iGEM 2023 — Team thessaloniki

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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## What should this page contain?

---

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

## Inspirations

---

* [2018 Munich](http://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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{% block title %}Engineering{% endblock %}
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![](https://static.igem.wiki/teams/4998/wiki/wiki-photos/pages-top/engineering-reviced-igemthess23.png)

* [Overview](#Overview-engineering)
* [Effects of IPTG induction](#engin1)
* [Choosing optimal strain](#engin2)
* [Optimal Promoters](#engin3)
* [Hydrogel](#engin5)

## Overview

---

Our project's success is largely due to our team's consistent dedication to the Design-Build-Test-Learn (DBTL) process.
Since we knew that DBTL is the key to realizing the full potential of our project, we were very driven to use it in
all part of our work. The cycle's various phases offer chances for development, invention, and improvement.
Applying DBTL was not just an option for us; it was a guiding philosophy that inspired us to take on difficulties,
learn from mistakes, and eventually develop solutions with the potential to transform the world. We are confident
that our commitment to DBTL will result in ground-breaking findings.

![Engineering cycle](https://static.igem.wiki/teams/4998/wiki/wiki-photos/engcycle-final-igemthess23.png)

**Figure 1:** Engineering Cycle

## Engineering success of Euphoresis

##### Effects of IPTG induction

##### Choosing optimal strain

##### Optimal Promoters

##### Hydrogel

## Effects of IPTG Induction

---

### Introduction

In our project environmental biosafety is of utmost concern and prevention of any genetically
modified organism escape is considered paramount. To address such risk, designing successfully
a kill switch strategy is considered crucial. Therefore a **multilayer kill switch strategy**
was designed in order to ensure both microorganisms’ death. We carefully selected **IPTG as the
trigger** that keeps cells alive and the kill switch activation to be induced by the LacI
repressor, that is available to repress in IPTG absence. However when building the design of
the kill switch mechanism we came across some disturbing findings about **cytotoxicity effects** of IPTG induction. Therefore investigation of LB/BG-11 media enriched with different IPTG
concentrations effect on cell density is crucial to be tested in order to make suitable adjustments
so that the IPTG inducer meets the aims of our system.

### Cycle 1

![Button design](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/designes-igemthess23.png)
DESIGN

![Button build](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/buildes-igemthess23.png)
BUILD

![Button test](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/testes-igemthess23.png)
TEST

![Button learn](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/learnes-igemthsedd23.png)
LEARN

IPTG is associated with cell deconstruction and cytotoxicity. In order to sufficiently
assess the impact of our inducer to cell density, we methodically examine a variety
of IPTG concentrations and their effect providing valuable results to maintain
cell viability.

The growth behavior of *B.subtilis* was determined by measuring cell absorbance at
600 nm (OD600). *B.subtilis* 3601 were inoculated in LB/BG-11 medium with
IPTG concentrations at 0, 0.05, 0.1, 0.2, 0.4 mM. Ηourly OD readings were taken
over 24 hours.

From the OD600 values we investigated the effect of IPTG variation in cell
density on the stationary phase when it hits its maximum. The control culture
(no IPTG in the media) showed, as expected, more exponential growth rate.
Cultures with 0.05 and 0.1 mM IPTG in the media had a small deviation from the
control, while for IPTG concentrations over 0.2mM the deviation was more distinct.

Our concerns about IPTG cytotoxicity were experimentally confirmed.
Higher concentrations, although yields in greater induction according
to the literature, also negatively affect cell density. We decided that the
optimal IPTG concentration in line with the objectives of our project is up
to 0.1mM. However, comparing recombinant hosts is considered crucial in order
to safely conclude that at that concentration IPTG, the inducer is suitable
without affecting cell growth and metabolism. Therefore more experiments about
metabolic burden needed to be performed in recombinant cultures.

### Cycle 2

![Button design](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/designes-igemthess23.png)
DESIGN

![Button build](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/buildes-igemthess23.png)
BUILD

![Button test](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/testes-igemthess23.png)
TEST

![Button learn](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/learnes-igemthsedd23.png)
LEARN

We took into account the measurements and results of cycle I for *B.subtilis* 3601.
To achieve optimal induction we should consider the highest IPTG concentrations yet
with the minimum, possible, negative effect of IPTG on cell growth. Therefore we
purpose 0.05 and 0.1mM IPTG as optimal concentrations in order to get the best
possible outcome. It is then vital to investigate how this concentration affects
individual *Nostoc* growth, considering the future coexistence with *B.subtilis*
within the consortium.

*Nostoc's* slow and multifactorial growth rate, did not allow us to investigate
IPTG and other factors variations separately due to time limitations.
Therefore the light density kept stably low simulating soil conditions.
The growth behavior of *Nostoc Oryzae* TAU-MAC 2710 was once again determined
by measuring cell absorbance at 600nm (OD600). Two cultures were inoculated
in LB/BG-11 medium with IPTG concentration at 0.1 mM (tested culture) and in
IPTG absence (control culture).

OD measurements were taken on a daily basis in order to test IPTG optimal
concentration and culture conditions. Test culture has grown very slowly over
20 days when *Nostoc* is still in its exponential phase. The growth curve of
tested culture exhibited slowed down progression of the exponential phase.
We suspected that either IPTG affected cell growth or the UV intensity
could also be considered as a decelerating factor.

Both IPTG and low light conditions were necessary for the success of Euphoresis.
Hence we decided to further test the culture in such harsh light conditions in
order to be able to thrive under soil as well as the second best IPTG concentration
of IPTG at 0.05mM. Furthermore, since we already knew about Nostoc’s slow growth
rates in such cases, the team decided to implement an alternative kill switch
circuit. In such design we coupled the circuit with a quorum sensing
mechanism so that *Nostoc’s* kill switch would be induced by the signaling
molecule that will be produced by *B.subtilis* (exhibiting high growth rate) while
its expression will be still IPTG inducible.

### Cycle 3

![Button design](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/designes-igemthess23.png)
DESIGN

![Button build](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/buildes-igemthess23.png)
BUILD

![Button test](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/testes-igemthess23.png)
TEST

![Button learn](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/learnes-igemthsedd23.png)
LEARN

In the redesign kill switch mechanisms we coupled the LuxR/LuxI quorum sensing.
Particularly, we propose that *B. subtilis* will contain the TetR gene controlled
by IPTG inducible promoter and when the inducer is present in the media TetR gene
is activated contributing to cell survival while in absence cell death.
Accordingly LuxI activation will produce AHL molecules that when diffused
into Nostoc cells contributes to cell survival, while in absence of IPTG cell death.
Cell survival in *Nostoc* will be achieved by expression of Antitoxin (VapB15) that
forms clusters with constitutively expressed toxin (VapC15) and deactivates it.
While for *B. subtilis* cell survival is achieved with TetR expression that represses
BsrG toxin production. Once the improved kill switch design was developed and the
optimal strain from the respective engineering cycle was selected, recombinant
cultures of *B. subtilis* WB800 needed to be tested in order to examine the metabolic
burden of recombinant hosts due to IPTG induction in the strain we selected.
Particularly when combined with recombinant hosts, IPTG - induction affects the
expressing conditions and adds further metabolic cost. Providing the least possible
metabolic burden while ensuring biosafety, is crucial for sufficient expression of
Laccases in *B. subtilis* and HetR in *Nostoc* in order to achieve phenolic compounds
degradation and nitrogen fixations in respect with biosafety, that is Euphoresis goal.

To investigate the additional metabolic burden on *B. subtilis* due to IPTG induction two cultures
were examined. *B. subtilis* 3601 (not recombinant) from the first OD600 measurements and *B. subtilis*
WB800 pJUMP29-1D’ (tested). Hourly OD readings were taken over 24 hours in a similar manner.
Unfortunately, Nostoc’s demanding transformation conditions did not allow us to investigate
the additional cost of metabolism in IPTG presence because of time limitations. However the
second purposed IPTG concentration was tested in a non recombinant host to examine IPTG impact
on growth. (Later we confirmed HetR production with an SDS page in those concentrations.
[See results](https://2023.igem.wiki/thessaloniki/results) )

*B.subtilis* tested cultures were inoculated in LB/BG-11 medium with IPTG concentration
at 0, 0.05, 0.1, 0.2 and 0.4 mM while *Nostoc* cultures were grown also in LB/BG-11 medium enriched
with 0.05mM of IPTG and low light conditions. To obtain results that can be properly evaluated according
to the first measurements, *B.subtilis* hourly OD readings were performed over 24 hours and *Nostoc* daily
OD measurements were conducted over 20 days.

As expected, *B.subtilis* pJUMP29-1D’-WB800 exhibits overall lower OD values compared to 3601
strain, but surprisingly with small deviation. pJUMP29-1D’-WB800 showed significant deviation between
up to 0.1mM IPTG, whose growth rates were similar to those without and over 0.2mM that the reduced
cell density is noticeable. According to the results for *Nostoc Oryzae* TAU-MAC 2710, at 0.05 mM no
significant deviation was observed compared to the IPTG concentration of 0.1 mM.
Thus it is concluded that we can test in our further experiments IPTG inducer concentrations of
0.05 and 0.1 mM for the two microorganisms to evaluate effectiveness of the improved kill switch design
and the genes of interest (laccases and HetR), while in the future more experiments need to be performed
on recombinant *Nostoc Oryzae* TAU-MAC 2710.

## Choosing optimal strain

---

### Introduction

One of the most important parts of our project was the expression of the laccase enzyme
produced by *B.subtilis*. Lacasse catalyzes the degradation of hydrophobic phenolic
compounds and lignin matrix in the soil that a high percent of microorganisms cannot achieve.
That matrix is formed after a wildfire and its degradation provides the soil with nutrients for
microorganisms development. Therefore enzymes are important to be expressed and also secreted
from *B. subtilis* in order to act on the soil’s ingredients passing through the pores
of our microspheres and hydrogel.

