 12.**  Restriction of cPCR product representing *ackA-pta* double knockout generation. *pta* gene have been amplified from genomic verified *ackA* knockout DNA and restricted
by BcuI. 2161 bp fragments represent wild type genotype, 1797 bp and 364 bp - while knockouts. 1 - wild type (negative control), 2 - no DNA added (wild type), 3 - *ackA-pta* knockout (1), 4 - *ackA-pta* knockout (2),
5 - *ackA-pta* knockout (3), 6 - *ackA-pta* knockout (4), 7 - *ackA-pta* knockout (5), 8 - *ackA-pta* knockout (6), 9 - *ackA-pta* knockout (7), 10 - *ackA-pta* knockout (8), 11 - *ackA-pta* knockout (9), 12 - *ackA-pta* knockout (10).

Our next move was to create *tyrP* knockout. Firstly, we have successfully obtained *tyrP* knockout (fig. 13).

![](https://static.igem.org/mediawiki/2021/0/0f/T--Vilnius-Lithuania--tyrP-knockout.png)

**Fig. 13.**  Restriction of cPCR product representing *tyrP* knockout generation. *tyrP* gene have been amplified from genomic DNA and restricted by BcuI. 1212 bp fragments represent
wild type genotype, 1014 bp and 184 bp - knockouts. 1 - wild type (negative control), 2 - *tyrP* knockout (1), 3 - *tyrP* knockout (2), 4 - *tyrP* knockout (3), 5 - *tyrP* knockout (4), 6 - *tyrP* knockout
(5), 7 - *tyrP* knockout (6), 8 - *tyrP* knockout (7), 9 - *tyrP* knockout (8), 10 - *tyrP* knockout (9), 11 - *tyrP* knockout (10).

However, we have not succeeded in creating double or triple knockouts (ackA-tyrP or ackA-pta-tyrP). As you can see in the fig. 14, restriction of cPCR product from randomly selected transformants do not show genomic modification in the *tyrP* gene. Interestingly, in the positive control line we can see very large DNA fragment (> 10 kbp). We could not explain this result without additional genomic analysis.

![](https://static.igem.org/mediawiki/2021/c/c2/T--Vilnius-Lithuania--double-tyrP-knockout1.png)

**Fig. 14.**  Restriction of cPCR product representing *tyrP* knockout generation. *tyrP* gene have been amplified from genomic DNA and restricted by BcuI. 1212 bp fragments represent
wild type genotype, 1014 bp and 184 bp - knockouts. 1 - wild type (negative control), 2 - *tyrP* knockout (positive controle obtain from previous experiments), 3 - *tyrP* knockout (1), 4 - *tyrP* knockout (2),
5 - *tyrP* knockout (3), 6 - *tyrP* knockout (4), 7 - *tyrP* knockout (5), 8 - *tyrP* knockout (6), 9 - *tyrP* knockout (7), 10 - *tyrP* knockout (8), 11 - *tyrP* knockout (9), 12 - *tyrP* knockout (10).

![](https://static.igem.org/mediawiki/2021/9/98/T--Vilnius-Lithuania--double-tyrP-knockout.png)

**Fig. 15.**  Restriction of cPCR product representing *tyrP* knockout generation. *tyrP* gene have been amplified from genomic DNA and restricted by BcuI. 1212 bp fragments represent
wild type genotype, 1014 bp and 184 bp - knockouts. 1 - negative control - no bacteria/DNA added, 2 - *tyrP* knockout (1), 3 - *tyrP* knockout (2), 4 - *tyrP* knockout (3), 5 - *tyrP* knockout (4), 6 - *tyrP* knockout (5), 7 - *tyrP* knockout (6), 8 - *tyrP* knockout (7), 9 - *tyrP* knockout (8), 10 - *tyrP* knockout (9), 11 - *tyrP* knockout (10).

We have repeated PCR from previously obtained *tyrP* knockouts and all knockouts had this one sharp fragment above 10 kbp ladder line (fig. 15). These surprising results might be obtained because of some unknown genomic reorganization of edited genomic locus. The exact reorganization output can be determined by genome sequence which was not in our focus. As we seek to avoid usage of undetermined changes containing *E. coli* Nissle 1917 strain, we decided to do not use *tyrP* knockout in further experiments. In addition, we do not succeed in obtaining *adhE* gene knockout even after testing two different sgRNAs.

### Naringenin evaluation

To evaluate the quantity of synthesis by our constructs/enzymes, we employed HPLC-MS to find naringenin and intermediate compounds. All enzymes were subjected to analysis first by themselves and further in different combinations. Both control for native cellular metabolism and with additional substrates were taken into account.

First, we created control chromatograms for naringenin and first enzymatic intermediate - *p*-coumaric acid (product of Tyrosine ammonia lyase (TAL) from naringenin synthesis pathway). By dissolving technical grade compounds in pure water we found retention times:

**Table 1.** Retention times for naringenin and *p*-coumaric acid

| Naringenin | *p*-coumaric acid |
| --- | --- |
| 6.88 min | 6.17 min |

![](https://static.igem.org/mediawiki/2021/9/90/T--Vilnius-Lithuania--naringenin.png)

**Fig. 16.**  Chromatogram at 309 nm. Naringenin control detection by HPLC-MS.

![](https://static.igem.org/mediawiki/2021/5/5d/T--Vilnius-Lithuania--p-coumaric_chrom_254.png)

**Fig. 17.**  Sample chromatogram at 254 nm. *p*-coumaric acid control detection by HPLC-MS with retention time 6.165 min.

