iGEM 2021 — Team Brno_Czech_Republic

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# Team:Brno Czech Republic/Notebook

Notebook

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* [ACHIEVEMENTS](https://2021.igem.org/Team%3ABrno_Czech_Republic/Achievements)

# Notebook

In this notebook, we summarize in quite some
detail each week we spent in the lab working on our iGEM
project. The notebook documents the chronological progress
of experiments, but does not include their individual
procedures and all results - these are described in detail
in the  [Protocols](https://2021.igem.org/Team%3ABrno_Czech_Republic/Protocols), [Experiments](https://2021.igem.org/Team%3ABrno_Czech_Republic/Experiments)
and  [Results](https://2021.igem.org/Team%3ABrno_Czech_Republic/Results)
sections.

## Week 1 (June 28 – July 2)

The first week in our lab is finally here! In
order to even start working in it, it was necessary for all
of us to complete online safety training. Our new team
members were introduced to the lab and its equipment. We
set aside these days for general preparation of the lab for
the coming weeks. From mixing LB media and agars, diluting
antibiotics to the necessary working concentration,
preparing plates for our bacteria, to testing
transformation of *E. Coli* DH5-ALPHA, *E.
coli* JM109, and *Bacillus subtilis* 168 competent
cells and PCR. We became familiar with the workspace to
make us feel at home while making sure our competent cells
and primers were working as they should. After all, as they
say, fortune favours the prepared!![](https://lh4.googleusercontent.com/6kX7J4ZzWhLhRO9SZm5c53YGIuRLUrHMRV7zeXGsRgmxEr-f6dLgBe_1gGFinvvqFJPAB6qYDyMH9vQJRBQDmFJgBtO6AmAsXj8H66MG_GSi53MMp6TlMmB_pdC__Q=s0 "notebook-img-1")
Figure 1: Preparing the lab for work

## Week 2 (July 5 – July 9)

However, the preparations are not over yet.
Some of the chemicals we had from last year, some of them
were brand new, so it was necessary to test them all
properly so that in the future we could rely on the
correctness of the experiments and their results. One of
the things that needed to be tested was the functionality
of the selection systems. This time we used a different
type of plasmid to transform *E. coli* and *B.
subtilis* than last week, so for *Bacillus* we had
to use a different antibiotic plate. The next day we could
only conclude that although the transformation of *E.
coli* came out with a beautifully clean negative control
plate, for *Bacillus* the negative control was
overgrown... Where was the problem? There were many
possibilities but one thing was certain. In the next few
days we will have to repeat the experiment and find out the
cause. We decided to make colony PCR from the transformants
and try a new intercalation dye, Midori Green, which is
non-toxic and so we will be able to manipulate it as well
as take pictures of the gels directly in our lab.

## Week 3 (July 12 – July 16)

We decided to divide this year's iGEM team
into three groups, which will rotate in the lab every week.
Each group also has a "leader" who has the most insight
into what is going on in the lab and is responsible for
passing on information to the next week's leader. So in the
following lines, for the sake of clarity, we will talk
about Míša's, Barča's, and Quency's groups. This week,
Barča's group was on "duty" in the lab. They started
testing the antibiotics which did not work last week and
prepared a new batch. This time, after the *B.
subtilis* transformation, the dish with the new
antibiotics with negative control was exemplary, without a
single colony. So probably the original antibiotics were at
the wrong working concentration. Subsequently, the colony
PCR conditions for *Bacillus* were optimized, and
since we had already run out of our construct B in the
commercial pUCIDT plasmid, *E. coli* JM109 was
transformed with it. The plasmid was isolated from the
transformants, restriction digested and put on a gel to
confirm the insertion. Awesome! We have plasmid with our
construct B in *E. coli* ! All that remains is to make
glycerol stocks, to serve as supply for future experiments
☺ .

## Week 4 (July 19 – July 23)

The Quency’s team took over the scepter in the
lab and the week started again with the transformation of
*E. coli* JM109, as we received constructs C and D in
commercial plasmids. The transformation was successful and
the results on the plates the next day were more than
interesting. In addition to the traditionally white
colonies, some of the plates were overgrown with pink
colonies. These were due to the production of the mScarlet
protein, which we have included in both construct C and D.
What is interesting, however, is the fact that this protein
is under the promoter for *B. subtilis* and also that
the colonies were visibly pink only on the ampicillin
plates. Again, it was necessary to establish overnight
cultures from several colonies, isolate the plasmid from
them, restriction-digest them, put them on a gel, and
analyze the products. This restriction analysis confirmed
the insertion of plasmids with both C and D constructs into
*E. coli* JM109, and we were able to make additional
glycerol stocks.![](https://lh6.googleusercontent.com/0BSzrpPEUg4pbqAZfVRuEjNaYfaCgRR_7PscvUIDap3qm_l3BfaFmOWxhdPGA-dr7vDyIi5ZRtVtv-vDuJPb6Nn5JKAZKXLq_aYNu5vBYmvc2SITmCBz9TDigOOz5Q=s0 "notebook-img-2")
Figure 2: The mystery of pink colonies

## Week 5 (July 26 – July 30)

With the new week, the services in the lab
have rotated again, this time a group led by Míša took up
residence. We started the week by isolating plasmids from
last week's validated *E. coli* transformants. The
following days we continued subcloning. We
restriction-digested the C and D constructs in IDT's
commercial plasmids, visualized them using gel
electrophoresis, and purified them. We ligated the digested
and purified constructs C and D with the destination vector
overnight. The next morning, we enthusiastically set about
transforming cloning *E. coli* with the aforementioned
ligation mixture. We were thrilled with the result. We grew
potential transformants on the selection plates. Those that
should contain the C construct, but also those that should
have the D construct. Are we getting excited
prematurely?

## Week 6 (August 2 – August 6)

The enthusiasm and joy of the successful
transformation late last week was only half broken by
Bára's announcement that construct C had been transformed
back into *E. coli* in a commercial vector from IDT.
The good news, however, is that construct D is already
present in the transformants in our destination vector.
Therefore, we did not have to wait for anything and
immediately started transforming *B. subtilis*. Then,
we saw something has grown on the selection plates again,
which might have suggested that we should not get our hopes
up prematurely. Leaving nothing to chance, we are going in
hard to test our potential success. We have set up the AmyE
assay and mixed up the mix for the LongPCR reaction. Both
experiments were successful and we can finally celebrate.
After a month of hard work, we managed to integrate our
first construct into the *B. subtilis*
chromosome!![](https://lh3.googleusercontent.com/1DLTPaoFvQMomSGJ6g11lr1eChRSCVqUy8txo12gi0kBC4C6auWqZyuMfZHPCAOB2iuICSPjUoYU1SNXnWZMTXBDMe8NXI3j3wdzn6OM8yFXZi4bUOTsuyjTsiz9Ag=s0 "notebook-img-3")
Figure 3: We were very pleased with the results of the AmyE
test

## Week 7 ( August 9 – August 13)

Quency is in charge this week. After the most
successful week we have had so far in this year's iGEM
competition, on Monday we started again from the beginning.
The goal now was to continue subcloning the B and C
constructs that are in pUCIDT plasmids. Hence, we set about
doing a restriction digestion of these constructs and
inserting them into our destination vectors. Unfortunately,
restriction digestion of these constructs did not listen to
us this week. The gels from the electrophoresis were uneasy
to read and we had a hard time guessing whether the
constructs were correctly digested or not. Simply put,
restriction digestion was playing a provocative game with
us. It was only on Friday that we were able to transform
*E. coli* with a ligated destination vector and the C
construct. On Saturday, Quency, full of anticipation, went
on campus to evaluate the transformation. The colonies are
on the plates, but will the correct plasmid with construct
C be in the transformants?