### Cycle 1

![Button design](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/designes-igemthess23.png)
DESIGN

![Button build](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/buildes-igemthess23.png)
BUILD

![Button test](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/testes-igemthess23.png)
TEST

![Button learn](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/learnes-igemthsedd23.png)
LEARN

Our goal was our *Bacillus subtilis* to produce extracellular laccases.
In order to ensure that our cells were producing laccases we had to verify their
extracellular expression. Although lacasses are endogenous proteins naturally
produced in *B.subtilis*, we had to ensure their exogenous production, as well as
to test their activity. Thus we designed the sequence of our peptide with a
TAT sequence that signals their extracellular transportation.

We selected the *Bacillus* 3601 strain that was available in our
laboratory. Therefore we designed laccase’s gene sequence along with TAT
signaling sequence. First we decided to follow Biobrick assembly because
of the technique’s reliability, compatibility with iGEM characterization
and flexibility in future designs. We performed both Biobrick assembly and the assembly with Mr
Giannakopoulos’ protocol due to unexpected results in other assemblies.
(see  [Optimal Promoters](#engin3) Engineering).
Next we consulted our instructor and
utilized his protocol in order to overcome the obstacles of the failed assembly.
Then we selected restriction enzymes that align with our digestion
(and do not destroy our gene of interest) and thus utilized E.coRI and PstI enzymes.
Next, following the protocol a mastermix was prepared using a ligation buffer
along with an enzymes-buffer ensuring that restriction enzymes will have the
optimal conditions to be activated at their maximum. Digestion and ligation
performed in one run and the cultures incubated in 37°C. We confirmed an
indication of assembly by PCR primers. Then we ran gel electrophoresis
and interpreted the corresponding bands. If we get the expected bands
then SDS page and Bradford is crucial to be performed in order to define
quantitatively endogenous and exogenous protein production respectively.

Agarose gel’s results were interpreted and successful assembly was achieved.
Therefore we proceed to SDS and Bradford experiments as planned. The two techniques
will provide us more accurate results reducing error margin. Knowing the molecular
weight of our laccase we were able to qualitatively examine its expression by comparing
gel bands between the control and tested samples. We expect the line of the molecular
weight that corresponds to lacasse’s molecular weight in the SDS page to be enhanced
compared to the control one. Additionally we expect an increase of total protein in
Bradford. Four cultures of *B.subtilis* 3610, that was available in our lab, were
inoculated with 0, 50, 75 and 100 μΜ CuSO4. Four samples from each culture were examined
on SDS page and Bradford.

According to the results of the gel bands provided, we observed no indication of lacasse
endogenous or exogenous protein. Surprisingly, we could not understand why, since the assembly
was tested and successful. Hence we started to investigate possible lab errors and reach out
for scientific feedback.

### Cycle 2

![Button design](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/designes-igemthess23.png)
DESIGN

![Button build](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/buildes-igemthess23.png)
BUILD

![Button test](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/testes-igemthess23.png)
TEST

![Button learn](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/learnes-igemthsedd23.png)
LEARN

After contacting PhD candidate in Biotechnology and Synthetic Biology Anargyros
Alexiou we realized that the problem was the proteases that our strain produces,
which destroy our laccase and that is the reason that our laccase was not
detected in the extracellular matrix. Following that valuable insight, we
decided to choose the strain of *Bacillus subtilis* WB800, originating
from *Bacillus subtilis* 168 strain with 7 genes of extracellular proteases
being knocked out. The extracellular proteases of *B. subtilis* possibly
destroyed our laccase.

Experimental design was in line with the previous one since the exact same procedures
will be tested in a different strain. So we transformed *B.subtilis* WB800 with the
lacasse recombinant gene and conducted Bradford and SDS pages to quantitatively
investigate protein expression. We also decided to test different concentrations
of CuSO4, since we were informed that it is crucial and increases lacasse activity
and production.

In four cultures of *B.subtilis* WB800 with concentrations of 0, 50, 75 and
100 μL CuSO4 Bradford and SDS page were conducted. We expected a higher band density
around 77kDa for SDS page as the concentration CuSO4 is rising, while in Bradford
increased extracellular total exogenous protein in respect with the rising of CuSO4.

According to the results from Bradford, with CuSO4 rise, the protein production
rises as well. CuSO4 is related with enzyme’s function. In SDS page results,
there is a notable protein concentration increase around 75kDa, which is around
laccasse’s molecular weight. With those two indications we can assume, a successful
laccasse production.

## Optimal Promoters

---

### Introduction

The original idea of our kill switch mechanism was to design two individual kill switches for each
microorganism with genes induced by IPTG in the media. The activation of genes ensures the genes
activation and cell survival, while in IPTG absence the deactivation of genes to cause cell death.
In Effects of IPTG Engineering, *B.subtilis* 3601 were tested on different concentrations of
IPTG in order to examine the negative effect of inducer in cell density. We proposed 0.05mM and
0.1 mM IPTG for optimal induction with minimum cell growth affection. Those values of IPTG were
tested in the same media that *Nostoc* was grown in. Due to Nostoc slow growth, we improved our kill
switch mechanism by coupling a quorum sensing system. The idea was that the genes triggered by IPTG
should located in *B.subtilis* to achieve a guaranteed IPTG induction according to high growth
rated of *B.subtilis* and deactivation of those genes to occur at the same time, again dependent
on *B.subtilis* growth. The quorum sensing system will ensure that AHL signaling molecules
will be produced by *B.subtilis* and be expressed constitutively, while *Nostoc* will not be
able to survive outside the consortium without *B.subtilis* (Design Cycle 1). Results from
cycle 1 assemblies failed and another protocol used to get the assemblies successfully (cycle 2).
However when experimenting with lacasse production on *B.subtilis* 3601, we came across unexpected
data that further investigated and proceeded with *B.subtilis* WB800 strain. Same IPTG concentrations
were tested on recombinant *B.subtilis* WB800 and compared with 3601 strain. We conclude
that still 0.05 and 0.1 mM IPTG values align with project objectives.

### Cycle 1

![Button design](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/designes-igemthess23.png)
DESIGN

![Button build](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/buildes-igemthess23.png)
BUILD

![Button test](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/testes-igemthess23.png)
TEST

![Button learn](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/learnes-igemthsedd23.png)
LEARN

According to the data derived from Effects of IPTG engineering, two values of IPTG
concentration are proposed in order to serve Euphoresis objectives. The improved kill
switch containing the activation genes in *B.subtilis* was designed and needed to
be tested in the particular values of IPTG. Unfortunately as the research continues,
we decided that maybe it should be more suitable to also use an IPTG inducible promoter
for LuxI activation, in order to be able to control both kill switches under the same
factor. Therefore, we redesigned *Nostoc’s* kill switch and placed a second order.
First we needed to follow carefully the techniques that best describe our objectives
before reaching the final step, that is IPTG induction.

Effectiveness of the circuit activation genes needed to be achieved performing
the suitable assembly. In this stage of the project, we chose to perform Biobrick
Assembly since it is a standard and easy assembly method that serves educational
purposes. Kill switch demonstration was in the initial phase and we had already
the first misguided choice, of choosing non IPTG inducible promoter (pGroE) when
there was a better option (Pgrac). Therefore utilizing Biobrick Assembly
we ensure that if further adjustments need to be performed in the future and
the genetic circuit to be more complex, with Biobrick we save precious time
because of the method’s universal prefix and suffix.

We used E.coRI and PstI restriction enzymes, after making sure that they didn’t cut
somewhere between the prefix and suffix where the genes of interest are located.
Afterwards we will demonstrate double digestion and ligation. The cultures have grown
successfully and PCR amplification followed. Gel electrophoresis was followed next,
in order to verify the molecular weights of each expected fraction by the restriction
enzyme according to the ladder. Then we may have sufficiently validated results.
Gel was not successful and no bands presented.

Although Biobrick has some advantages that we wanted to exploit, it comes with
certain disadvantages that we had already been briefed from iGEM. Especially about
the method’s yield of success. We suspected that the method's overall yield was the
problem, since no band was observed.

### Cycle 2

![Button design](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/designes-igemthess23.png)
DESIGN

![Button build](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/buildes-igemthess23.png)
BUILD

![Button test](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/testes-igemthess23.png)
TEST

![Button learn](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/learnes-igemthsedd23.png)
LEARN

Taking into consideration the results from the previous failed assembly and
iGEM’s notification about the method’s yield we needed to examine if the
composite parts assembly was successful before proceeding to further investigations.

Driven by our instructor’s advice Mr Giannakopoulos, we utilize an original
protocol made by himself in order to overcome such barriers. According to that
protocol, an additional buffer is included in the mastermix that ensures that
restriction enzymes will have the optimal conditions to be activated at their
maximum. The additional presence of the enzyme buffer along with the ligase buffer
is one of their major differences. Furthermore, when utilizing the thermocycler for
the assembly reaction mixture, the three distinguished steps contribute to background
digestion.

Similarly, our aim was to first verify a successful assembly before moving to
further investigations regarding gene expression and inducer variation. In order
to achieve that, we selected EcorI and PstI , performed digestion and ligation in
one run according to Mr. Giannakopoulos’ protocol. After cultures’ successful growth,
we performed PCR quantification and ran agarose gel, screen fractions’ molecular
weights and interpreted the data.

According to the results, we achieved a successful assembly and agarose gel’s
bands correspond to the expected MW of the fractions digested with the restriction
enzymes. We suggest other iGEM teams facing similar obstacles with the BioBrick
assembly to use it [(see Contribution)](https://2023.igem.wiki/thessaloniki/contribution).

### Cycle 3

![Button design](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/designes-igemthess23.png)
DESIGN

![Button build](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/buildes-igemthess23.png)
BUILD

![Button test](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/testes-igemthess23.png)
TEST

![Button learn](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/learnes-igemthsedd23.png)
LEARN

We therefore decided to follow Mr Giannakopoulos protocol to assemble the
composite part containing the LuxI gene under control of Pgrac promoter,
that arrived belatedly. Unfortunately, although the assembly was achieved,
we were not able to perform experiments designed for quantification of
fluorescence. On the other hand, the assembly composite part of the ptRc-02
derivative promoter controlling TetR expression was successfully completed
and needed to be investigated in regard to efficient BsrG repression due to the
TetR repressor.