![](https://static.igem.org/mediawiki/2021/b/b6/T--Vilnius-Lithuania--p-coumaric_chrom_309.png)

**Fig. 18.**  Sample chromatogram at 309 nm. *p*-coumaric acid control detection by HPLC-MS with retention time 6.166 min.

This information enabled us to search for compounds in more complex mixtures, in particular LB medium from overnight cultures. Furthermore, we were able to distinguish our products based on retention time, m/z and UV-Vis absorption spectrum.

Using the HPLC-MS method we analyzed the media samples of cultures containing pTRKH2 vectors with TAL gene and J23101 Anderson or surface layer protein A (slpA) promoters. The data of the experiments showed that plasmid with J23101 Anderson promoter and TAL encoding sequence determines an efficient synthesis of *p*-coumaric acid in our transformants, nevertheless analogous processes have not been identified in the samples containing slpA promoter. We hypothesized that the reason for this data non-reproducibility may be a possible mutation in pTRKH2 vector containing slpA promoter, the hypothesis later was approved by Sanger sequencing.

Similar intermediate compound detection strategy was applied for constructs containing 4CL and CHS encoding sequences. We supplied cultures with *p*-coumaric acid as a substrate for their specific reactions and used HPLC-MS method to detect the consumption of *p*-coumaric acid. The experiments were conducted with cultures containing pTRKH2 vectors with 4CL gene and J23101 Anderson or slpA promoters, as well as linked (linkers: GSG, GGGGS, (GGGGS)2, (GGGGS)3, EAAAK, (EAAAK)2, (EAAAK)3) 4CL and CHS genes under the same promoters. However, none of the aforementioned constructs have been found to demonstrate distinct enzymatic activity by consummation of the given substrate.

In the hopes of finding further intermediates we searched for a few additional m/z as a result of accumulation. *p*-coumaroyl-CoA and naringenin chalcone were chosen as the ones who could give us more information. However, fusion protein samples did not show any signs of naringenin chalcone. Moreover, we found the same m/z of *p*-coumaroyl-CoA in both control and sample from the desired construct medium when supplied with additional *p*-coumaric acid. This suggested to us that we could not precisely determine the quantity of synthesized *p*-coumaroyl-CoA because we do not know detailed information about internal processes. We hypothesize that control *E. coli* DH5 alpha has 4CL homology enzymes for forming carbon-sulfur bonds as acid-thiol ligases and thus synthesis of *p*-coumaroyl-CoA by both recombinant and native enzymes overshadow one another. We even cannot be sure about synthesis of a particular compound because it needs further analysis by NMR as chromatograms and UV-Vis spectrum lack structural information.

Detailed reports from HPLC-MS are referred to in table 2.

**Table 2.** HPLC-MS reports and data.

| Name | Description | File |
| --- | --- | --- |
| Technical grade *p*-coumaric acid | Technical grade *p*-coumaric acid was dissolved in water for reference chromatogram and other specifications. | [**Download**](https://static.igem.org/mediawiki/2021/e/e9/T--Vilnius-Lithuania--p-coum_pure.PDF) |
| TAL1 lysate | First enzyme Tyrosine ammonia lyase (TAL) in pTRKH2 plasmid with supplied *p*-coumaric acid to LB medium. Culture was lysed to check for compounds inside cells. | [**Download**](https://static.igem.org/mediawiki/2021/a/a7/T--Vilnius-Lithuania--TAL_lys.PDF) |
| TAL1 LB medium | First enzyme Tyrosine ammonia lyase (TAL) in pTRKH2 plasmid with supplied *p*-coumaric acid to LB medium. Only LB medium was subjected to HPLC-MS analysis. | [**Download**](https://static.igem.org/mediawiki/2021/a/a4/T--Vilnius-Lithuania--TAL_nonlys.PDF) |
| DH5alpha with *p*-coumaric acid | Only DH5alpha without any plasmid cells were grown in LB medium with supplied *p*-coumaric acid. | [**Download**](https://static.igem.org/mediawiki/2021/7/71/T--Vilnius-Lithuania--DH5a.PDF) |
| LB with *p*-coumaric acid | LB medium with *p*-coumaric acid. | [**Download**](https://static.igem.org/mediawiki/2021/7/7a/T--Vilnius-Lithuania--LB_with_p-coum.PDF) |
| LB without *p*-coumaric acid | LB medium without *p*-coumaric acid. | [**Download**](https://static.igem.org/mediawiki/2021/7/72/T--Vilnius-Lithuania--LB_without_p-coum.PDF) |
| GS1 LB medium without *p*-coumaric acid | Fusion protein (4CL and CHS) construct with GGGGS linker in pTRKH2 plasmid without supplied *p*-coumaric acid to LB medium. | [**Download**](https://static.igem.org/mediawiki/2021/c/cf/T--Vilnius-Lithuania--GS1_LB_without_p-coum.PDF) |
| GS1 lysate with *p*-coumaric acid | Fusion protein (4CL and CHS) construct with GGGGS linker in pTRKH2 plasmid without supplied *p*-coumaric acid to LB medium. | [**Download**](https://static.igem.org/mediawiki/2021/d/d7/T--Vilnius-Lithuania--GS1_lys.PDF) |
| GS1 lysate with *p*-coumaric acid | Fusion protein (4CL and CHS) construct with GGGGS linker in pTRKH2 plasmid without supplied *p*-coumaric acid to LB medium. | [**Download**](https://static.igem.org/mediawiki/2021/d/d7/T--Vilnius-Lithuania--GS1_lys.PDF) |
| GS1 LB medium with *p*-coumaric acid | Fusion protein (4CL and CHS) construct with GGGGS linker in pTRKH2 plasmid with supplied *p*-coumaric acid to LB medium. | [**Download**](https://static.igem.org/mediawiki/2021/b/b7/T--Vilnius-Lithuania--GS1_LB_with_p-coum.PDF) |
| GSG LB medium with *p*-coumaric acid | Fusion protein (4CL and CHS) construct with GSG linker in pTRKH2 plasmid with supplied *p*-coumaric acid to LB medium. Culture was lysed to check for compounds inside cells. | [**Download**](https://static.igem.org/mediawiki/2021/5/52/T--Vilnius-Lithuania--GSG.PDF) |
| Naringenin | Technical grade naringenin dissolved in distilled water. | [**Download**](https://static.igem.org/mediawiki/2021/f/fa/T--Vilnius-Lithuania--Naringenin_HPLC.PDF) |