## Week 8 (August 16 – August 20)

A new week started and with it another group
came to work again. Now under the leadership of Míša. On
Monday morning, construct B in the pUCIDT plasmid was
restriction digested and at the same time the presence of
construct C in plasmid 3661 in *E. coli* was tested by
cPCR. Unfortunately, both experiments came out negative.
The next day we repeated the cPCR, again unsuccessfully =
construct C probably failed to ligate into pDG3661 and
transform *E. coli*. Nothing left to do but
restriction-digest again, verify products on
electrophoretic gels, isolate them from gel, then ligate
and transform. On Wednesday, we isolated the plasmids
needed for restriction cloning from overnight cultures of
*E. coli*. This time we were let down by the
equipment, the thermostat set at 37°C only warmed up to
28°C overnight for some unknown reason. Therefore, the
concentrations of the isolated plasmids were miserably low.
In addition, on this day we transformed competent *E.
coli* JM109 with hopefully successfully ligated products
from yesterday. The following morning we evaluated the
transformation. One of the ligated products (pDG3661 +
construct C) yielded many transformants on the plates.
However, we rejoiced prematurely. After performing cPCR of
more than 30 randomly selected colonies and gel
electrophoresis, we are sad to say that our ligated product
is not present in any colony. This week, perhaps,
everything that was planned went wrong. We were exhausted
and unmotivated. We hoped that Barča's group will do much
better next week!![](https://lh4.googleusercontent.com/3W3ThKIIOHvztJ1xSqUux7DpUZj3-WFBT066RYKkGp7VsVM4vxAQmDbnrDjKjMwthUX_cau9aj_LyPGJlMMd_N0MmeLw1JvXTHxb-Z8PhTGGJqQCMYadO-O1t3DuFA=s0 "notebook-img-4")
Figure 4: Even if you work hard and accurately, experiments
can just fail.

## Week 9 (August 23 – August 27)

On Monday, Barča's group started lightly. The
functionality of the new polymerases was tested and
overnight cultures were established. On Tuesday, it was
possible to confirm with 100% certaintibility that the
putative ligated products from last week are actually still
our initial pUCIDT+C. Restriction cloning of this construct
from the pUCIDT vector is probably not the way to go, so
primers for PCR cloning will be designed. In addition,
restriction digestion of plasmid 1664 and plasmid pUCIDT
with the B construct was successful and will be ligated and
subsequently used to transform *E. coli*. The
following day, work was done on transforming cloning *E.
coli* with the ligation mixture from the previous day.
Work was also done to test the functionality of the AmyE
assay. On Thursday, the transformation was verified by
cPCR. However, this did not turn out the best, apparently
we inadvertently used the wrong primers. Therefore, on
Friday we decided to verify the transformants one more
time, but now by using restriction analysis. It turned out
that the transformation was successful and construct B was
located in the destination vector pDG1664. Finally, we can
transform *B. subtilis*!

## Week 10 (August 30 – September 3)

Quency's group had a big thing coming up on
Monday. After a long time, we again set about transforming
*B. subtilis*, but this time with plasmid pDG1664
containing construct B. In addition, the functionality of
the polymerases for LongPCR was verified. The latter is
important to amplify the transition between the *B.
subtilis* chromosome and our integrated constructs,
which could be sent for sequencing. In the morning, we
spotted *B. subtilis* colonies on agar plates. At that
moment, we were all a little breathless, hoping that the
transformation had been successful and we would be able to
excitedly announce to our PIs that we had managed to
integrate a second construct into the chromosome. However,
we were unable to verify it by PCR. We were trying to
optimize the conditions of the PCR reaction, trying to use
different primers, even annealing temperatures. It was only
on Friday that we were able to successfully verify the
presence of the B construct in the *B. subtilis*
chromosome for the first time.![](https://lh4.googleusercontent.com/dHfe42LJgClvFWaGN-GIP9O5sWZjeF5L6Cff4BhHNksXKrbV6p3FbZ4o35fh0DyiN0JZDFmQQ9zTd5PMvWXFZzX4a2dh5Gizu3mS4clWXbQfwOAquGCJ63xq4IAPcA=s0 "notebook-img-5")
Figure 5: Proof of successful integration of B construct
into *B. subtilis´*; chromosome

## Week 11 (September 6 – September 10)

Míša's group decided to leave nothing to
coincidence and try again to verify the transformation of
*B. subtilis* by construct B. After all, those
verification PCR reactions last week did not quite come out
the way they were supposed to. So we tried to repeat the
success of the Quency group using their optimization
tweaks. Unfortunately, our first verification did not turn
out positive. After visualization by electrophoresis, we
saw only narrow faint bands on the gel. The next day we
repeated the PCR reaction again, again unsuccessfully. We
are beginning to worry if the positive result from last
week was significant. It was not until the third day and
the third attempt that we were also able to successfully
verify that the transformation was successful. We sent the
purified PCR amplicons for sequencing on Thursday. On the
same day, we visited the Loschmidt lab to measure the
spectra absorbed by *B. subtilis* samples with
construct B and D after IPTG induction. On the last day of
this week, we finally started working on PCR cloning of
construct C. We were able to successfully amplify and
purify it. We also prepared glycerol stocks of *B.
subtilis* with construct B and stored them in a -80°C
freezer.