We therefore designed an SDS page in order to be able to determine the variation
of IPTG effect on BsrG expression. Additionally, we investigated *in silico* the
effect of IPTG induction under different concentrations on promoter induction.
More specifically the production of proteins under control of IPTG inducible
promoters was tested in different circumstances: with varying IPTG concentration,
inducer association constant and plasmid copy number. At the same time, according
to *in silico* analysis, when pGraC and pTrC02 derived promoters are used to control
LuxI and TetR production respectively, there are a few ways to accomplish coordinated
induction. Those variables are about plasmid copy number and promoter association
constant. However in both *silico* analysis about BsrG (toxin) and VapB15
(nostoc antitoxin) under different copy numbers, they achieved desirable outcomes
utilizing medium vectors.

According to the data, one band had no correlation with IPTG when supposed
to and control and tested cultures did not show the expected bands according
to their molecular weight.

According to the data, we observed that BsrG toxin production in *B.subtilis*
is not regulated effectively by IPTG. Therefore, we conclude that utilizing the
same IPTG inducible promoter Pgrac to both genes LuxI and TetR that trigger the kill
switch, could be proved suitable, since our vector is suitable.

### Cycle 4

![Button design](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/designes-igemthess23.png)
DESIGN

![Button build](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/buildes-igemthess23.png)
BUILD

![Button test](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/testes-igemthess23.png)
TEST

![Button learn](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/learnes-igemthsedd23.png)
LEARN

The improved design contained the LuxI gene under control of an IPTG inducible promoter pGraC
and the TetR gene under control of another IPTG inducible pGraC promoter. We constructed reporter
plasmids in order to include process controls, and fluorescent proteins at each step of the kill
switch in order to efficiently quantify TetR, BsrG for *B.subtilis* and LuxI, LuxR, Antitoxin production
for *Nostoc*. However due to time limitations, quantification of the kill switches mechanisms could
not be completed and need to be investigated in the future.

## Hydrogel

---

### Introduction

A hydrogel was designed entrapping the microspheres containing the synthetic consortium.
Deeply motivated by sustainable goals and circular economy, we designed the hydrogel matrix with two
co-polymers chitosan and pectin, derived from industrial waste and one synthetic peptide. The aim of
our biopolymer is to improve the soil's water capacity hence increasing its hydrophilicity since after
a wildfire, soil becomes hydrophobic and cannot retain water. Therefore our hydrogel should have good
swelling ability to align with Euphoresis goals. Additionally, it should be able to survive long enough
into the soil since it provides an extra layer of protection and further enhances biosafety of the project.
Before adding the synthetic peptide in our formula, different ratios of the two polysaccharides needed to
be examined, in order to conclude the optimal polymer consistency with the desired properties. We needed
to ensure high water absorbance ability and a lifespan of at least one month.
More specifically, after contacted with Professor Peppas we concluded to first tested the ratios 1⁄2 and 1⁄3

### Cycle 1

![Button design](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/designes-igemthess23.png)
DESIGN

![Button build](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/buildes-igemthess23.png)
BUILD

![Button test](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/testes-igemthess23.png)
TEST

![Button learn](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/learnes-igemthsedd23.png)
LEARN

In our design we investigated both pectin and chitosan properties regarding the desired
characteristics of the final product. Pectin provides high swelling ability,
while chitosan enhances stability. After consulting with Professor Peppas, we chose the ratio
of CS2%PE1%, CS3%PE1% so that we will be able to exploit both polymers characteristics, while
avoiding the risk of pectin’s instability when it is in excess.

When a new material is designed, its desired properties should be followed by its
corresponding physicochemical properties in order for the characterisation to be accurate
and valid. Hence viscosity measurement tests were crucial to conduct first, along with swelling
tests. This investigation will provide us a comprehensive overview of the crosslink network for
each proposed ratio.

Since at pH = 7 pectin’s charge is negative, while chitosan’s charge is positive, the two
polymers crosslink was conducted by addition of fully dissolved pectin in chitosan at 50°C
in solution of 0.1 M hydrochloric acid. 0.5 mL of pure acetic acid was then added in each
beaker and left under stirring for 24h. Viscosity was determined utilizing an electronic
rheometer at temperatures 25°C and 37°C, 5 days after the formation. After the samples
were dried for 2-3 days, swelling tests were performed in time = 0, 10, 20, 30, 60, 120
minutes and 24 hours by measuring initial dry mass of each sample and the dry mass of tested
samples after swelling.

Swelling ability of CS2%PE1%, CS3%PE1% exhibits similar values. In pH 7.3 they
have already reached swelling equilibrium below 200 minutes and by 1400 minutes they
still present swelling. While for pH 5 they seem not to have reached swelling
equilibrium at 1400 minutes, indicating a much higher swelling capacity at those conditions.

### Cycle 2

![Button design](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/designes-igemthess23.png)
DESIGN

![Button build](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/buildes-igemthess23.png)
BUILD

![Button test](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/testes-igemthess23.png)
TEST

![Button learn](https://static.igem.wiki/teams/4998/wiki/wiki-photos/dbtl/learnes-igemthsedd23.png)
LEARN

The choice of the correct ratio in our hydrogel was dependent on the degradation
rate/stability of our samples. Therefore we needed to test all hydrogel samples in
a soil environment within 20 days. Degradation of the hydrogel matrix is highly
affected by stability in soil, so such an examination is considered vital for our
desired goal in order to evaluate hydrogel properties based on their lifetime.

In order to test the degradation rate/stability of our hydrogel was tested in
soil. The dried sample of the hydrogel was placed inside the soil. It was weighed
at the start and then every 7 days for a month to predict its lifespan. The sample
were at room temperature

Soil’s pH was measured at pH=6.85 , a value within the range of greek forests’
soil, as said by Prof. Tsantilas. By tracking each sample's mass loss, the produced
hydrogels' soil stability was assessed. Each dry scaffold's part was carefully divided
and then put into a plastic cup that had been partially filled with soil samples.
The soil was then added to the cup until it was full.The samples' weight was measured
once more after each interval and stability/degradation was determined.

According to the data, we can fairly conclude that the CS2%PE1% hydrogel can absorb
external moisture and hasn't degraded throughout the course of the 20 days of the studies
due to no mass loss. These measurements lead us to choose CS2%PE1% hydrogel
as the best material for our project.

# References

1. Dvorak, P., Chrast, L., Nikel, P.I. et al. Exacerbation of substrate toxicity by IPTG in Escherichia coli BL21(DE3) carrying a synthetic metabolic pathway. Microb Cell Fact 14, 201 (2015). https://doi.org/10.1186/s12934-015-0393-3.

![forest](https://static.igem.wiki/teams/4998/wiki/wiki-photos/forestfooterneon-igemthess23.png)

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![](https://static.igem.wiki/teams/4998/wiki/wiki-photos/sustainability/experiments-big-final-4-igemthess23.png)

* [Overview](#Overview-experiments)
* [Peptide’s Experiments](#exp-1)
* [Hydrogel’s Experiments](#exp-2)
* [Microorganisms’ Experiments](#exp-3)

## Overview

---

During our work we studied, followed and documented dozens of protocols widely known in the field or more specific and adapted to our own needs. Here we present all of them that we utilized and that guided us in our (sought-after) lab work concerning our 3 basic modules : the engineered peptide’s expression, the hydrogel’s preparation and lastly, the creation, recombination, and characterisation of the bacterial consortium and its products .

#### Peptide’s Experiments

Our desired engineered sequence was purchased by Genscript biotech Corporation, New Jersey and shipped to us cloned into a pEX-A128 plasmid vector using Xho1/Nde1 restriction enzymes. The below procedures were executed :

**Transfer of the desired sequence into pET-29c plasmid vector**

### Transformation of TOP10 E.Coli cells with the plasmid vector pEX-A128

##### Introduction

---

This procedure aims to transform E.Coli bacteria cells so that we can amplify our purchased plasmid pEX-A128. TOP10 bacterial cells are known as the most popular E.Coli strain engineered for high transformation efficiency. They are suitable for cloning supercoiled DNA and allow stable replication of high-copy number plasmids.

### Alkaline Lysis of Bacteria Cells

##### Introduction

---

This procedure aims to isolate the now amplified purchased plasmids pEX-A128

### Digestion of plasmid vectors pEX-A128 and pET-29c(+)

##### Introduction

---

This procedure aims to digest both of the plasmids so that we can isolate and transfer our insert in between them. This method utilizes the restriction enzymes Nde1 and Xho1.

### DNA Extraction from Agarose Gel Using NucleoSpin Gel and PCR Clean-up Kit

##### Introduction

---

This procedure aims to extract and isolate our desired insert from the Agarose Gel. Here, this method acts as an intermediate step into cloning the desired nt sequence from the purchased engineered plasmid (pEX-A128) to the target plasmid (pET-29c).

### Ligase Reaction for joining DNA Segments

##### Introduction

---

This procedure aims to recombine the plasmid vector pET-29c with our desired insert. This technique is commonly used to ligate the ends of linear DNA molecules or combine different linear DNA molecules. The reaction is catalyzed by DNA ligase, with T4 DNA ligase used in this case. This protocol specifically details the ligation of DNA segments, such as PCR products or digested fragments, into the plasmid vector pET-29c(+).

### Transformation of TOP10 E.Coli cells with pET-29c(+) vector

##### Introduction

---

This procedure aims to transform E.Coli Bacteria Cells with our desired recombined vector to be able to amplify and store it. The same procedure described above is followed.

### Alkaline Lysis of Bacteria Cells

##### Introduction

---

This procedure aims to isolate the now amplified recombinant plasmid pET-29c

### Digestion of plasmid vector pET-29c(+)

##### Introduction

---

This procedure is diagnostic and it is used so that we can be sure of the efficient recombination of pET-29c. This method utilizes the restriction enzymes Nde1 and Xho1.