In parallel, we have also introduced linked 4CL-CHS and CHI (5th mutant) encoding genes into the *E. coli* Nissle 1917 genome (fig. 19, 20). cPCR of transformants probably containing TAL encoding gene in the *colicin* gene has not shown clear results as additionally to the expected 1.7 kp band there were and two a bit smaller bands. For this reason, we conducted enzymatic activity measurement by identifying *p*-coumaric acid appearance in growth medium in order to validate TAL encoding gene insertion into *E. coli* Nissle 1917 genome.

![](https://static.igem.org/mediawiki/2021/c/c9/T--Vilnius-Lithuania--_4CL-CHI.png)

**Fig. 19.**  Linked 4CL-CHS encoding genes insertion into *colicin* gene. L - GeneRuler Express DNA Ladder, 1 - wild type (negative control), 2 - negative 4CL\_GGGGS\_CHS mutant (1), 3 - positive
4CL\_GGGGS\_CHS mutant (2), 4 - positive 4CL\_GGGGS\_CHS mutant (3), 5 - positive 4CL\_GGGGS\_CHS mutant (4), 6 - positive 4CL\_GGGGS\_CHS mutant (5), 7 - positive 4CL\_(GGGGS)2\_CHS mutant (1), 8 - positive 4CL\_(GGGGS)2\_CHS mutant (2),
9 - positive 4CL\_(GGGGS)2\_CHS mutant (3), 10 - positive 4CL\_(GGGGS)2\_CHS mutant (4), 11 - positive 4CL\_(GGGGS)2\_CHS mutant (5), 12 - positive 4CL\_(GGGGS)3\_CHS mutant (1), 13 - positive 4CL\_(GGGGS)3\_CHS mutant (2), 14 - negative
4CL\_(GGGGS)3\_CHS mutant (3), 15 - negative 4CL\_(GGGGS)3\_CHS mutant (4), 16 - positive 4CL\_(GGGGS)3\_CHS mutant (5), 17 - positive 4CL\_GSG\_CHS mutant (1), 18 - negative 4CL\_GSG\_CHS mutant (2), 19 - positive 4CL\_GSG\_CHS mutant
(3), 20 - positive 4CL\_GSG\_CHS mutant (4), 21 - positive 4CL\_GSG\_CHS mutant (5), 22 - positive 4CL\_EAAAK\_CHS mutant (1), 23 - positive 4CL\_EAAAK\_CHS mutant (2), 24 - positive 4CL\_EAAAK\_CHS mutant (3), 25 - positive 4CL\_EAAAK\_CHS
mutant (4), 26 - positive 4CL\_EAAAK\_CHS mutant (5), 27 - negative 4CL\_(EAAAK)2\_CHS mutant (1), 28 - negative 4CL\_(EAAAK)2\_CHS mutant (2), 29 - positive 4CL\_(EAAAK)2\_CHS mutant (3), 30 - negative 4CL\_(EAAAK)2\_CHS mutant (4),
31 - negative 4CL\_(EAAAK)2\_CHS mutant (5), 32 - positive 4CL\_(EAAAK)3\_CHS mutant (1), 33 - negative 4CL\_(EAAAK)3\_CHS mutant (2), 34 - positive 4CL\_(EAAAK)3\_CHS mutant (3), 35 - positive 4CL\_(EAAAK)3\_CHS mutant (4), 36 - positive
4CL\_(EAAAK)3\_CHS mutant (5).

![](https://static.igem.org/mediawiki/2021/7/78/T--Vilnius-Lithuania--_CHS.png)

**Fig. 20.**  CHI encoding gene insertion into *nupG* gene. L - GeneRuler 1 kbp DNA Ladder, 1 - wild type (negative control), 2 - wild type *nupG*, 3 - wild type *nupG*, 4 - wild type *nupG*, 5 - wild type *nupG*, 6 - *nupG* mutant with CHI insert, 7 - wild type *nupG*, 8 - wild type *nupG*.

As we have identified TAL enzymatic activity in previous experiments, we measured TAL enzymatic activity, where TAL encoding gene is inserted into *E. coli* Nissle 1917 genome.
We have grown cocultures composed of wild type Nissle producing TAL, *ackA-pta* double Nissle knockout producing linked 4CL-CHS enzymes and wild type Nissle producing CHI. After 48 hours cocultures inoculation in LB medium at 37°C HPLC-MS measurement has been done. It revealed that only TAL was enzymatically active from our naringenin metabolic pathway.

#### Kill-switch

VapXD kill-switch accuracy was firstly evaluated with a serial dilution spotting method. Sets of agar plates with and without bile salts were incubated in different temperatures of 37 °C, 30 °C, and room temperature (24 °C). However, the distinction between plates with and without bile salts supplementation was only seen at room temperature (figures 21 and 22).