## Week 12 (September 13 – September 17)

This week has started off challenging. On
Monday the semester at the university started and so in the
next few weeks we will not be able to be in the lab with
the full line-up. The older members of the team already
have to work hard on their diploma theses and the younger
ones have lectures almost every day. Nevertheless, we have
decided that we still want to gather the last remnants of
strength and try to get the C construct into the shuttle
vector pDG3661 and then transform *B. subtilis* with
it. So on Monday, the amplicon of product C and pDG3661
were restriction-digested, the products on gel were
purified and ligated overnight. The next morning, the
ligation mixture was used to transform competent *E.
coli* JM109 cells. This was the last chance. Either it
works or it does not, and we will have to resign ourselves
to the fact that the C construct will forever remain only
in the pUCIDT plasmid. On Wednesday morning, we arrived at
the lab with anticipation, took the plates out of the
incubator... and there were the transformants! But we knew
we had not won yet. We have had several instances where
construct C has, for reasons we do not understand, ligated
again with the pUCIDT plasmid, which carries the same
antibiotic resistance as our shuttle vectors. So we rushed
to verify the colonies by colony PCR. This time, for each
colony, we verified not only the presence of construct C in
pDG3661, but also the presence of the pUCIDT plasmid. In
the first batch of colonies that we tested, all had a
positive product signal corresponding to construct C in the
pUCIDT plasmid. All but one - the colony with the assigned
number 1. The faint band corresponding to the product size
of construct C in pDG3661 gave us hope. And so the next day
we tested another batch, including colony #1, for
confirmation. And there it was! Even in the other two
colonies. And so, after two months of trying, our dream
became a reality. We have construct C in the shuttle vector
pDG3661! The week was capped off by transforming the *E.
coli* BL21(DE3) expression strain with constructs B and
D in pUCIDT plasmids.![](https://lh5.googleusercontent.com/DalNfoMfXtOY1KP71diRUrhEBnGRvXNPrmybU6i4PhBdqZKHneFsFyvnwj9SmiVECUHQM3NIQ8QWVGP0rXxwW7X2vV2pXRty1Wb3Z1RYfJAx_ChoN0gA6EiKL6ym9A=s0 "notebook-img-6")
Figure 6: And so, after two months of trying, our dream
became a reality. We have construct C in the shuttle vector
pDG3661!

## Week 13 (September 20 – September 24)

After isolating plasmid pDG3661 with construct
C from an ON culture of *E. coli*, we finally
attempted to transform our *B. subtilis*. However, due
to the low concentration of plasmid DNA and a minor
transformation error, we were unsuccessful the first time.
So we established another ON culture of *E. coli* and
the next day we tried to isolate the plasmids again. This
time we had beautiful concentration and purity. The
transformation process went smoothly, the plates were put
in the incubator and were looked at the next day. At the
same time, we were able to successfully verify the
transformed *E. coli* BL21(DE3) with constructs B and
D in the pUCIDT plasmids using colony PCR. And not to
forget, we also received a shipment from Dr. Krásný from
Prague with various strains of *B. subtilis* and *E.
coli* that we will use as controls in the following
experiments. So at the end of the week, we made glycerol
stocks of these strains and the AmyE test with the *B.
subtilis* transformants was set up. And sure enough, it
turned out great!

## Week 14 (September 27 – October 1)

The AmyE test came out above all our
expectations, suggesting that *B. subtilis* had a
broken AmyE site in its chromosome and our C construct
integrated into it. However, verification is not yet
complete. On Sunday evening, ON cultures were established
from *B. subtilis* transformants and on Monday we set
about isolating the chromosome. Subsequently, long PCR was
performed. One of the primers was complementary with the
interface of the chromosome and part of the plasmid
pDG3661, which was integrated. The other one was
complementary on some part of the construct. The PCR came
out positive and the products of the expected sizes shone
on the gel! We then additionally purified the PCR mixtures
and sent the samples for sequencing. The latter will have
the last word. Thursday was scheduled to be undoubtedly the
most logistically complex experiment of the season.
Everyone who could was involved in thinking up and
executing it. And every hand was more than needed. On this
day we were scheduled to measure at Tecan in Loschmidt's
labs, and in the afternoon we had arranged to go finally
see our beloved bacteria under the fluorescence microscope
at CEITEC. Starting next week there will be lectures in our
iGEM lab so we will have limited access there.![](https://lh6.googleusercontent.com/48fwHbAqmvDjo2uqSEAzQybFSwEIGm9a6HP2VXxVckXhMSz4Z0owVDicIDs_zv0b9n82fad0lIXa3WKhi0StOfu8wFYPls_jiJ3B3rJE6dHJPCL8Du6WlI2KaUI5xQ=s0 "notebook-img-7")
Figure 7: Bacterial culture of *E. coli* producing
mScarlet protein

## Week 15 (October 4 – October 8)

This week was reserved for writing the wiki.
The wiki freeze is inexorably approaching, so it is time to
write down everything we have been working on for the last
few months. There is currently blood and coffee flowing
through our veins :).

![](https://static.igem.org/mediawiki/2021/8/8d/T--Brno_Czech_Republic--A-Icon-kalendar.svg "notebook-ic-1")![](https://static.igem.org/mediawiki/2021/2/26/T--Brno_Czech_Republic--A-Icon-falkona.svg "notebook-ic-2")![](https://static.igem.org/mediawiki/2021/2/24/T--Brno_Czech_Republic--A-Icon-miska.svg "notebook-ic-3")![](https://static.igem.org/mediawiki/2021/d/d5/T--Brno_Czech_Republic--A-Icon-pipoeta.svg "notebook-ic-4")![](https://static.igem.org/mediawiki/2021/d/d4/T--Brno_Czech_Republic--A-Icon-ELFO.svg "notebook-ic-5")![](https://static.igem.org/mediawiki/2021/7/72/T--Brno_Czech_Republic--A-Icon-mozek.svg "notebook-ic-6")![](https://static.igem.org/mediawiki/2021/3/39/T--Brno_Czech_Republic--Test-Icon.svg "notebook-ic-7")

Igem Team Brno, Czech Republic 2021

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# Team:Brno Czech Republic/Engineering

Engineering Success

![](https://static.igem.org/mediawiki/2021/8/8f/T--Brno_Czech_Republic--uvodka-Engineering.jpg "engineering-uvodka")

# Engineering success

Notice: This page is only a summary of our engineering success and the path
to it. All the detailed information about the design, parts, experiments
and results can be found at wiki pages [Theory](https://2021.igem.org/Team%3ABrno_Czech_Republic/Theory), [Project Design](https://2021.igem.org/Team%3ABrno_Czech_Republic/Design), [Wet lab](https://2021.igem.org/Team%3ABrno_Czech_Republic/Experiments) and [Results](https://2021.igem.org/Team%3ABrno_Czech_Republic/Results).

# Research

For a large portion of our team, our iGEM journey started almost two years
ago as we have decided to participate in iGEM 2020 as just the second team
from Czech Republic ever. That year we decided to focus on cyanobacteria.
As most of us had spent a lot of time in Brno - a city with a famous dam -
water-bloom was a topic we were all familiar with and passionate about.
That year we opted to design a system for directly reducing the population
of cyanobacteria by lysing their cell wall and for the degradation of some
of their toxins. While researching the topic of blue-green algae
overabundance, one thing kept coming up - phosphate pollution of surface
waters. As we discussed the topic further with various experts and even
asked the general public (see more on page [Human practices](https://2021.igem.org/Team%3ABrno_Czech_Republic/Human_Practices)), it became clear
that phosphate accumulation was truly a perfect
direction for our project.