**Transformation of E.Coli BL21**

### Transformation of E. coli strain BL21(DE3) with recombinant plasmids pET-29c(+)

##### Introduction

---

This procedure aims to transform E.Coli Bacteria Cells with our desired recombined vector to be able to express our desired insert. The BL21(DE3) strain is widely used for recombinant protein production due to its engineered capacity to produce T7 polymerase and its deficiency in Lon and OmpT proteases.

### Induction of peptide’s gene’s expression

##### Introduction

---

This procedure aims to induce our insert’s expression with the use of IPTG. IPTG is a molecular analog of allolactose, which removes a repressor from the lac operon to induce gene expression. Here, bacteria of E. coli strain BL21(DE3), previously transformed with the recombinant plasmids pET-29c (+), are used to induce our peptides expression

**Isolation and Purification of produced peptide**

### Protein Isolation by Affinity Chromatography (native conditions)

##### Introduction

---

This procedure aims to isolate our produced peptide due to its interaction of its (6xHis)-COOH Tag with a Ni-Column. Affinity chromatography is based on the specific and reversible binding of proteins to ligand molecules attached to an inactive chromatography material.

**Identification of produced product - peptide**

### Bradford Assay

##### Introduction

---

We followed this procedure in order to quantify the expressed peptide’s concentration in samples so we could prepare the SDS-PAGE samples to be run in the next procedure.

### Protein electrophoresis on polyacrylamide gel in the presence of SDS (SDS-PAGE)

##### Introduction

---

This procedure aims to identify the expression of our desired insert (peptide) with the qualitative method of SDS-PAGE. This technique allows the separation of proteins based on their molecular weight.

### Protein staining in polyacrylamide with Coomassie Brilliant Blue R-250

##### Introduction

---

This procedure aims to stain the bands of the peptide created from the SDS-Page procedure. This method is based on the interaction of Coomassie Brilliant Blue R-250 dye with proteins, leading to the formation of a coloured complex of cyan colour

### (6xHis)-Tag Removal Using TEV-Protease

##### Introduction

---

TEV Protease is recommended for cleavage of a His-tag following purification by Affinity Chromatography (Ni Spin Columns). It is noted that while designing our peptide sequence we must have incorporated the amino acid sequence recognized by the protease enzyme appropriately

#### Hydrogel’s Experiments

### Hydrogel’s Preparation

##### Introduction

---

This procedure is used for the polymerization of chitosan, pectin and chitosan and pectin respectively under acidic conditions so that our control hydrogels can be formed.

#### Microorganisms’ Experiments

### Preparation of LB Growth Media and pouring LB Agar plates

**Amplification of plasmid vector**

### Resuspension of plasmid DNA and transformation of competent E.Coli TOP10 and DH5a competent cells with plasmid

##### Introduction

---

This procedure aims to transform E.Coli bacteria cells so that we can amplify plasmid vector. TOP10 and even DH5a bacterial cells are known E.Coli strains with high transformation efficiency. They are suitable for cloning supercoiled DNA and allow stable replication of high-copy number plasmids.

### Minipreps and NanoDrop

##### Introduction

---

This technique is used for the extraction of plasmids I8, I8’ C12, C12’, E12, E12’, K8, K8’, M8, M8’, E9, E9’, G8, G8’, A12, A12’ of the iGEM Kit, from host E. coli cells. Below the  [Monarch® Plasmid Miniprep Kit](https://dx.doi.org/10.17504/protocols.io.bg9qjz5w) is followed so that we can isolate our plasmid vector that has been amplified.

**Construction recombinant plasmid vectors, amplification and isolation :
Our part’s DNA sequences were designed and purchased from Twist Bioscience, California. We then inserted them into a plasmid vector () , cloned them into E.Coli Systems and isolated them.**

### Biobrick assembly protocol

##### Introduction

---

### Golden gate assembly protocol

##### Introduction

---

### Modified assembly protocol with the use of two ristriction enzymes

##### Introduction

---

### Amplification Polymer Chain Reaction (PCR)

##### Introduction

---

This procedure is carried out to determine and identify the results of the plasmid’s recombination. We used kb markers and tried to match the bands that occurred after this method’s execution to our expected nt size.

### Agarose Gel Electrophoresis

##### Introduction

---

Agarose Gel Electrophoresis is carried out as a diagnostic procedure for confirmation of the successful transformation of the E. coli competent cells, for the determination of the success of our fragments’ amplification and purity, after PCR etc.

### SDS-PAGE : Mini-PROTEAN® TGX™ gel in the Mini-PROTEAN Tetra cell

##### Introduction

---

Mini-PROTEAN TGX Precast Protein Gels is used for separation of polypeptides ~2–400 kD. It is noted that long-life TGX (Tris-Glycine eXtended) Gels have a novel formulation and can be used for both standard denaturing protein separations as well as native electrophoresis.

### BLUE SILVER Coomassie Colloidal Blue Stain

##### Introduction

---

This procedure is based on (Candiano et al Electrophoresis 2004) and has a duration of 5 – 13 hrs. It's used so that the bands that occured for the SDS-PAGE procedure can be stained and observed. This protocol concerns a modified Neuhoff's colloidal Coomassie Blue G-250 stain with considerably higher sensitivity, approaching the one of conventional silver staining.

### Transformation of Bacillus Subtilis (WB800N) with our recombinant vector

### Transformation of Nostoc Oryzae TAU-MAC 2710 with our recombinant vector

### Induction of genes’ expression with IPTG

### Bradford assay

![forest](https://static.igem.wiki/teams/4998/wiki/wiki-photos/forestfooterneon-igemthess23.png)

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![](https://static.igem.wiki/teams/4998/wiki/wiki-photos/pages-top/results-final-igemthess23.png)

## Overview

---

This year Euphoresis team decided to deal with a project which combines classical molecular biology techniques, polymer chemistry and innovative synthetic biology-based methodologies. Thus, knowledge from different scientific fields including molecular biology, biochemistry, computer science and polymer-technology was employed.

In this page, you can find all the results from producing, purifying, and characterizing our parts while it is also noted that analytical information regarding the strategies that were employed for the execution of each step is provided on the experiments page.

![Button Image 1](https://static.igem.wiki/teams/4998/wiki/wiki-photos/results/hydrogel-button-igemthess23.png)

### Hydrogel

![Button Image 2](https://static.igem.wiki/teams/4998/wiki/wiki-photos/results/microbes-button-igemthess23.png)

### Microbes

### Hydrogel Workflow

To be able to construct the final proposed hydrogel material, multiple basic modules
had to be developed from scratch with the goal to be efficiently assembled as a functional novel material
for soil-conditioning and priming purposes. Our team, after conceptualizing and designing our project’s
components, worked in a laboratory to be able to present a basic proof of concept.

![Hydrogel results workflow](https://static.igem.wiki/teams/4998/wiki/wiki-photos/results/workflow-hydrogel.png)

# Viscosity measurement test

## Viscosity measurement test

#### Aim:

Hydrogel’s characterisation-Determine the viscosity of each hydrogel sample
(CS3%,CS2%PE1%, CS3%PE1%) in temperatures of 25°C and 37°C.

#### Methods:

It is noted that our proposed hydrogels consisting of different polysaccharide ratios,
were compared to the control hydrogel CS3% only, and not with CS3% and PE1% hydrogel due
to faults in its preparation. This procedure is preceded from the other assays as it is the
first step to characterize and verify the production of a novel hydrogel material such as
our proposed one.
The viscosity of each hydrogel was measured with the electronic rheometer with temperature
control BGD 175/ from Biuged Laboratory Instruments (Guangzhou) Co., Ltd.The spindle used
for our samples was the SC4-21. Measurements were taken on day 5 after hydrogel preparation.

#### Results:

![pp](https://static.igem.wiki/teams/4998/wiki/wiki-photos/results/rheometer.png)

**Figure 1:** Rheometer used for the viscosity determination assays.

![Execution of viscosity measurement via the respective rheometer’s program.                                ](https://static.igem.wiki/teams/4998/wiki/wiki-photos/results/rheometer-working.png)

**Figure 2:** Execution of viscosity measurement via the respective rheometer’s program.

##### The procedure described above, resulted in the following diagrams:

![pp](https://static.igem.wiki/teams/4998/wiki/wiki-photos/results/25-viscosity.png)

**Graph 1:** Overall hydrogels Viscosity (CS3%, CS2%PE1% , CS3%PE1%) at 25°C

![pp](https://static.igem.wiki/teams/4998/wiki/wiki-photos/results/37-viscosity.png)

**Graph 2:**Overall hydrogels Viscosity (CS3%, CS2%PE1%, CS3%PE1%) at 37°C

#### Conclution:

From the diagrams taken above we conclude that physical crosslink was
carried out successfully between chitosan and pectin and thus our material
was formed effectively. Nevertheless, the assays carried out afterwards are
necessary for the selection of the desired optimal ratio to be used.

# Swelling efficiency test

## Swelling efficiency test

#### Aim:

Determine the swelling capacity of each hydrogel (CS3%,CS2%PE1%, CS3%PE1%) in room temperature

#### Methods:

Based on this method we can pick out the sample that has the optimal water adsorption
capacity, appropriate for our desired goal. Swelling ability of the prepared hydrogels
was evaluated by measuring the amount of water sorption aptitude of dH2O (pH=7.3)
and acidic buffer (pH=5.5) . Part of each dry scaffold was carefully cut, weighed
(Wd) and placed into a beaker filled with dH2O (or buffer) at room temperature.
Measurements were held at predetermined times (10 min, 20 min, 30 min, 1 h, 2 h, and 24 h).
After each respective time the samples were placed on filter paper to remove the excess
surface water and their weight (Ws) was measured again. Swelling ratio was calculated
according to equation (1) [1] . It is noted that the measurement of each sample was held
in triplicates and that our proposed hydrogels with different polymer ratios were compared
to the control hydrogel CS3% only, and not with CS3% and PE1% hydrogel due to faults in its preparation.