![](https://static.igem.org/mediawiki/2021/2/25/T--Vilnius-Lithuania--VapXD_Kill-switch_without_bile.png)

**Fig. 21.**  Serial dilution spotting assay on M9 medium without bile salts.

![](https://static.igem.org/mediawiki/2021/6/6a/T--Vilnius-Lithuania--VapXD_Kill-switch_with_bile.png)

**Fig. 22.**  Serial dilution spotting assay on M9 medium with bile salts.

Results showed bile-regulated and cold-inducible VapXD kill-switch and bile-regulated and p-slpA VapXD kill-switch work on agar plates at room temperature, as with lower OD600 values they inhibited cell growth.

Further VapXD kill-switch performance was tested in liquid medium, as OD600 measurements were performed. First of all, VapD toxin ([BBa\_K3904000](http://parts.igem.org/Part%3ABBa_K3904000)) activity was characterized while regulating its production with cold-induced promoter ([BBa\_K3904003](http://parts.igem.org/Part%3ABBa_K3904003)). Graphs at the top of figure 23 illustrate bacteria growth without toxin and graphs at the bottom with the toxin in different temperatures. While comparing obtained data in 37 and 24 °C, temperature change can be seen as inducing greater toxin production and cell death. On the other hand, VapX activity is not fully accurate due to the leakage of the promoter during the temperature change from 37 to 24 °C.

![](https://static.igem.org/mediawiki/2021/6/62/T--Vilnius-Lithuania--vapxd-1.png)

What is more, VapXD with the bile-induced promoter before antitoxin and with the cold-induced promoter before toxin was characterized. Graphs in the top of X figure demonstrate bacteria growth with and without bile salts supplementation
in media at 24 °C, as graphs in the bottom at 37 °C. It can be seen that OD600 in the presence of bile salts and 37°C bacteria grow more exponentially than without bile salts and in 24 °C. In the ideal
case, no antitoxin should be produced in the absence of bile salts and 24 °C, and toxin synthesis should be induced. However, the results indicate that in such conditions, bacteria growth is only slightly repressed.

![](https://static.igem.org/mediawiki/2021/d/d2/T--Vilnius-Lithuania--vapxd-2.png)

When results in 37 °C obtained with different OD600 values were averaged (Fig. 25. Mean comparison with/no bile 37 °C), the difference between measurements with and without bile salts appeared to be mathematically
insignificant.

![](https://static.igem.org/mediawiki/2021/7/7e/T--Vilnius-Lithuania--vapxd-3.png)

The activity of different promoters before VapX toxin was compared without bile salts supplementation in media at 37 °C. From promoters’ strength evaluation measurements (fig. 26), it was seen that the p-slpA promoter ([BBa\_K3904712](http://parts.igem.org/Part%3ABBa_K3904228)) is the strongest in our inventor. VapXD assessment also showed that under this promoter, toxin production is more significant than under other promoters. The sloping graph rise illustrates this because more toxin is produced, and bacteria growth is inhibited. However, after the results were averaged, no significant difference between different promoters was seen.

![](https://static.igem.org/mediawiki/2021/7/7d/T--Vilnius-Lithuania--vapxd-4.png)

**Fig. 26.**  Comparison of VapXD kill-switch with different promoters before toxin (without bile salts in 37 °C).

While comparing results with different promoters in 24 °C, no significant difference can be seen (fig. 27).

![](https://static.igem.org/mediawiki/2021/e/ed/T--Vilnius-Lithuania--vapxd-5.png)

**Fig. 27.**  Comparison of VapXD kill-switch with different promoters before toxin (without bile salts in 24 °C).

Furthermore, we successfully inserted the VapXD system construct into *E. coli* Nissle 1917 genome (fig. 28, 29). Both of these VapXD systems constructs have been inserted into the *E. coli* Nissle 1917 genome with 100 percent efficiency and proved by DNA sequencing.

![](https://static.igem.org/mediawiki/2021/3/3d/T--Vilnius-Lithuania--VapXD-cold-bile.png)

**Fig. 28.**  cPCR of *E. coli* Nissle 1917 transformants. 1.3 kb PCR product formation from genomic DNA proves VapXD construct occurrence in the *colicin* genomic locus. All except 6th
colony are proved to be mutants harboring the VapXD system, where toxin encoding gene translation is induced by cold and antitoxin - by bile acids. L - GeneRuler 1 kb Ladder, 1 - VapXD-cold&bile mutant (1), 2 - VapXD-cold&bile
mutant (2), 3 - VapXD-cold&bile mutant (3), 4 - VapXD-cold&bile mutant (4), 5 - VapXD-cold&bile mutant (5), 6 - negative control (wild type *E. coli* Nissle 1917).

![](https://static.igem.org/mediawiki/2021/5/58/T--Vilnius-Lithuania--VapXD-slpA-bile.png)

**Fig. 29.**  cPCR of *E. coli* Nissle 1917 transformants. 1.3 kb PCR product formation from genomic DNA proves VapXD construct occurrence in the *colicin* genomic locus. All colonies
are proved to be mutants harboring the VapXD system, where toxin encoding gene translation is induced by constitutive PslpA promoter, while antitoxin - by bile acids inducible promoter. L - GeneRuler 1 kb Ladder, 1 - negative
control (wild type *E. coli* Nissle 1917), 2 - VapXD-cold&bile mutant (1), 3 - VapXD-cold&bile mutant (2), 4 - VapXD-cold&bile mutant (3), 5 - VapXD-cold&bile mutant (4), 6 - VapXD-cold&bile mutant (5), 7 - VapXD-cold&bile
mutant (6), 8 - VapXD-cold&bile mutant (7), 9 - VapXD-cold&bile mutant (8), 10 - VapXD-cold&bile mutant (9), 11 - VapXD-cold&bile mutant (10), 12 - VapXD-cold&bile mutant (11), 13 - VapXD-cold&bile mutant (12).