This year we want to target the cause of the problem instead of its
symptoms. So instead of focusing on cyanobacteria themselves, we have opted
to try and combat the increase in phosphate concentrations in water bodies.
Phosphates are a vital source of nutrition for cyanobacteria and the large
amounts of phosphate fertilizers which end up being washed from the fields
into surface waters is one of the main contributors to the increasing
incidence of water-bloom.

Another reason why phosphate accumulation is rather important is the ever
increasing rate at which phosphates are used up - not just in agriculture
but also in many different industries as well as consumer's homes.
Phosphates are a finite resource which we will not be able to replace with
an alternative any time soon. So the recycling of accumulated phosphates
has definitely been one of our priorities.

# Imagine

So now we had a goal in mind and it was finally time to think about the
specifics.

When attempting to accumulate phosphates in bacteria - or any other
organism for that matter - one must keep in mind that every living thing
needs phosphates to live and thus has some way to intake and store this
substance. That can be equally an advantage and a disadvantage. On one
hand, we can be almost certain that our bacteria will take in phosphates
from its surroundings even without our involvement. On the other hand,
these phosphates will not stay inertly floating inside of its cytoplasm
forever - they will be used to produce a variety of molecules and
structures which in turn could be re-metabolised and phosphates could be
released outside again.

## BMCs

So in order to increase the rate at which our bacteria of choice retains
phosphates, we have decided to use bacterial microcompartments or BMCs -
large proteinous structures which encapsulate a biochemical pathway to
provide ideal conditions for it, and to stop external influences.
Phosphates should be able to easily pass through the semipermeable wall of
the BMC where it would be incorporated into a larger structure which would
no longer be able to get outside.

To store phosphates in larger molecules, we have decided to encapsulate the
enzyme polyphosphate kinase or PPK into the BMCs. This enzyme links
phosphates into large chains called polyphosphates. Polyphosphates in BMCs
would be safe from being digested by other enzymes and would thus remain in
our system and would not reenter into circulation.

PPK can be easily localized to BMCs by fusing this protein to a couple of
N-terminal amino acids from one of the proteins which are naturally
encapsulated in these microcompartments.

## Response to phosphate concentrations

BMCs sounded like an ideal means of improving phosphate accumulation. But
there was another issue. BMC production requires the expression and
assembly of large amounts of heterologous proteins and can thus decrease
the vitality of modified bacteria. This issue could be partially solved by
ensuring that BMC expression would only be triggered under specific
conditions. Making BMC production tied into the changes in phosphate levels
was an obvious solution.

As mentioned above, all organisms need to take in phosphates. That means
that all organisms must have a way of responding to changes in phosphate
concentrations. In many other bacteria, this task is performed by the Pho
pathway, where the phosphorylation (or lack thereof) of the PhoR receptor
leads to changes in gene expression from the PPho promoter. This pathway is
activated by a lack of phosphates and downstream of this promoter are thus
located genes which mediate phosphate intake and ensure that the bacteria
gets enough of this vital nutrient. So by placing a repressor of a promoter
controlling BMC production under the PPho promoter native to the bacteria,
we can create a system which responds to higher phosphate concentrations by
producing BMCs with encapsulated polyphosphate kinase.

## Bacillus subtilis

One of the easier decisions was choosing an organism to work with. Last
year we opted for *Bacillus subtilis* and this year we decided to not break
that pattern. As opposed to E. coli, *B. subtilis can thrive in lower* *E. coli*
temperatures of water bodies while also producing a decent amount of
proteins. It has also been already demonstrated that heterologous BMC
production in these bacteria is possible and the Pho pathway and the
sequences of its genes were also documented thoroughly for this organism.

More details about the theory behind our project and our ideas can be found
on the page [THEORY](https://2021.igem.org/Team%3ABrno_Czech_Republic/Theory).

# Design

So now we had an idea of how our system should look and what genes we
needed to express in our modified *B. subtilis*. It was the time to design
and pick our parts and carefully put them together into composite parts
which would not only serve our purpose but also allow us to test their
functionality and visualize what is happening in our system. We have
designed four composite parts to test various components of our system.
Parts of three of these constructs can be assembled together later on to
form a functioning system which responds to the increase in phosphate
concentrations by BMC production. The last construct was designed for the
production of a protein which would be encapsulated in these BMCs.

More detail about each composite part can be found on the page [PROJECT DESIGN](https://2021.igem.org/Team%3ABrno_Czech_Republic/Design).

## BMC production and protein encapsulation

Composite part A was designed to test and characterise the production and
assembly of BMCs in *Bacillus subtilis*. We opted for the heterologous
production of BMC shell proteins from the Pdu operon of the bacteria
Parageobacillus thermoglucosidasius which is closely related to B.
subtilis. Heterologous production and assembly of Pdu BMC in *B. subtilis*
has been demonstrated in a study by Wade *et al*. (2019), where they have
identified five essential shell proteins. In composite part A, we place all
five essential Pdu BMC shell proteins downstream of an IPTG inducible
promoter in order to effectively induce BMC production without fearing that
it would drastically decrease the vitality of our bacteria. This promoter
would later on be replaced by a modified promotor from composite part D,
which should be able to bind cI repressor and thus stop the expression of
BMCs in the presence of this molecule.

![](https://static.igem.org/mediawiki/2021/0/0e/T--Brno_Czech_Republic--A.jpg "engineering-pic-4")
Figure 1:
Composite part A

In that study, they have used 24 N-terminal amino acid residues from PduP
protein as a tag to encapsulate protein inside of Pdu BMCs. PduP tag binds
to C-terminal helixes of shell proteins of BMC and mediates incorporation
of fused protein, in our case GFP, into the BMC. In composite part B, we
have fused this PduP tag with the green fluorescent protein GFP. The
localization of the fluorescent signal into the BMC, instead of it being
dispersed through the entire cell, should thus indicate correct assembly of
BMCs as well as the encapsulation of protein on the inside.

![](https://static.igem.org/mediawiki/2021/1/1c/T--Brno_Czech_Republic--B.jpg "engineering-pic-5")
Figure 2:
Composite part B

The fluorescent protein GFP could be replaced by PPK enzyme further down
the line to create a functional system for sequestering and storing
phosphates in BMCs in a form of polyphosphates.