Swelling ratio%=(Ws-Wd)/Wd\*100 (1)

#### Results:

![pp](https://static.igem.wiki/teams/4998/wiki/wiki-photos/results/swelling-experiment.png)

**Figure 1:** Experimental procedure of determination of swelling
ratio of each hydrogel. Top picture (A) depicts dry state hydrogel and bottom (B) swollen hydrogel
through the experiment’s duration.

##### The procedure described above, resulted in the following diagrams:

![pp](https://static.igem.wiki/teams/4998/wiki/wiki-photos/results/swelling-ratio-ph7-3.png)

**Graph 1:** Swelling Ratio of CS3%, CS2%PE1%, CS3%PE1% at room
temperature in dH2O (pH= 7.3)

![pp](https://static.igem.wiki/teams/4998/wiki/wiki-photos/results/swelling-ratio-ph5-5.png)

**Graph 2:**Swelling Ratio of CS3%, CS2%PE1%, CS3%PE1% at room
temperature in buffer (pH= 5.5)

#### Conclution:

From the tests above, it can be concluded that the control CS3% hydrogel has the
best swelling ratio among the hydrogels tested while CS2%PE1% and CS3%PE1% hydrogels have
comparable swelling ratios in both 7.3 and 5.5 pH values. It should be also noted that,
for CS2%PE1% and CS3&PE1% hydrogels’ swelling ratios in ddH2O of the one-day measurements
still show a rising diagram’s trend which means that they have not yet reached their water saturation phase.
This can also be concluded for CS3% hydrogel who displays a great increase of swelling ratios in buffer 5.5.
Nevertheless it shall be noted that in the progress of our measurements CS3% hydrogel exhibited low stability
and began to lose its structure early in the experiment.

# Hydrogel’s degradation assay in soil

## Hydrogel’s degradation assay in soil

#### Aim:

Determine the degradation rate / stability of each hydrogel (CS3%, CS2%PE1% , CS3%PE1%) in
a soil environment within 20 days.

#### Methods:

Based on this method we can pick out the sample that has the optimal stability in soil,
appropriate for our desired goal. Soil’s pH was measured as a suspension in dH2O (1.5% w/v)
in triplicates and was found at 6,85 (σ=0.04) by Prof. Theodora Matsi.

Soil stability of the prepared hydrogels was evaluated by measuring the mass loss of each
sample. Part of each dry scaffold was carefully cut, weighed (Wd) and placed into a plastic
cup filled halfway with soil. Then the cup was filled completely with soil. It is noted
that the hydrogel pieces were secured into loose net material. Measurements were held at
predetermined times (0,8 and 20 days). After each respective time the samples’ weight (Ws)
was measured again. Stability was calculated according to equation (2) [2]. It is noted
that the measurement of each sample was held in triplicates and that hydrogels with the
proposed polysaccharide ratios were compared to the control hydrogel CS3% only, and not
with CS3% and PE1% hydrogel due to faults in its preparation.

Canonical Dry Mass %: \( \frac{W\_s}{W\_d} \times 100 \)

#### Results:

![ Execution and preparation of soil stability assays for the hydrogels (CS3%, CS2%PE1%, CS3%PE1%) day 0. ](https://static.igem.wiki/teams/4998/wiki/wiki-photos/results/stability-tests.png)

**Figure 1:** Execution and preparation of soil stability assays for the hydrogels (CS3%, CS2%PE1%, CS3%PE1%) day 0.

##### In a timespan of 20 days our results are summarized in the following diagram:

![ Stability Assay of Hydrogels: Canonical Dry Mass of each hydrogel % measured in the span of 20 days left in soil.                                ](https://static.igem.wiki/teams/4998/wiki/wiki-photos/results/degradability.png)

**Graph 1:** Stability Assay of Hydrogels: Canonical Dry Mass of each hydrogel % measured in the span of 20 days left in soil.

#### Conclution:

From the diagrams retrieved above it can be concluded that CS3% hydrogel degrades as expected into neutral pH
soil (pH=6.85 , a value within the range of greek forests’ soil, as said by Prof. Tsantilas).
For CS3%PE1% hydrogel (potentially due to its high CS ratio) the diagram exhibits a similar image,
but it can be concluded that physical crosslink with pectin aids hydrogel’s activity. Lastly, concerning
the CS2%PE1% hydrogel, as diagram 6 shows, its mass remains unaltered, and we can safely conclude that
this material a) can absorb environmental moisture and b) hasn’t entered the degradation state in the 20
days timespan of the experiments. These measurements rend CS2%PE1% hydrogel as our optimal material for
the project’s purposes.

### Peptide Workflow

![Hydrogel results workflow](https://static.igem.wiki/teams/4998/wiki/wiki-photos/results/workflow-peptide.png)

# Transfer of peptide’s sequence from pEX-A128 to pET-29c plasmids

## Transfer of peptide’s sequence from pEX-A128 to pET-29c plasmids

#### Aim:

Determinate the success of the insert’s transfer from the delivered plasmid to our expression vector pET-29c

#### Methods:

As mentioned, after designing our proposed peptide we purchased it cloned into an intermediate plasmid vector (pEX-A128). It is noted that our engineered peptide reverse-translated consists of around 100-130 base pairs. In order to determine its successful insertion into the final vector (pET-29c) and before transforming TOP10 cells with it, we carried out Agar Electrophoresis experiments: one concerning the digestion of the 2 plasmids (pEX-A128 containing our insert and pET-29c) and one after plasmid isolation from TOP10 E.Coli cells (recombinant pET-29c) . After carrying out these experiments the images below were obtained:

#### Results

![ Images after digested samples (lane 2) electrophoresed through Agar (1.5%). Red arrows bands correspond to our insert and to digested vector pET-29c (~100 bp and ~5400 bp respectively). The DNA marker sizes are shown left from each electrophoresis image.                            ](https://static.igem.wiki/teams/4998/wiki/wiki-photos/results/peptide-digestions.png)

**Figure 1:** Images after digested samples (lane 2) electrophoresed through Agar (1.5%). Red arrows bands correspond to our insert and to digested vector pET-29c (~100 bp and ~5400 bp respectively). The DNA marker sizes are shown left from each electrophoresis image.

![Marker 100-3000 bp (left) was electrophoresed in lane 1 and marker 0.5-10 kb (right) was electrophoresed in lane 3 in picture shown in B. B] Agar Electrophoresis: Markers were electrophoresed in lanes 1 and 3. In lane 2 the sample of the digested (with Xho1 and Nde1) recombinant pET-29c was loaded. The bands shown correspond to the pET-29c (~ 6 kb) and our insert (~100 bp) respectively.      ](https://static.igem.wiki/teams/4998/wiki/wiki-photos/results/peptide-digestion-agar-1-lane.png)

**Figure 2:** Marker 100-3000 bp (left) was electrophoresed in lane 1 and marker 0.5-10 kb (right) was electrophoresed in lane 3 in picture shown in B. B] Agar Electrophoresis: Markers were electrophoresed in lanes 1 and 3. In lane 2 the sample of the digested (with Xho1 and Nde1) recombinant pET-29c was loaded. The bands shown correspond to the pET-29c (~ 6 kb) and our insert (~100 bp) respectively.

#### Conclution:

We concluded that the digestions and so the transfer of our insert was carried out successfully between the 2 plasmids and therefore we could continue our process to transform BL21 bacterial cells so that we can induce our insert’s expression. It is noted that the bands appeared dim because of the low level of expression or the small molecular weight of our peptide’s nt sequence.

# SDS-Page for determination of peptide’s expression

## SDS-Page for determination of peptide’s expression

#### Aim:

Determine the expression of the peptide after IPTG induction before and after purification with Affinity Chromatography.

#### Methods:

After we transformed *E. Coli* BL21 cells and induced the expression of our desired insert we carried
our Bradford assays and run SDS-PAGE electrophoresis experiments of 4 bacterial cultures (it is noted
that culture whose sample was electrophoresed in lane 2 and 9 was not induced with IPTG) so that we
could detect a band corresponding to the peptide’s size (around 4 kDa). The same procedure was repeated
once more for the culture who appeared to express most highly the peptide (Sample 3, lane 6) after the
bacterial lysis and affinity Chromatography experiments so that we can detect it more efficiently.
It is noted that in the second SDS-PAGE crude samples as well as 3 elusion samples were loaded (not shown).
After carrying out these experiments the images below were obtained:

#### Results

![ Images after digested samples (lane 2) electrophoresed through Agar (1.5%). Red arrows bands correspond to our insert and to digested vector pET-29c (~100 bp and ~5400 bp respectively). The DNA marker sizes are shown left from each electrophoresis image.                        ](https://static.igem.wiki/teams/4998/wiki/wiki-photos/results/sds-before-and-after-iptf-peptide.png)

**Figure 1:** SDS-PAGE of whole bacterial cells (lysed with lysozyme)
before and after IPTG induction of our insert’s expression. 4 Samples collected from liquid bacterial
cultures were electrophoresed. It is noted that protein markers are loaded at lanes 1 and 10. Lanes
4, 6 and 8 depict samples of IPTG induced bacterial cells. Faint bands can be seen in all those lanes
with the clearest to appear in lane 6 at the anticipated protein weight.

![Marker 100-3000 bp (left) was electrophoresed in lane 1 and marker 0.5-10 kb (right) was electrophoresed in lane 3 in picture shown in B. B] Agar Electrophoresis: Markers were electrophoresed in lanes 1 and 3. In lane 2 the sample of the digested (with Xho1 and Nde1) recombinant pET-29c was loaded. The bands shown correspond to the pET-29c (~ 6 kb) and our insert (~100 bp) respectively.      ](https://static.igem.wiki/teams/4998/wiki/wiki-photos/results/purified-peptide.png)

**Figure 2:** SDS-PAGE of eluent 1 (lane 2) after Affinity Chromatography with Ni-Columns

#### Conclution:

We concluded that our desired protein was indeed produced from the recombinant BL21 cells at its
expected molecular weight (~ 4 kDa) and that its purification with Affinity Chromatography was carried
out successfully. It is noted that the bands appeared dim because of the low level of expression or the
small molecular weight of our peptide. Finally at Fig. 9 our produced peptide appears higher than expected
(~ 12 kDa) and that's potentially because of peptide’s dimerization or trimerization. Thus, it is safe to
conclude that our proposed peptide can indeed be polymerized (as designed) and further additional pre-treatment
should be carried out (more intense denaturation conditions such as longer boiling of samples or higher
concentrations of b-mercaptoethanol) so that it can be observed at its anticipated size via the SDS-PAGE
procedure.