VapXD kill-switch inserted in *E.coli* Nissle 1917 genome was also characterized by OD600 measurements. The results of the genomic construct were compared to the plasmid construct.

The first observation from fig. 30 was that bacteria containing construct in the genome grow more exponentially. It does not have to maintain antibiotic resistance and the burden for the cell lowers.

![](https://static.igem.org/mediawiki/2021/5/55/T--Vilnius-Lithuania--genome-functional-1.png)

As shown in fig. 31 no significant difference in the VapXD kill-switch performance was seen while comparing results of the genomic and plasmid constructs with the same promoters.

![](https://static.igem.org/mediawiki/2021/8/8d/T--Vilnius-Lithuania--genome-functional-3.png)

While comparing results obtained during the measurements with and without bile salts (fig. 32), no significant difference was seen. This once again indicates promoter leakage and the system's inaccuracy.

![](https://static.igem.org/mediawiki/2021/7/79/T--Vilnius-Lithuania--genome-functional-2.png)

**Fig. 32.**  VapXD system performance in the environment with and without bile salts.

#### Further suggestions for VapXD kill-switch implementation

As it can be seen from the results discussed before, there are some places for the VapXD kill-switch improvement. One of them could be to search for alternative (weaker or constitutive) promoters instead of bile-induced. What is more, a bigger variety of promoter could be tested in order to obtain 2:1 stoichiometry of antitoxin and toxin in a more accurate way.

### Detection

#### *Entamoeba histolytica* recombinant protein synthesis

In order obtain suitable biomarkers for *Entamoeba histolytica* detection, we needed to produce recombinant *Entamoeba histolytica* proteins - pyruvate, phosphate dikinase (PPDK) and cysteine proteinase 5 (CP5). The CP5 gene was cloned into a pET-28a(+) vector which is used to tag proteins with a histidine tag on both C and N terminus. The PPDK gene was first cloned into a pET-28a(+) plasmid, that we modified by removing extra amino acids before the histidine tags. However, constructs kept mutating so we decided to clone it into the default pET-28a(+) plasmid to keep the chance of mutation at minimum. The conditions of biomass growth were similar for both proteins, the induction was carried out in the BL21 strain of *E. coli* for 3 hours in 37°C at concentration of 0.6 mM IPTG for CP5 and 1 mM for PPDK. The bacteria containing the PPDK plasmid also were grown in TB medium and the CP5 containing transformants were grown in LB medium.

![](https://static.igem.org/mediawiki/2021/6/6e/T--Vilnius-Lithuania--PPDK_raiska.png)

**Fig. 33.**  SDS gel showing succesfull production of PPDK in TB medium after induction with 1mM IPTG for 3 hours in 37°C.

![](https://static.igem.org/mediawiki/2021/a/a4/T--Vilnius-Lithuania--CP5_raiška.png)

**Fig. 34.**  CP5 SDS gel showing the comparrison beetwen 1mM and 0.6mM IPTG induction in LB medium for 3 hours in 37°C.

Since active protease can lyse this protein producing organism, the CP5 gene contains an inactive and insoluble pro-enzyme site that has a N-terminal pro-sequence, which can be cut off by the enzyme itself outside the cell. Therefore, the purification of CP5 was supposed to be done in three distinct steps: denaturation, renaturation and activation. Unfortunately, the yield of the protein was too low and the process itself was too costly to repeat it several times in order to optimize the purification conditions. We could only see the same pre-enzyme band in the SDS gel after the final step. According to the relevant literature we speculate that the most important factor in the process is the ratio between the protein and the refolding buffer. This was the only thing we could not check during our experiments, as we could not manage to successfully concentrate the reforded protein.

![](https://static.igem.org/mediawiki/2021/2/29/T--Vilnius-Lithuania--CP5_perlankstymas.png)

**Fig. 35.**  CP5 after purification (lane 1) with CP5 after refolding and activation (lane2)

Since we could not purify the active and soluble protein we had to find a way to stabilise the denatured protein in a buffer suitable for aptamer selection i.e. without urea or other strong denaturants. We tried out several compounds with protein stabilising potential and discovered that denaturated CP5 pro-enzyme is stabilised by CuCl2. Unfortunately, the stabilising effect was not as significant as we hoped and there was not enough of the protein in our soluble fraction to use for our further experiments.

![](https://static.igem.org/mediawiki/2021/c/c8/T--Vilnius-Lithuania--CP5_stabilizacija.png)

**Fig. 36.**  SDS gel showing the CP5 stabilisation experiment. 1-0.375M arginine, 2-0.75M trechalose, 3-0.5M proline, 4-0.5M manitol, 5- 10mM CuCl2.

Although we failed to produce the CP5 protein, we still had another possible biomarker - PPDK. The purification of this protein was supposed to be much simpler based on the literature, however we faced the same problems here as well. Although we found some of our target protein in the soluble fraction, the amount was too insufficient compared to what we needed to evolve the aptamers. It also seemed to have formed insoluble inclusion bodies. We hypothesise that it happens because of the extra amino acids in the pET-28a(+) backbone. We managed to increase the solubility by lysing the cells in the presence of detergents - Triton X-100 and NP-40. However, the protein was still barely visible on the Western blot membrane.