## Response to phosphate concentration

Composite part C was designed to test the response of PPho promoter in
 *Bacillus subtilis* to the changes in phosphate levels in the medium. As
mentioned above PPho is a native promoter of  *B. subtilis* and it is
activated by the Pho signaling pathway which is in turn active in low
phosphate concentrations. This promoter was described in a study by Hulett
et. al. We used the predicted sequence from this study and placed a
reporter protein mScarlet-I downstream of this promoter to monitor its
function. The cI repressor has also been placed under the control of PPho promoter.

![](https://static.igem.org/mediawiki/2021/2/26/T--Brno_Czech_Republic--C1.jpg "engineering-pic-6")
Figure
3: Composite part C

In low phosphate concentrations in minimal medium, modified bacteria should
produce mScarlet-I and fluorescent signal should be present. In high
phosphate concentrations, the promoter should be inactive and no
fluorescent signal should be measured.

## Modified promotor and its repression

There are not many efficiently repressible promoters which would also work
well in  *B. subtilis*. To create our switch system, we opted to modify the
Pgrac promoter. Original Pgrac can be repressed by  *lacI* repressor which can
be removed from the operator of this promoter by IPTG. We took out the lacO
sequence and replaced it by the cI operator (OcI) sequence. This design
should allow the strong promoter to constitutively initiate transcription
until the cI repressor binds to OcI and the expression is stopped.

Repressor cI is natively produced by phage lambda where it controls the
decision between initiating lytic or lysogenic cycle. This repressor binds
to the O1, O2 and O3 operators, forms multimers and bends the DNA molecule,
preventing transcription initiation from the pR promoter. We added one of
the operator sequences to the Pgrac promoter to create Pgrac-OcI which
should thus be repressed by the cI repressor. As cI repressor is a peptide,
it can be easily produced by  *B. subtilis*, allowing our system to regulate
on its own.

To test the functionality of this switch system, we have placed sequences
coding mScarlet-I and cI repressor downstream of the IPTG inducible
Phyperspank promoter and GFP gene downstream of our modified Pgrac-OcI. All
proteins were fused with SNV degradation tags to ensure that our system
will be able to react quickly to induction and there would be no proteins
which would remain in the cytoplasm long after their promoter was turned
off.

![](https://static.igem.org/mediawiki/2021/9/98/T--Brno_Czech_Republic--D.jpg "engineering-pic-7")
Figure 4:
Composite part D

Without IPTG induction, cI repressor as well as the reporter fluorescent
protein mScarlet-I is not produced so there is nothing stopping Pgrac-OcI from initiating
transcription of GFP and we should thus be able to measure
typical GFP fluorescence. After IPTG induction, the Phyperspank can
initiate the expression of mScarlet-I and cI repressor. The repressor
should bind to Pgrac-OcI and repress the production of GFP. After a while,
there should be no or very little GFP fluorescence and a lot of signal
coming from mScarlet-I.

## Putting it all together

Composite part Z has been combined from previously mentioned parts A, C,
and D to create a functional system which responds to increase in phosphate
levels by initiation of BMC production and assembly.

This construct will be assembled by modifying composite part D using
restriction digestion. Promoter Phyperspank would be replaced by the PPho promoter from
composite part C which responds to the phosphate
concentration in the growth medium. Additionally, the GFP sequence would be
replaced by the sequences of the Pdu BMC shell proteins from composite part
A. This modification would be done using restriction sites NdeI and XhoI.

![](https://static.igem.org/mediawiki/2021/9/92/T--Brno_Czech_Republic--Z.jpg "engineering-pic-8")
Figure 5:
Composite part Z

At low phosphate concentrations, PPho promoter should activate the
transcription of the cI sequence and lead to the production of the cI
repressor. This repressor inhibits transcription initiation from the Pgrac
Oc promoter and thus prevents the formation of more BMCs. This system
ensures that BMCs are produced only when the bacteria encounter high
phosphate concentrations.

# Build

In our project we used plasmids pDG1664 and pDG3661. They are shuttle
vectors, which means that they are designed to be used in two different
organisms - in our case E. coli and *B. subtilis. As they are designed for* *E. coli*
ectopic integration into the chromosome of *B. subtilis*, they only carry
replication origin for *E. coli*. This ensures that we are able to store and
replicate our plasmids in *E. coli*, while ensuring that colonies of *B. subtilis* which exhibit
resistance to corresponding antibiotics should have
the target sequence integrated into its chromosome.

Vector pDG1664 was utilized for composite part B. It carries a gene
encoding resistance to ampicillin for selection in *E. coli* and a gene for
resistance to erythromycin for selection of transformants in *B. subtilis*
which is thus located in the segment which will be integrated into the
chromosome of *B. subtilis*, more specifically into the thrC sequence.

![](https://static.igem.org/mediawiki/2021/b/be/T--Brno_Czech_Republic--pDG1664.png "engineering-pic-10")
Figure 6: Schematic depiction of pDG1664 plasmid

Vector pDG3661 was utilized for composite parts A, C, D and Z. It carries a
gene encoding resistance to ampicillin for selection in *E. coli* and a gene
for resistance to chloramphenicol for selection of transformants in B.
subtilis which is thus located in the segment which will be integrated into
the chromosome of *B. subtilis*, more specifically into the amyE sequence.

![](https://static.igem.org/mediawiki/2021/a/a5/T--Brno_Czech_Republic--pDG3661.png "engineering-pic-11")
Figure 7: Schematic depiction of pDG3661

We ordered our constructs to be synthesized and delivered in plasmids from
company IDT. They were sent to us in pUCIDT plasmids which contain a
replication origin for *E. coli* and encode resistance to ampicillin.

When ordering the constructs for synthesis, we have encountered a big
problem - composite part A had a high level of homology due to the
similarities in sequences of Pdu shell proteins. We have opted to order the
rest and then split composite part A into two constructs and change the
design so it could be ligated into one construct with Golden Gate cloning.
That has however taken a log of time as we needed to research the method
first. In the end, the two halves of composite part A arrived too late and
we did not have the time to work with it and achieve results.

Therefore, everything described below was only done with composite parts B,
C and D.

## Cloning into shuttle vectors and transformation of E. coli

First we transformed non-expression *E. coli* JM109 strain with pUCIDT
plasmids containing our constructs to store and replicate these vectors.

Then we needed to clone our constructs into the vector mentioned above. We
performed restriction digestion of our constructs in pUCIDT plasmids as
well as the empty vectors using enzymes BamHI and HindIII. Correct size of
fragments was verified using electrophoresis and the fragments were then
cut out and purified from the gel.

Digested constructs were ligated with corresponding plasmids and were
subsequently used for the transformation of chemocompetent *E. coli* cells
which were then inoculated onto agar plates containing ampicillin. We have
transformed two E.coli strains - *E. coli* JM109 and expression strand
BL21(DE3), which has a gene for *lacI* inserted into its chromosome and can
thus be used for IPTG induction. Transformation of *E. coli* was confirmed
using restriction digestion and colony PCR.