## Review

On a final note, the experiments were conducted successfully and our team was able to confirm part of
its proposed original design. However, it should be mentioned that due to tight time constraints, experiments
concerning the construction and characterization of the microspheres, encapsulation of the consortium in them,
antimicrobial tests with our peptide (as well as with the original one : 2L21) and the preparation and study
of hydrogels containing our peptide are missing and thus should and will be carried out additionally and
complementary. It is noted once again that due to faults at PE1% hydrogel, comparisons with pectin-only hydrogel
need to be executed as well. Finally, it would be ideal to execute pilot-applications of our proposed material
in affected soils (under strict and certain conditions).

### Microbes Workflow

![Microbes results workflow](https://static.igem.wiki/teams/4998/wiki/wiki-photos/results/workflow-microorganisms.png)

# IPTG and   *Bacillus subtilis*  - Initial experiments

## IPTG and *Bacillus subtilis*

#### Aim:

The selection of the correct IPTG concentration

#### Methods:

Isopropyl β-D-1-thiogalactopyranoside (IPTG) is a molecular biology reagent, a mimic of allolactose
(lactose metabolite). IPTG controls the expression of the promoters related to the quorum sensing system,
and the kill switches. However, IPTG in high concentrations has toxic action in cells. So, we had to test
the effect of different IPTG concentrations in the bacteria population.
At the start of our experiments, we conducted the assemblies using the strain Bacillus subtilis 3601.
Before the parts for the assemblies arrived, we had to check the IPTG variation effect. The cultures were
grown ON at 30°C, in BG/LB medium. The next day, we conducted spectrophotometry OD600 measurements after
inducing them with 0.05mM, 0.1mM, 0.2mM, 0.4mM, and one control sample with no IPTG. The collection of
the data was done every hour for 24h.

#### Results:

**Table 1:** *Bacillus subtilis* #3601 growth rates

|  | 1h | 2h | 3h | 4h | 5h | 6h | 7h | 8h | 9h | 10h | 11h | 12h | 13h | 14h | 15h | 16h | 17h | 18h | 19h | 20h | 21h | 22h | 23h | 24h |
| control | 0.086 | 0.097 | 0.113 | 0.140 | 0.166 | 0.193 | 0.215 | 0.248 | 0.252 | 0.280 | 0.321 | 0.366 | 0.441 | 0.490 | 0.560 | 0.614 | 0.679 | 0.726 | 0.759 | 0.790 | 0.821 | 0.844 | 0.859 | 0.867 |
| 0.05 mM | 0.090 | 0.091 | 0.114 | 0.138 | 0.161 | 0.192 | 0.221 | 0.254 | 0.276 | 0.296 | 0.321 | 0.346 | 0.420 | 0.445 | 0.485 | 0.525 | 0.564 | 0.604 | 0.644 | 0.683 | 0.723 | 0.763 | 0.803 | 0.835 |
| 0.1 mM | 0.088 | 0.089 | 0.114 | 0.147 | 0.161 | 0.196 | 0.229 | 0.262 | 0.264 | 0.297 | 0.322 | 0.347 | 0.391 | 0.416 | 0.447 | 0.477 | 0.508 | 0.538 | 0.594 | 0.633 | 0.676 | 0.719 | 0.795 | 0.853 |
| 0.2 mM | 0.092 | 0.095 | 0.115 | 0.146 | 0.186 | 0.191 | 0.225 | 0.264 | 0.278 | 0.303 | 0.328 | 0.354 | 0.379 | 0.404 | 0.430 | 0.455 | 0.461 | 0.485 | 0.509 | 0.514 | 0.557 | 0.529 | 0.540 | 0.549 |
| 0.4 mM | 0.089 | 0.094 | 0.116 | 0.161 | 0.160 | 0.189 | 0.216 | 0.253 | 0.273 | 0.292 | 0.316 | 0.340 | 0.364 | 0.388 | 0.413 | 0.437 | 0.461 | 0.485 | o.509 | o.514 | 0.557 | 0.529 | 0.540 | 0.549 |

![pp](https://static.igem.wiki/teams/4998/wiki/results/growth-rates-3601.png)

**Graph 1:**Graphical representation of *Bacillus subtilis'* growth rate

#### Conclution:

After examining the growth rates we realized that IPTG in concentration higher than 0.1 mM
has a negative effect on *B. subtilis* growth. The following experiments for the growth rates
of  *Nostoc* were conducted in the range of 0-0.1

# Growth rate of cyanobacterium   *Nostoc oryzae*   TAU-MAC 2710

## Growth rate of cyanobacterium *Nostoc oryzae* TAU-MAC 2710

#### Aim:

IPTG induction in Nostoc Oryzae TAU-MAC 2710

#### Methods:

Originally, our plan was to design the kill switch of the cyanobacterium in a similar way with the
*B. subtilis.* More specifically, we wanted to have the same induction system: an IPTG induced system. We decided on the use strain of Nostoc Oryzae TAU-MAC 2710 with the guidance of our PI Prof. Gkelis. After choosing the strain we had to test its growth rates on different IPTG concentrations. Having found out that concentrations more than 0,1 mM is toxic for B. subtilis, we tested the cyanobacteria cultures in 0.05 mM, and 0.1 mM and one control with no IPTG. The cultures were grown in 30oC, in LB/BG medium. Because the cyanobacterium metabolic rhythm is affected by the light conditions, we made sure that our cultures were in limited light to simulate the conditions in the first layer of the soil. We measure our samples at 680, 750, and 800 nm, every two days.

#### Results:

**Table 1:** *Nostoc Oryzae*  TAU-MAC 2710 growth rates without IPTG

|  | 0d | 2d | 4d | 6d | 8d | 10 | 12 | 14 | 16 | 18 | 20 |
| 680nm | 0.175 | 0.206 | 0.48 | 0.67 | 1.2 | 1.45 | 1.808 | 2.14 | 2.456 | 2.545 | 2.898 |
| 750nm | 0.164 | 0.186 | 0.408 | 0.649 | 0.96 | 1.374 | 1.544 | 2.065 | 2.397 | 2.504 | 2.85 |

**Table 2:** *Nostoc Oryzae*  TAU-MAC 2710 growth rates with 0.05 mM IPTG IPTG

|  | 0d | 2d | 4d | 6d | 8d | 10d | 12d | 14d | 16d | 18d | 20d |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 680nm | 0.255 | 0.312 | 0.309 | 0.414 | 0.45 | 0.645 | 0.77 | 0.978 | 1.345 | 1.587 | 1.897 |
| 750nm | 0.221 | 0.27 | 0.265 | 0.395 | 0.425 | 0.639 | 0.745 | 0.955 | 1.295 | 1.52 | 1.844 |

**Table 3:** *Nostoc Oryzae*  TAU-MAC 2710 growth rates with 0.1 mM IPTG IPTG

|  | 0d | 2d | 4d | 6d | 8d | 10d | 12d | 14d | 16d | 18d | 20d |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 680nm | 0.099 | 0.143 | 0.114 | 0.345 | 0.255 | 0.458 | 0.609 | 0.871 | 1.17 | 1.648 | 1.88 |
| 750nm | 0.099 | 0.124 | 0.111 | 0.294 | 0.229 | 0.449 | 0.597 | 0.794 | 1.045 | 1.612 | 1.846 |

![pp](https://static.igem.wiki/teams/4998/wiki/results/growth-rates-cyanobacterium-no-iptg.png)

**Figure 1:** Graphical representation of *Nostoc Oryzae* TAU-MAC 2710 growth rate without IPTG

![ Graphical representation of Nostoc Oryzae TAU-MAC 2710 growth rate with 0.1 mM IPTG  ](https://static.igem.wiki/teams/4998/wiki/results/growth-rates-cyanobacterium-iptg-0-05.png)

**Figure 2:** Graphical representation of *Nostoc Oryzae* TAU-MAC 2710 growth rate with 0.05mM IPTG

![ Graphical representation of Nostoc Oryzae TAU-MAC 2710 growth rate with 0.1 mM IPTG](https://static.igem.wiki/teams/4998/wiki/results/growth-rates-cyanobacterium-iptg-0-1.png)

**Figure 3:** Graphical representation of *Nostoc Oryzae* TAU-MAC 2710 growth rate with 0.1 mM IPTG

#### Conclution:

After those results, we realized that the growth of our cyanobacterium is really slow, as expected in those harsh conditions due to low light density and the natural slower growth rate compared to *B. subtilis*. Comparing the controlled culture to the two tested cultures the growth rates were not significantly slower. The general slow rate of cyanobacterium does not make it suitable for it to maintain a quick on-off IPTG induced kill-switch system. After this, we redesigned our project so that the kill-switch of cyanobacterium was controlled by a quorum sensing system. Additionally, between the two concentrations of IPTG (0.05 mM, 0.1mM) there was no great difference in the growth of Nostoc.

# Choosing Plasmid Vector

## Choosing Plasmid Vector

#### Aim:

The selection of the most suitable plasmid vector by reviewing the copy numbers.