![](https://static.igem.org/mediawiki/2021/5/5e/T--Vilnius-Lithuania--BLOTAS.png)

**Fig. 37.**  Western blot of PPDK. Red border highlights EhPPDK.

Next step was PPDK stabilisation, we found out that denatured PPDK can be stabilised in a buffer containing 0.375 M arginine. PPDK was then further purified through nickel affinity chromatography and used for further experiments.

![](https://static.igem.org/mediawiki/2021/a/ad/T--Vilnius-Lithuania--Netirpus_tirpus.png)

**Fig. 38.**  Fractions of EhPPDK protein synthesis induction and solubility.

#### SELEX

To perform systematic evolution of ligands by exponential enrichment (SELEX) in vitro we used the basic SELEX protocols using magnetic beads. A protein with a His-tag is attached to the beads and after the incubation of target protein in the aptamer pool we can wash off the residual non-attached oligonucleotide sequences and perform a PCR reaction by loading the beads directly to the PCR mixture. Although the SELEX process itself seemed to be rater simple, we did have to optimise several selection parameters. The PCR reaction only worked when we substituted the TE buffer for water as the aptamer storage solvent, since even minimal amounts of EDTA in the aforementioned buffer tended to prevent Taq polymerase from amplifying the target DNA strands. The magnetic beads themselves also seemed to inhibit the amplification process when it was performed using Taq polymerase, therefore we decided to compare different polymerases and settled on Phusion polymerase, since it displayed the highest reaction efficiency.

![](https://static.igem.org/mediawiki/2021/c/cb/T--Vilnius-Lithuania--5th_SELEX.png)

**Fig. 39.**  Example of non-specific fragments appearing in 16-20 PCR cycles generated from 5th round of SELEX.

Examplery SELEX inputs and outputs by rounds are depicted in table 3.

| Round | Input DNA (µg) | Beads + Target structure (µg) (1:1) | Binding Incubation Time (min) | Washing Steps | Number of PCR Cycles | Output - Yield (µg) | Input/Output ratio (I/O) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 29 | 80 | 90 | 1x 500 µl | 12 | 1.816 | 6.3% |
| 2 | 0.92 | 40 | 60 | 1x 500 µl | 17 | 1.335 | 145.1% |
| 3 | 0.72 | 40 | 60 | 2x 500 µl | 24 | 0.964 | 134.8% |
| 4 | 0.82 | 40 | 60 | 2x 500 µl | 20 | 1.096 | 133.2% |
| 5 | 0.83 | 40 | 60 | 2x 500 µl | 14 | 5.772 | 695.4% |
| 6 | 0.78 | 40 | 45 | 2x 500 µl | 16 | 4.840 | 622.9% |
| 7 | 1.50 | 40 | 45 | 2x 1000 µl | 13 | 3.540 | 236.0% |
| 8 | 1.12 | 40 | 30 | 2x 1000 µl | 18 | 3.236 | 290.2% |
| 9 | 1.43 | 40 | 30 | 2x 1000 µl | 16 | 3.640 | 254.2% |
| 10 | 2.37 | 40 | 15 | 2x 1000 µl | 16 | 6.762 | 285.2% |

#### Emulsion PCR (ePCR)

We found creation of emulsion an easy task, nonetheless few problems occured. Firstly, the initial version of emulsion showed irresistivity for thermal cycling and micelles broke after 15 cycles of PCR, when according to the relevant literature, it was supposed to happen after approximately 30 cycles. For this we tried creating emulsions in colder conditions and by mixing them for longer. None of these strategies showed better results.
Additionally, comparison of ePCR and open PCR (oPCR) products was done. In figure 40 different cycle count was used and from this data we can see that oPCR started generating non-specific fragments after 25 cycles and emulsion PCR lagged at overall production of fragments. What is more, In the end both methods did not create the same fragments.

![](https://static.igem.org/mediawiki/2021/6/6f/T--Vilnius-Lithuania--ePCR_vs_oPCR.png)

**Fig. 40.**  Initial testing with ePCR.

##### ePCR v1

We observed produced micelles using fluorescent microscopy (400x Magnification) with purified GFP shown in figures 41 and 42.

![](https://static.igem.org/mediawiki/2021/a/a4/T--Vilnius-Lithuania--emulsion_fluor_1.png)

**Fig. 41.**  Fluorescence microscopy with GFP in micelles.

![](https://static.igem.org/mediawiki/2021/2/23/T--Vilnius-Lithuania--emulsion_fluor_2.png)

**Fig. 42.**  Fluorescence microscopy with GFP in micelles.

Micelles were stable at room temperature while observing them.

##### ePCR v2

We tested updated composition emulsion and it did not show any signals of breakage even after 50 PCR cycles. The main problem with this is when we want to check nonspecific PCR products in electrophoresis. It is not an easy task to break emulsion with neither 1-butanol, nor isopropanol. However when used in PCR purification kit the emulsion has gone from cloudy to clear from binding buffer and centrifugation at 20.000 rcf.

To recreate both versions of emulsions see instructions in our [**protocols and materials**](https://static.igem.org/mediawiki/2021/e/e0/T--Vilnius-Lithuania--protocols_and_materials.pdf).

##### Observations

Distribution among PCR tubes by 50 µl leaves quite visible mineral oil smear on pipette tips. It is better to produce more of the overall mixture for higher yield.
As per visual examination emulsion breaks even after 10 cycles of PCR which suggests that different emulsifiers should be used.