Transformed JM109 *E. coli* cells were used for storage and replication of
our plasmids. We made glycerol stocks of *E. coli* transformants to be kept
at -80°C. We have inoculated some of the verified cultures into liquid LB
media and let them grow overnight. Afterwards we performed plasmid
isolation to obtain enough shuttle vectors pDG3661 and pDG1664 containing
our constructs for transformation of *B. subtilis* and integration into its
chromosome. Transformed BL21 (DE3) *E. coli* colonies were used to test the
functionality of our composite parts.

Further information and results can be found on the page [RESULTS](https://2021.igem.org/Team%3ABrno_Czech_Republic/Results) in the section [PREPARATION OF E. COLI CLONING HOST](https://2021.igem.org/Team%3ABrno_Czech_Republic/Results).

## Transformation of Bacillus subtilis

We have transformed *B. subtilis* 168 cells with pDG1664 and pDG3661 plasmids
containing our constructs which were isolated from *E. coli* JM109 colonies.
Vector pDG1664 was designed for integration into the sequence of thrC and
vector pDG3661 was designed for integration into the sequence amyE. A
combination of antibiotics erythromycin and lincomycin were used for
selection of successfully transformed colonies transformed with pDG1664\_B
vectors. The antibiotic chloramphenicol was used for selection of
transformants containing pDG3661\_D.

Successful transformation and the insertion into the chromosome of B.
subtilis was verified by AmyE test and also by long PCR with one primer
complementary to a sequence on the chromosome of *B. subtilis* in the
proximity of the insertion site while the other primer annealed to our
composite part. The amplicons containing a sequence spanning from the
chromosome of *B. subtilis* to the beginning of our composite part were then
sent for sequencing for further proof.

Further information and results can be found on the page [RESULTS](https://2021.igem.org/Team%3ABrno_Czech_Republic/Results) in the section [INTEGRATION OF SYNTHETIC CONSTRUCTS INTO THE
CHROMOSOME OF B. SUBTILIS](https://2021.igem.org/Team%3ABrno_Czech_Republic/Results).

### Composite part C

There were some issues with cloning Composite part C into pDG3661 After
many tries where the construct remained inside of the pUCIDT plasmid, we
have decided to first amplify the sequence of construct C using long PCR
and then perform restriction digestion and ligation using only the
amplicon, hoping that it would not re-ligate to pUCIDT this time. This
approach ended up working but transformation of construct C into *E. coli*
and then *B. subtilis* ended up taking significantly more time and we did not
have time to functionally characterize this construct.

If we had time to characterize composite part C we would have used the
following approach. *B. subtilis* cells with construct C in the chromosome
would be first grown in LB medium. The cultures would then be washed and
inoculated into minimal MOPS media adjusted for *B. subtilis* and modified in
a way where all phosphate components are replaced by Tris-HCl. Phosphates
can be then added to the medium in varying amounts to create media with
different phosphate concentrations. These cultures would then be pipetted
onto plates and both OD and mScarlet-I fluorescence would be continuously
measured using Tecan fluorometer. In low phosphate concentrations, the PPho should be turned on,
mScarlet should be produced and the fluorescence
intensity should increase.

![](https://static.igem.org/mediawiki/2021/b/b0/T--Brno_Czech_Republic--Experiment-schema.svg "engineering-pic-12")
Figure 8:
Scheme of our experimental approach

# Test

So now that we had strains of both *B. subtilis and BL21(DE3)* *E. coli*
containing each of our constructs, it was time to test out the
functionality of our constructs. One issue we encountered was that the
strain of *B. subtilis which we were using did not contain the gene for* *lacI*
repressor and thus couldn't be used for IPTG induction without inserting
this gene into its chromosome. At the beginning of our project, we assumed
that lacI was already inserted into the chromosome of our *B. subtilis* *lacI*
strain and we only found out otherwise later on. All of our IPTG inducible
promoters thus worked as constitutive in our *B. subtilis* cells. It was too
late to perform another transformation, so we decided to use the
aforementioned expression strand of *E. coli* BL21(DE3) to test the response
of our composite parts to IPTG induction.

## Characterization of constructs B and D

After successful transformation of E. coli and *B. subtilis, we designed the* *E. coli*
following experiments to demonstrate the functionality of the different
components of our project design.

First we determined the growth curve for each culture from regular
measurements of OD600. We tested how the system responds to the presence of
IPTG in the expression strain of E.coli BL21(DE3) cells. In *B. subtilis*
cells we presumed that IPTG inducible promoters would behave as
constitutive.

We used several different methods to detect the presence of the reporter
proteins GFP and mScarlet-I. We used a Tecan plate fluorometer for
simultaneous and continuous measurement of both OD600 and fluorescence of
the cultures. Additionally, we used FluoroMax Plus from Horiba Scientific
for more sensitive measurement of fluorescence spectrum in cuvettes at one
point in time. We then verified the results by fluorescence microscopy.

We should keep in mind that in *B. subtilis*, our constructs were integrated
into its chromosome and thus only one copy was present in each cell. E.coli
BL21(DE3) cells on the other hand contained our constructs inserted into
multicopy pUCIDT plasmids. The expression of the target proteins was
therefore much stronger in E.coli.

### Table 1: E. coli cultures used in following experiments

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Experiment | Constructs | Fluorescent proteins | Induction (IPTG) | Label |
| *E. coli* BL21 (DE3) | None - controls | - | - | BL21(DE3) without IPTG |
| At OD 0.6 | BL21(DE3) IPTG 0.6 |
| At inoculation | BL21(DE3) IPTG overnight |
| Composite part B | GFP | - | BL21(DE3)\_PUCIDT\_B without IPTG |
| At OD 0.6 | BL21(DE3)\_PUCIDT\_B IPTG 0.6 |
| At inoculation | BL21(DE3)\_PUCIDT\_B IPTG overnight |
| Composite part D | GFP mScarlet-I | - | BL21(DE3)\_PUCIDT\_D without IPTG |
| At OD 0.6 | BL21(DE3)\_PUCIDT\_D IPTG 0.6 |
| At inoculation | BL21(DE3)\_PUCIDT\_D IPTG overnight |
| *E. coli* BL21 (DE3) | None - controls | - | - | BL21(DE3) without IPTG |
| At OD 0.8 | BL21(DE3) IPTG |
| Composite part B | GFP | - | BL21(DE3)\_PUCIDT\_B without IPTG 0.8 |
| At OD 0.8 | BL21(DE3)\_PUCIDT\_B IPTG 0.6 |
| Composite part D | GFP | - | BL21(DE3)\_PUCIDT\_D without IPTG 0.8 GFP |
| At OD 0.8 | BL21(DE3)\_PUCIDT\_D IPTG 0.8 GFP |
| mScarlet-I | - | BL21(DE3)\_PUCIDT\_D without IPTG 0.8 mScarlet\_I |
| At OD 0.8 | BL21(DE3)\_PUCIDT\_D IPTG 0.8 mScarlet\_I |

### Determination of growth curves by continuous measurements of OD by Tecan fluorometer

Firstly, we have visualized the growth of each of our cultures to determine
if the expression of our target proteins of each of our constructs poses a
metabolic burden on transformed cells.