#### Methods:

For the conduction of our experiments we needed a plasmid backbone with medium copy number. According to our model results a plasmid with medium copy number is proposed to satisfy at the same time the needs for our antitoxin production, the coordination of TetR and LuxI, and the production of the Bacillus toxin. We decided to use the plasmids with medium copy numbers from the iGEM Distribution Kit. We transformed TOP10 E.Coli cells with the plasmids I8, C12, E12, K8, M8, E9, G8, and A12 and then the cells were plated and incubated at 37oC, overnight. Every plasmid contained a resistant gene for the antibiotic of kanamycin so, in order to detect the transformed E.coli cells, the plating was done in LB agar with kanamycin. Only the cells that have received the plasmid survived and formed colonies in the LB Agar. The transformation of E.Coli cells was successful for all the eight plasmids.

We inoculated into liquid growth media one culture of each plate and we grew the cells at 37°C
overnight. Then we proceeded to plasmid extraction followed by NanoDrop spectrometry.

NanoDrop spectrometry is used to determine the purity of the plasmids.

All nucleotides absorb at 260 nm, meaning that absorbance in a different wavelength may indicate
the presence of other molecules, like a protein. The 260 nm/280 nm ratio can be used as a primary
indicator of the purity of DNA. A ratio of ~1.8 is indicative for “pure” DNA. The 260/230 ratio is a
secondary indicator of DNA purity and indicates “pure” DNA in the 2.0-2.2 rates.

#### Results:

From the NanoDrop spectrometry, the following results were obtained (C equals plasmid concentration
in ng/μL):

**Table 1:**  *NanoDrop sprectometry*

|  | I8 | I8' | C12 | C12' | E12 | E12' | K8 | K8' | M8 | M8' | E9 | E9' | G8 | G8' | A12 | A12' |
| C | 33 000 | 26 500 | 29 250 | 27 550 | 25 100 | 19 450 | 20 400 | 24 050 | 31 350 | 32 050 | 7 750 | 8 150 | 26 000 | 29 300 | 23 800 | 27 050 |
| 260/280 | 2.336 | 2.459 | 2.442 | 2.699 | 2.521 | 1.741 | 2.442 | 2.699 | 2.801 | 2.789 | -7.409 | -9.549 | 3.038 | 3.123 | 4.057 | 3.981 |
| 260/230 | 3.154 | 3.239 | 2.681 | 3.112 | 2.856 | 3.484 | 2.681 | 3.112 | 1.235 | 2.546 | 3.611 | 2.866 | 2.170 | 1.764 | 1.847 | 1.972 |

#### Conclution:

In order to conduct the assembly we needed an efficient plasmid concentration. All of the plasmid concentrations seemed to be satisfying, apart from the E9-E9’ concentration, which was below 20,000 ng/μL. However, 260/280 and 260/230 ratios indicated impurities in all the plasmids. Taking all those into account, we chose to use the C12 plasmid for our parts assembly, which seemed to have the best combination of 260/280 and 260/230 ratios, as well as a concentration that is considered satisfactory.

# Original design assembly - Biobrick assembly

## Original design assembly - Biobrick assembly

#### Aim:

The successful recombination of our genes of interest inside the plasmid vector and the successful
transformation of the cells.

#### Methods:

We designed our sequences, with the respective prefix and suffix, to be compatible with the
requirements of BioBrick assembly. At our first attempt, we tried to assemble our parts following
the protocol of New England Biolab for the double digestion with restriction enzymes. We used the
restriction enzymes E.coRI and PstI. After the plasmid was digested, we continued with the ligation
according to the T4 DNA Ligase NEB protocol. After the two protocols, we used the recombinant plasmids
to transform *E. coli* TOP 10 cells. The cultures of the cells were an initial proof of the
successful transformation.

We designed the suitable primers for every desirable part and continued with the PCR procedure to
amplify our genes of interest. We continued with the electrophoresis gel to validate their transformation.

#### Results:

Despite what we anticipated, the results of the gel electrophoresis showed that the transformation
was not successful for the assembly with multiple fragments.

![pp](https://static.igem.wiki/teams/4998/wiki/laccase-assembly-1.png)

**Figure 1:** Electrophoresis of DNA parts after biobrick assembly

#### Conclution:

We decided to try to modify the protocol for the assembly, using the same restriction enzymes to achieve assembly with multiple fragments.

# Original design assembly - Modified assembly protocol

## Original design assembly - Modified assembly protocol with the use of two restriction enzymes

#### Aim:

The successful recombination of our genes of interest inside the plasmid vector and the successful
transformation of the cells.

#### Methods:

Knowing from the iGEM protocols that BioBrick assembly does not have high succession rate, and by
following the consultation of our team’s instructor Christos Giannakopoulos we decided to follow a
modified assembly protocol. More specifically we used a specific Golden Gate Assembly Protocol and
instead of BsaI we added the restriction enzymes of BioBrick Assembly. At this point of our project
the design for the promoter of LuxI changed because we wanted the immediate induction of cyanobacteria
to be also IPTG induced. So we only recombined the composite parts regarding the Bacillus subtilis kill
switch, laccase gene, and HetR gene. More specifically for our kill-switch we did an assembly of three
composite parts, while for the other two it was one composite part assembly. The plasmid mixture was used
to transform competent *E. coli* Top10 cells. Then we used the primers that we designed and we proceeded to
the PCR procedure to amplify the desired genes. We continued with the electrophoresis gel to validate their
transformation.

#### Results:

![pp](https://static.igem.wiki/teams/4998/wiki/laccase-assembly-2.png)

**Figure 1:**Electrophoresis of DNA parts after biobrick assembly

#### Conclution:

The assembly was successful. The impressive part was that we managed to have a successful triple
assembly for our kill-switch mechanism in one recombinant plasmid with [our new protocol!](https://static.igem.wiki/teams/4998/wiki/wiki-photos/experiments/modified-assembly-protocol-with-the-use-of-two-ristriction-enzymes-benchling.pdf)

# Transformation of   *Bacillus subtilis*  3601

## Transformation of *Bacillus subtilis* 3601

#### Aim:

To start the expression of our laccase gene and our kill switch system in  *B. subtilis* in order to start the characterization of our parts

#### Methods:

We followed the protocol of minipreps and nanodrops from our successfully transformed
with the desired recombinant  *E. coli*cells for *B. subtilis* 3601 transformation of the
composite parts of laccase and *Bacillus* kill switch mechanism. After the transformation
we prepared plate cultures in LB agar broth medium with kanamycin.

#### Results:

The transformation was successful, as we managed to have cultures grow in the plates

![Plate of transformed Bacillus subtilis cells](https://static.igem.wiki/teams/4998/wiki/results/transformation.png)

**Figure 1:**Transformed *Bacillus subtilis* bacteria

#### Conclution:

We started the characterization of our parts.

# Initial laccase detection - Bradford assay

## Initial laccase detection - Bradford assay

#### Aim:

To determine the total protein in the extracellular matrix. We are expecting the modified *B. subtilis*
3601 to produce more extracellular proteins than the control one.

#### Methods:

After the successful transformation of our *B. subtilis* cells we inoculate cultures from the
plates with the transformed *B. subtilis* into a liquid growth medium. We incubate them in 37°C for
48h for the laccase production with 3 different concentration of CuSO4 at 50 μΜ, 75 μΜ, 100μΜ,
one transformed *B.subtilis* with 0 mM sample CuSO4 and one control non transformed *B. subtilis*.
After those 48h we centrifuge our samples and took 2 ml of the supernatant. First we calibrated
with standard concentrations and then we calculated the concentration of our samples.

#### Results:

The resulting concentrations of our samples compared with the controlled ones were approximately the same.
Although we conducted an SDS page in case we could detect a higher OD in the band of the molecular weight
of our laccase in our samples compared to the controlled ones.

#### Conclution:

Although the results of Bradford were not showing any enhanced protein production
and extraction, we decided to conduct an SDS-page to see if there would be any significant changes in the
expression of proteins in the molecular weight of our laccase.

# Initial laccase detection - SDS-PAGE

## Initial laccase detection - SDS-PAGE

#### Aim:

To detect higher absorbance in the bands with laccase’s molecular weight in our samples
compared with the control

#### Methods:

Following the protocol for the SDS page we used the same supernatant from the same samples as above in
Bradford (3 different concentration of CuSO4 at 50 μΜ, 75 μΜ, 100μΜ, one transformed *B.subtilis*
0 mM sample CuSO4 and one control non transformed *B. subtilis*). Then we scan the resulted gel.

#### Results:

The protein volume in the band of the molecular weight of our laccase compared to
the control sample had no significant difference.

![SDS-Page gel of secreted laccases](https://static.igem.wiki/teams/4998/wiki/results/unsuccessful-sds.png)

**Figure 1:**SDS-PAGE of secreted laccases

#### Conclution:

Because the recombination and transformation were successful we had to troubleshoot on what could interfere
with the successful expression of our laccase. After contacting PhD candidate Anargyros Alexiou we realized
that the problem was the extracellular protease that our strain was producing. He suggested the *B. subtilis*
WB800N that has its extracellular proteases knocked out

# IPTG and Bacillus subtilis- After changing strain

## IPTG and Bacillus subtilis- After changing strain

#### Aim:

The selection of the correct IPTG concentration

#### Methods:

After changing the strain of *B. subtilis* we decided to calculate the growth rate after we transformed
the *B. subtilis* WB800N to have more accurate results about its toxicity (because the extra plasmid
increases the metabolic burden). We transformed our strain with pJUMP29-1D’ plasmid. Again, the cultures
were grown ON at 30°C. The next day, we conducted spectrophotometry OD600 measurements after inducing them
with 0.05mM, 0.1mM, 0.2mM, 0.4mM and control with no IPTG. The collection of the data was done every hour
for 24h.

#### Results:

According to the results, we observe that cell density with IPTG inducer concentrations up
to 0.1mM is similar to the control culture. On the contrary, cultures with IPTG above 0.2mM
exhibit low cell density indicating slow metabolism and potential cytotoxicity.