##### Recommendations for future usage

* Prepare 20 percent more overall volume to have desired final reaction coverage.
* Use ABIL EM 90 instead of Span 80 for better emulsion preservation during PCR. Keep in mind that using ABIL EM 90 will require additional materials for emulsion breakage.
* Break emulsions with a binding buffer from your PCR purification kit. To ensure breakage test your kit on sample.

#### Aptamer Sanger sequencing

6th, 8th and 10th rounds of SELEX were selected for Sanger sequencing of aptamers. PCR products from each round were blunt-end ligated into pUC19 for transformation. Molar ratio of aptamers and vector as 50:1 (aptamers:vector) for more efficient multi insert ligation. Each round was plated on different LB medium plate with ampicillin. 50 colonies from each round were selected for colony PCR and delivered for further direct sequencing. Last round results were aligned by CLUSTAL multiple sequence alignment (MUSCLE 3.8) and with gathered data generated WebLogo with website from University of California, Berkeley.

![](https://static.igem.org/mediawiki/2021/0/03/T--Vilnius-Lithuania--10_weblogo.png)

The results were filtered using Perl scripts from any unspecific fragments, and their frequency was calculated using local BLAST. Consequently, two sequences with the highest frequency are shown in table 4.

**Table 1.** Most abundant aptamers from Sanger sequencing without added primers.

| No. | Aptamer sequence | Count |
| --- | --- | --- |
| 1 | AGAGTCGGTCGATAGTACTATCGACCGACTCT | 8 |
| 2 | GAGTCGGTCGATAGTACTATCGACCGACTCT | 4 |

As we saw from sequencing results that aptamer No. 2 is a subsequence of aptamer No. 1. However they have different secondary and tertiary structures. For both of them structures are depicted in figures 44 and 45. Secondary structure was folded using The UNAFold Web Servers [mFold](http://www.unafold.org/mfold/applications/dna-folding-form.php) application at 20°C. Furthermore, tertiary structures were created using [RNAComposer](http://rnacomposer.cs.put.poznan.pl/) from Vienna formatting. We acknowledge that RNA structure is not the same as DNA, however in previous researches showed that composition of DNA and RNA is highly similar and thus 3D RNA structure can be used as DNA.

![](https://static.igem.org/mediawiki/2021/1/1f/T--Vilnius-Lithuania--No_1.png)

**Fig. 44.**  Secondary structure of No. 1 aptamer sequence.

![](https://static.igem.org/mediawiki/2021/d/d8/T--Vilnius-Lithuania--No_2.png)

**Fig. 45.**  Secondary structure of No. 2 aptamer sequence.

![](https://static.igem.org/mediawiki/2021/4/41/T--Vilnius-Lithuania--No_1_tert.png)

**Fig. 46.**  Tertiary structure of No. 1 aptamer sequence.

![](https://static.igem.org/mediawiki/2021/d/df/T--Vilnius-Lithuania--No_2_tert.png)

**Fig. 47.**  Tertiary structure of No. 2 aptamer sequence.

Furthermore we decided to check whether our structures are influenced by primer sequences used in SELEX. We added them to sequences as if they were extracted using PCR. Also secondary and tertiary structures were done using previous websites.

**Table 1.** EFBA generated aptamers for albumin and EhPPDK with controls

| No. | Aptamer sequence | Count |
| --- | --- | --- |
| 1.2 | TGACATTGCCACTCTGAACCAGAGTCGGT CGATAGTACTATCGACCGACTCTACTATCGACCGACTCTGATC | 8 |
| 2.2 | TGACATTGCCACTCTGAACCGAGTCGGTC GATAGTACTATCGACCGACTCTACTATCGACCGACTCTGATC | 4 |

![](https://static.igem.org/mediawiki/2021/8/82/T--Vilnius-Lithuania--No_1.2.png)

**Fig. 48.**  Secondary structure of No. 1 modified aptamer sequence.

![](https://static.igem.org/mediawiki/2021/9/9e/T--Vilnius-Lithuania--No_2.2.png)

**Fig. 49.**  Secondary structure of No. 2 modified aptamer sequences.

![](https://static.igem.org/mediawiki/2021/b/b4/T--Vilnius-Lithuania--No_1.2_tert.png)

**Fig. 50.**  Tertiary structure of No. 1.2 modified aptamer sequence.

![](https://static.igem.org/mediawiki/2021/4/44/T--Vilnius-Lithuania--No_2.2_tert.png)

**Fig. 51.**  Tertiary structure of No. 2.2 aptamer sequence.

#### Aptamer NGS sequencing

In order to sequence our aptamers by NGS Illumina MiSeq platform we designed primers for extending aptamers with needed indexes and adapters. Using Illumina Experiment manager we created sample sheet containing crucial instrumental data.
All used primers for indexing are uploaded to part registry are shown in table 5.

![](https://static.igem.org/mediawiki/2021/3/38/T--Vilnius-Lithuania--NGS_1_opt.png)