![](https://static.igem.org/mediawiki/2021/7/72/T--Brno_Czech_Republic--Figure27.jpg)

Figure 9: Growth
curves of *E. coli* BL21 (DE3) strains

We can extrapolate from the growth curves that the *E. coli* BL21 (DE3)
cultures grow more slowly after IPTG induction. This is particularly
apparent in the cultures which were induced during inoculation. This
demonstrates a slight toxicity of IPTG for our *E. coli* cultures as this
effect was observed even in our controls to which IPTG was added.

The growth of cultures transformed with construct B was not significantly
different from the growth of our controls, so it does not seem to carry
increased metabolic burden. The growth of cultures transformed with
construct D was slowed down as these cultures remained in the lag phase far
longer than the controls.

### Measurement of emission spectra of mScarlet-I and GFP in E. coli BL21(DE3) using FluoroMax Plus

We have also used fluorospectrometer FluoroMax Plus from Horiba Scientific,
which is more sensitive and allows the measurement of signals with
different wavelengths through the spectrum of light. The measurements can
however not be done continuously and were thus performed at one point in
time - 4 hours after IPTG induction.

![](https://static.igem.org/mediawiki/2021/0/09/T--Brno_Czech_Republic--Results-figure30.png)

Figure 10:
Emission spectra of GFP from construct B in *E. coli* BL21 (DE3)

![](https://static.igem.org/mediawiki/2021/2/20/T--Brno_Czech_Republic--Results-figure33.png)

Figure 11:
Emission spectra of GFP from construct D in *E. coli* BL21 (DE3)

![](https://static.igem.org/mediawiki/2021/a/a4/T--Brno_Czech_Republic--Results-figure34.png)

Figure 12:
Emission spectra of mScarlet-I from construct D in *E. coli* BL21
(DE3)

Typical fluorescent signal profile of corresponding fluorescent protein as
well as an increase in fluorescence intensity compared to the controls was
recorded both in cultures carrying construct B (GFP signal) and construct D
(sf GFP and mScarlet-I signals). This indicates that each fluorescent
protein in corresponding constructs is being expressed and is functional.

There was however no significant difference in fluorescence intensity of
cultures which were induced with IPTG and cultures where IPTG was not
added. This is in accordance with the measurement of mScarlet-I relative
fluorescence in the Tecan fluorometer. This further demonstrates that the
IPTG inducible promoters are functioning constitutively.

Plasmids from the pETM line, which are most commonly used for the
transformation of *E. coli BL21 (DE3) typically contain an additional* *lacI*
gene to ensure that all inducible promoters are sufficiently repressed. We
however didn't have these plasmids available on short notice and thus we
used pUCIDT plasmids which do not carry the gene for *lacI* repressor. It is
therefore possible that the cells were not able to produce sufficient
amount of *lacI* repressor from one gene integrated into their chromosome to
effectively stop the expression from our IPTG inducible promoter which were
located on multicopy plasmids.

Moreover, if the expression from our IPTG inducible promoters is truly
constitutive it would mean that in construct D, protein mScarlet-I as well
as the repressor cI should be expressed continuously. Repressor cI should
then repress the modified promoter PGrac-OcI which controls the expression
of GFP. The fluorescence intensity of GFP from construct D is significantly
lower than of GFP from construct B. This might suggest that our modified
promoter can truly be somewhat repressed by the repressor cI. There are
however other explanations why the expression from construct D would be
lower - construct D is bigger and poses a larger metabolic burden on the
cells so the cultures might not have had the same OD at the time of
measurement. So this is only a small indication which requires further
testing.

### Observation of E. coli BL21(DE3) cultures under fluorescence microscope

Findings from previous measurements were confirmed using fluorescence
microscopy. More information about our experimental setup can be found in
the section WET LAB and RESULTS.

![](https://static.igem.org/mediawiki/2021/1/19/T--Brno_Czech_Republic--Results-figure31.png "engineering-pic-18")

![](https://static.igem.org/mediawiki/2021/4/48/T--Brno_Czech_Republic--Results-figure32.png "engineering-pic-19")

![](https://static.igem.org/mediawiki/2021/a/a1/T--Brno_Czech_Republic--Results-figure35.png "engineering-pic-20")

### Table 2: B. subtilis cultures used in our experiments

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Experiment | Construct | Fluorescent protein | Induction | Label |
| Tecan fluorometer | None - control | - | - | *B. subtilis* 168 |
| GFP gene in chromosome  + control | GFP | Xylose induction | *B. subtilis* positive control GFP in chromosome |
| Composite part B | GFP | - | *B. subtilis* \_B |
| Composite part D | GFP mScarlet-I | - | *B. subtilis* \_D |
| FluoroMax Plus | None - control | - | - | *B. subtilis* 168 |
| GFP gene in chromosome  + control | GFP | Xylose induction | *B. subtilis* positive control GFP in chromosome |
| Composite part B | GFP | - | *B. subtilis* \_B |
| Composite part D | GFP | - | *B. subtilis* \_D GFP |
| mScarlet-I | - | *B. subtilis* \_D mScarlet\_I |

### Determination of growth curves by continuous measurements of OD by Tecan fluorometer

Firstly, we have visualized the growth of each of our cultures to determine
if the expression of our target proteins of each of our constructs poses a
metabolic burden on transformed cells.

![](https://static.igem.org/mediawiki/2021/6/69/T--Brno_Czech_Republic--Figure285.jpg)

Figure 16: Growth
curves of *B. subtilis* 168 strains

The growth rate of cultures transformed with constructs B and D decreases
down a little during the later phase of their growth compared to the
controls. This indicates that both constructs pose a metabolic burden on *B. subtilis* 168 cultures.

Expression of fluorescent proteins from *B. subtilis* was a lot smaller than
from *E. coli* and we were therefore unable to measure any fluorescent signal
using the less sensitive Tecan fluorometer. Fluorescence in *B. subtilis* was
later recorded using FluoroMax Plus.

### Measurement of emission spectra of mScarlet-I and GFP in B. subtilis 168 using FluoroMax Plus

We have also used fluorospectrometer FluoroMax Plus from Horiba Scientific,
which is more sensitive and allows the measurement of signals with
different wavelengths through the spectrum of light. The measurements can
however not be done continuously and were thus performed at one point in
time - 4 hours after the xylose induction of positive control.