**Table 1:** *Bacillus subtilis* #3601 growth rates

| 1h | 2h | 3h | 4h | 5h | 6h | 7h | 8h | 9h | 10h | 11h | 12h | 13h | 14h | 15h | 16h | 17h | 18h | 19h | 20h | 21h | 22h | 23h | 24h |
| control | 0,092 | 0,094 | 0,120 | 0,154 | 0,153 | 0,181 | 0,194 | 0,212 | 0,246 | 0,256 | 0,275 | 0,294 | 0,313 | 0,332 | 0,352 | 0,371 | 0,390 | 0,409 | 0,447 | 0,496 | 0,547 | 0,644 | 0,692 | 0,764 |
| 0.05 mM | 0,096 | 0,093 | 0,132 | 0,139 | 0,158 | 0,172 | 0,206 | 0,216 | 0,242 | 0,256 | 0,275 | 0,294 | 0,313 | 0,332 | 0,351 | 0.369 | 0.388 | 0.445 | 0.464 | 0.483 | 0.502 | 0.587 | 0.645 | 0.695 |
| 0.1 mM | 0,098 | 0,092 | 0,122 | 0,132 | 0,160 | 0,162 | 0,200 | 0,224 | 0,226 | 0,249 | 0,267 | 0,285 | 0,303 | 0,321 | 0,340 | 0,358 | 0,401 | 0,458 | 0,477 | 0,496 | 0,515 | 0,600 | 0,658 | 0,713 |
| 0.2 mM | 0,100 | 0,096 | 0,123 | 0,135 | 0,167 | 0,176 | 0,191 | 0,270 | 0,222 | 0,227 | 0,250 | 0,247 | 0,269 | 0,244 | 0,266 | 0,273 | 0,259 | 0,278 | 0,294 | 0,319 | 0,298 | 0,278 | 0,261 | 0,269 |
| 0.4 mM | 0,096 | 0,096 | 0,129 | 0,136 | 0,157 | 0,178 | 0,196 | 0,208 | 0,214 | 0,250 | 0,273 | 0,270 | 0,292 | 0,267 | 0,289 | 0,296 | 0,282 | 0,301 | 0,317 | 0,342 | 0,321 | 0,301 | 0,284 | 0,311 |

![](https://static.igem.wiki/teams/4998/wiki/results/growth-rates-wb800.png)

**Graph 1:**Graphical representation of *Bacillus subtilis'* growth rate in different concentrations of IPTG

#### Conclution:

The growth rates of *B. subtilis* WB800N present decrease in cell density in IPTG concentrations higher than 0,1 mM.

# Transformation of  *Bacillus subtilis*  WB800N

## Transformation of *Bacillus subtilis* WB800N

#### Aim:

To start the expression of our laccase gene and our kill switch system in *B. subtilis* in order to
start the characterization of our parts

#### Methods:

After changing the strain and testing the growth rates, we proceeded to transform this strain with the
composite parts of laccase and the kill switch mechanism. We followed the protocol of minipreps and
nanodrops from our successfully transformed *E. coli* cells with the desired recombinant plasmids for
*B. subtilis* 3601 transformation of the composite parts of laccase and *Bacillus* kill switch mechanism.
After the transformation we prepared plate cultures in LB agar broth medium with kanamycin.

#### Results:

The transformation was successful, as we managed to have cultures grow in the plates

#### Conclution:

We started the characterization of our parts.

# Laccase detection after changing strain - Bradford assay

## Laccase detection after changing strain - Bradford assay

#### Aim:

To determine the total protein in the extracellular matrix. We are expecting the modified *B. subtilis* WB800N
to produce more extracellular proteins than the control one.

#### Methods:

After the successful transformation of our *B. subtilis* cells we inoculate cultures from the plates
with the transformed  *B. subtilis* into a liquid growth medium. We incubate them in 37°C for 48h for
the laccase production with 3 different concentration of CuSO4 at 50 μΜ, 75 μΜ, 100μΜ, one transformed
*B.subtilis* with 0 mM sample CuSO4 and one control non transformed *B. subtilis*. After those 48h we
centrifuge our samples and took 2 ml of the supernatant. First we calibrated with standard concentrations
and then we calculated the concentration of our samples.

#### Results:

The resulting concentrations of our samples compared with the controlled ones had differences compared with the controlled ones

![Bradford calibration table](https://static.igem.wiki/teams/4998/wiki/the-original-bradford-calibration-table.png)

**Table 1:**Bradford calibration table

![Bradford calibration graph](https://static.igem.wiki/teams/4998/wiki/calibration.png)

**Graph 1:**Bradford calibration graph

![The results of Bradford](https://static.igem.wiki/teams/4998/wiki/bradford-calibration-table.png)

**Table 2:** The results of Bradford

![The results of Bradford Graph](https://static.igem.wiki/teams/4998/wiki/bradford-diagram.png)

**Graph 2:** The results of Bradford Graph

#### Conclution:

As it is proved, the raise of CuSO4 concentrations cause a protein production rise. Since CuSO4
is related to the copper functioning laccase enzyme, it can be implied that a rise of laccase production is detected
when this molecule is present in higher concentrations.

# Laccase detection after changing strain - SDS-PAGE

## Laccase detection after changing strain - SDS-PAGE

#### Aim:

To detect higher protein volume in the bands with laccase’s molecular weight in our samples compared with the control

#### Methods:

Following the protocol for the SDS page we used the same supernatant from the same samples as above
in Bradford (3 different concentration of CuSO4 at 50 μΜ, 75 μΜ, 100μΜ, one transformed *B.subtilis*
with 0 mM sample CuSO4 and one control non transformed *B. subtilis*). Then we scan the resulting gel.

#### Results:

The protein volume in the band of the molecular weight of our laccase compared to the control sample had significant differences

![](https://static.igem.wiki/teams/4998/wiki/sds.png)

**Table 1:**SDS-PAGE for the lac7 enzyme in the transformed *B. subtilis* WB800N strain, under different CuSO4 concentrations.
The bands at the ~75 kDa imply lac7 presence in the extracellular *B. subtilis* region

![The results of Bradford Graph](https://static.igem.wiki/teams/4998/wiki/protein-volume.png)

**Graph 1:** SDS-PAGE lac7 quantification diagram

#### Conclution:

A notable protein concentration raise, in the range of 75 kDa, was detected compared to the controls,
implying a boosted laccase production as CuSO4 concentration rises. Thus the production of our laccase
can be considered successful an has indications that correlates with CuSO4 concentration

# Kill switch mechanism

## Kill switch mechanism

#### Aim:

To detect an adj. volume in toxin’s molecular weight band according to the variations of IPTG concentration.

#### Methods:

After the transformation of *B. subtilis* WB800N we inoculated into liquid growth medium with different
concentrations of IPTG. More specifically we had liquid cultures 0 mM, 0,05 mM, 0,1 mM and one control
non modified *B. subtilis* WB800N culture. We incubated the cultures for 24h at 37°C. We calculated the
absorbancy in the band of the molecular weight of the toxin BsrG.

#### Results:

**Table 1:** *Bacillus subtilis* #3601 growth rates

| IPTG |  |
|  | Adj. Volume |
| Control | 679530.3333 |
| 0 mM | 1670656 |
| 0 mM | 1126005.333 |
| 0 mM | 1272054 |
| 0 mM | 1798577 |
| 0.05 mM | 1317322.667 |
| 0.05 mM | 1155572.667 |
| 0.1 mM | 1851681 |
| 0.1 mM | 553558.6667 |

![pp](https://static.igem.wiki/teams/4998/wiki/results/sds-hetr-toxin.png)

**Figure 1:**

#### Conclution:

There was no immediate correlation between the concentrations of IPTG and Our kill switch system.
The desired result would be to have lower protein volume as the IPTG concentration increases.
The results do not prove that. So, we decided to change the promoter of TetR with Pgrac, as
it was derived from a *B. subtilis* endogenous promoter and could have better results.

# Transformation of  *Nostoc Oryzae*  TAU-MAC 2710

## Transformation of *Nostoc Oryzae* TAU-MAC 2710

#### Aim:

To transform to *Nostoc Oryzae* TAU-MAC 2710 to start the expression of HetR (in order to see in
the next steps the increase in the number of heterocysts)

#### Methods:

*Nostoc oryzae* TAU-MAC 2710 was used for electrotransformation. The cells were selected in
the log growth phase because the DNA can easily integrate foreign genes. We conducted plasmid extraction
following the protocol of minipreps and nanodrops in order to extract the successful recombinant plasmids
containing HetR composite part from E.coli transformation. After the transformation via electroporation
and the inoculation of the cells into liquid growth medium with kanamycin, we conducted PCR and electrophoresis
to prove the transformation.

#### Results:

The transformation of the *Nostoc oryzae* TAU-MAC 2710 was unsuccessful, as we did not see the fragment
of our gene in the electrophoresis.

#### Conclution:

After multiple failures in our tries to transform the cyanobacterium we decided to just try
and see if the HetR protein is expressed in the *E. coli* cells

# HetR - SDS page

## HetR-SDS page

#### Aim:

We wanted to detect higher adj. volume in the band of molecular weight of our HetR

#### Methods:

After the failure to transform the cyanobacterium with our recombinant plasmid we decided to detect
if the protein of HetR was being expressed in the competent *E. coli* cells. We centrifuge our sample
and keep the cell pellet. We proceeded to cell lysis and then followed the protocol for the SDS page.
We had samples from two liquid cultures with successfully transformed *E. coli* in LB medium with kanamycin
and one control non transformed *E. coli*.

#### Results:

The results were showing higher protein volume in the band of the molecular weight of HetR in our samples than in our control.

**Table 1:** *Bacillus subtilis* #3601 growth rates

|  | Adj. volume |
| Control | 732220 |
| 1 | 896060 |
| 2 | 1112298 |

![pp](https://static.igem.wiki/teams/4998/wiki/results/sds-hetr-toxin.png)

**Graph 1:**SDS-page gel for the detection of HetR produced

#### Conclution:

There is a possibility, because of the notable difference between the absorbance in samples and control
that the promoter of HetR is functional as a promoter in *E. coli*, too. So, it is possible that the HetR
protein was being produced.

![forest](https://static.igem.wiki/teams/4998/wiki/wiki-photos/forestfooterneon-igemthess23.png)

{% endblock %}