**Fig. 52.**  NGS first indexing optimization. 3rd cycle aptamers were degraded.

**Table 5.** SELEX round indexing for Illumina sequencing

| No | 5' end | 3' end |
| --- | --- | --- |
| 1 | [Seq\_In\_1.1](http://parts.igem.org/Part%3ABBa_K3904501) | [Seq\_In\_2.1](http://parts.igem.org/Part%3ABBa_K3904511) |
| 2 | [Seq\_In\_1.2](http://parts.igem.org/Part%3ABBa_K3904502) | [Seq\_In\_2.2](http://parts.igem.org/Part%3ABBa_K3904512) |
| 3 | [Seq\_In\_1.3](http://parts.igem.org/Part%3ABBa_K3904503) | [Seq\_In\_2.3](http://parts.igem.org/Part%3ABBa_K3904513) |
| 4 | [Seq\_In\_1.4](http://parts.igem.org/Part%3ABBa_K3904504) | [Seq\_In\_2.4](http://parts.igem.org/Part%3ABBa_K3904514) |
| 5 | [Seq\_In\_1.5](http://parts.igem.org/Part%3ABBa_K3904505) | [Seq\_In\_2.5](http://parts.igem.org/Part%3ABBa_K3904515) |
| 6 | [Seq\_In\_1.6](http://parts.igem.org/Part%3ABBa_K3904506) | [Seq\_In\_2.6](http://parts.igem.org/Part%3ABBa_K3904516) |
| 7 | [Seq\_In\_1.7](http://parts.igem.org/Part%3ABBa_K3904507) | [Seq\_In\_2.7](http://parts.igem.org/Part%3ABBa_K3904517) |
| 8 | [Seq\_In\_1.8](http://parts.igem.org/Part%3ABBa_K3904508) | [Seq\_In\_2.8](http://parts.igem.org/Part%3ABBa_K3904518) |
| 9 | [Seq\_In\_1.9](http://parts.igem.org/Part%3ABBa_K3904509) | [Seq\_In\_2.9](http://parts.igem.org/Part%3ABBa_K3904519) |
| 10 | [Seq\_In\_1.10](http://parts.igem.org/Part%3ABBa_K3904510) | [Seq\_In\_2.10](http://parts.igem.org/Part%3ABBa_K3904520) |

All 20 sequences have the same Tm for PCR.

#### PDA synthesis with aptamers

The first STEP in PDA synthesis is 10,12-Tricosadiynoic acid (**TCDA**) ester activation with NHS. After that it can react with an amine group and form covalent bond. To recreate both versions of emulsions see instructions in our [**protocols and materials**](https://static.igem.org/mediawiki/2021/e/e0/T--Vilnius-Lithuania--protocols_and_materials.pdf).

##### Materials:

**Table 6.** Materials used in polydiacetylene synthesis

| Name | M (g/mol) |
| --- | --- |
| 10,12-Tricosadiynoic acid (**TCDA**) (91445) | 346.55 |
| 10,12-pentacosadiynoic acid (**PCDA**) (76492) | 374.60 |
| N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (**EDC-HCl**) (E6383) | 191.70 |
| Ethanolamine (411000) | 61.08 |
| N-hydroxysuccinimide (**NHS**) (130672) | 115.09 |
| Methylene chloride (Dichloromethane - DCM) | - |
| Dimethyl-2-(dimethylphosphino)ethylphosphine (**DMPE)** | 150.14 |

After dissolving the final fatty acid and aptamer conjugated compounds mixture in chloroform PVDF membrane was dipped 3 times into it and dried between each immersion. Finally the membrane was dried thoroughly and placed under direct UV light (254 nm) for 1 minute. The end product is a blue colored PDA inside of PVDF.

#### For further testing aptamers

##### Anti-HSA aptamer with PDA

In aptamer database we found a sequence for human serum albumin (HSA) and ordered it with amine modification on 5’ end of ssDNA. Overall sequence and secondary structure is shown in figure 53.

![](https://static.igem.org/mediawiki/2021/9/99/T--Vilnius-Lithuania--HSA_Apt-06.png)

It was tested in conjugation with PDA and showed positive results. Both negative control (non-native BSA) and distilled water did not show color change. From this we can say that the chosen anti-HSA aptamer is specific and suitable for diagnostic test as positive blood control.

![](https://static.igem.org/mediawiki/2021/a/ae/T--Vilnius-Lithuania--PDA_results.png)

**Fig. 54.**  Testing of anti-PPDK and anti-HSA aptamers by conjugating with PDA. a - PDA with anti-HSA aptamer shows positive selection for native form BSA, b - PDA with SELEX developed anti-PPDK aptamer
shows positive selection for all types of solutions, c - PDA with anti-PPDK aptamer after humid air-blow shows positive selection in all area, d - PDA without aptamer as negative control.

##### Anti-PPDK aptamer with PDA

Results from Sanger sequencing suggested us to order specific aptamers with amine modified 5’ end for coupling with TCDA. Both aptamer and aptamer with primer sequences were coupled and polymerized. The reaction with pyruvate phosphate kinase solution showed positive results as well as reaction with control buffer and bovine serum albumin. No visual difference was seen.

For more detailed control reactions water and ethanol were chosen. Both of them showed positive results. Color developed on PDA by ethanol was different from those with water based solutions - a little bit on the darker side with a pinch of brown discoloration (figure 54. spot 5).

In our case we think that evolutionized and sequenced aptamers that were synthesized for polymerization do not meet criteria needed for positive selection of PPDK. Further analysis of SELEX sequences should be done.

#### IT aptamer evaluation

We evaluated the affinity of the first three **neural network model** (TEA) generated sequences with **aptamer-based Western blot**. For each target protein **four cases** were studied: the first one - **a control**, for which the protein of interest was taken without any aptamer and other three cases for **each of the chosen aptamer** from the list.

We did not observe any significant signal in the blot with albumin aptamers, although there was a rather bright band at the position of 100 kDa for the first and second aptamer of EhPPDK, which might indicate **interaction** between the aptamer and the protein. Due to the lack of time, we could not investigate aptamer binding affinity by applying another method. In order to make more confident statements about TEA's ability to produce affine aptamer sequences, more **target cases** should be studied, different **scoring functions** and different **validation methods** should be applied.

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