![](https://static.igem.org/mediawiki/2021/0/0d/T--Brno_Czech_Republic--Figure40.jpg)

Figure 17:
Emission spectra of GFP from construct B in *B. subtilis*

In *B. subtilis*, typical fluorescent signal profile of corresponding
fluorescent protein as well as an increase in fluorescence intensity
compared to the negative controls was only recorded in cultures with
construct B in their chromosome (GFP signal). This indicates that
fluorescent protein GFP in construct B is being expressed and is
functional.

There was however no significant increase in fluorescence intensity
compared to the negative controls in both GFP and mScarlet-I in cultures
with construct D in their chromosome. This is however contradicted by the
observations made using fluorescence microscopy, so we cannot reach a
concrete conclusion in regards to the expression of GFP and mScarlet-I from
construct D in
B. subtilis
.

### Observation of B. subtilis cultures under fluorescence microscope

B.subtilis strains with constructs B and D in their chromosome as well as
the control strain with no integration, were also observed using
fluorescence microscopy. More information about our experimental setup can
be found in the section [WET LAB](https://2021.igem.org/Team%3ABrno_Czech_Republic/Experiments)
and [RESULTS](https://2021.igem.org/Team%3ABrno_Czech_Republic/Results).![](https://static.igem.org/mediawiki/2021/d/da/T--Brno_Czech_Republic--Results-figure43.png "engineering-pic-23")![](https://static.igem.org/mediawiki/2021/a/a5/T--Brno_Czech_Republic--Results-figure44.png "engineering-pic-24")![](https://static.igem.org/mediawiki/2021/4/42/T--Brno_Czech_Republic--Results-figure45.png "engineering-pic-25")

When compared to the control - *B. subtilis* 168 with no insert in its
chromosome - there is an indication of fluorescence in cultures transformed
with both construct B and D. In culture with construct D in its chromosome
there is an indication of both GFP and mScarlet-I fluorescence. This
contradicts the measurements of emission spectra made with FluoroMax Plus
and these results are thus inconclusive and need more experimal backing.

# Summary

We have obtained the growth curves of both E. coli and *B. subtilis cultures* *E. coli*
transformed with constructs B and D using Tecan fluorometer. While there
were some apparent effects of higher metabolic burden on the growth of E.
coli carrying construct B and *B. subtilis* with either construct in its
chromosome, the presence of our constructs does not seem to be too
detrimental to the culture.

We have managed to confirm the expression and functionality of all
fluorescent proteins from constructs B and D in *E. coli* with measurements
from Tecan fluorometer (only mScarlet-I), FluoroMax Plus and fluorescence
microscopy.

In *E. coli* BL21(DE3), there was no difference in the expression of all
fluorescent proteins from constructs B and D after IPTG induction. Both of
our IPTG inducible promoters seem to be initiating transcription
constitutively. This might be the result of lower expression of *lacI*
repressor which is inserted in bacteria's chromosome compared to the number
of IPTG inducible promoters which are present in the cell in multicopy
plasmids.

Fluorescence intensity of GFP produced by *E. coli* carrying construct D was
significantly lower than of GFP produced by *E. coli* carrying construct B.
In construct D, GFP expression is controlled by PGrac-OcI promoter which
was modified to respond to repressor cI. This repressor is also produced by
construct D where it is placed downstream from an IPTG inducible promoter
PHyperspank and its expression should thus roughly correspond to the
expression of mScarlet-I which is also controlled by this promoter. So if
IPTG inducible promoters are not properly repressed by *lacI*, the lower
fluorescence intensity of GFP from construct D could be caused by the
constitutive expression of repressor cI. This would indicate that our
modified promoter can indeed be somewhat repressed by cI. This is however
not conclusive and still requires further testing.

The expression and functionality of all GFP from constructs B in B.
subtilis was also confirmed with measurements from FluoroMax Plus and
fluorescence microscopy.

Fluorescence of GFP and mScarlet-I in *B. subtilis* cultures transformed with
construct D was however only detected by fluorescence microscopy. It is
thus still unclear whether the expression and correct folding of mScarlet-I
and GFP is taking place in *B. subtilis* cultures transformed by construct D
and further testing is necessary to confirm our findings.

# Improve

Our project is far from being completed as there are many aspects which
could be improved in the future.

One large part is the expression and assembly of BMCs which was not tested
as we were not able to perform Golden Gate cloning of composite part A. One
takeaway from this situation is to consider the level of homology between
the sequences of parts which the constructs are composed of. If they are
quite similar, it might be wise to split the composite part into multiple
constructs designed to be joined by Golden Gate cloning or other such
method straight away, so we would wait for the verdict of the company from
which we order synthetic constructs.

As is, it is likely that IPTG induction would not work for any of our
constructs. The functionality of IPTG induction in *B. subtilis* should be
enabled by integrating the gene for *lacI* repressor controlled by a strong
constitutive promoter into the chromosome of *B. subtilis* strains which
already have our composite parts inserted into the chromosome.
Transformation of *B. subtilis with* *lacI* gene could also be done using
plasmids designed for stable expression in this bacteria. However, we do
not have any experience working with them and chromosomal integration was
recommended to us by experts working with *B. subtilis*. As both options have
their pros and cons, it will most likely take more research to decide which
would work the best for us.

In *E. coli* BL21(DE3) we can clone the constructs into a plasmid which is
designed for IPTG induced expression in *E. coli* BL21(DE3) - such as the pET
plasmids. These plasmids should contain an extra *lacI* gene to ensure the
repression of our genes in the absence of IPTG and this could improve the
functionality of IPTG induction in *E. coli* BL21(DE3).

When subcloning construct C from pUCIDT plasmid into pDG3661, we struggled
to get rid of the contamination by pUCITD plasmid. This problem was largely
caused by the fact that we have used the same selection marker for both
plasmids, so colonies containing pUCIDT instead of pDG3661 could grow on
our plate. This is definitely a mistake we will not repeat in the future.
The functionality of construct C also remains to be tested. This construct
does not require IPTG induction to function fully so we should be able to
test it out without further transformation of *B. subtilis* strain with this
construct in the chromosome.

Another aspect of our project to consider is that the level of expression
of our reporter proteins from the chromosome of *B. subtilis* might not be as
high as from multicopy plasmids. This might be a problem, especially for
the expression and correct assembly of BMCs, as the heterologous expression
of BMC was most commonly performed and studied in *E. coli* with plasmids
carrying the genes of the shell proteins. In our experiments, the
expression of GFP from *B. subtilis* strain transformed with construct B was
however rather good so that might not be an issue. Additionally,
overexpression can also harm the cell and slow down the growth of the
culture, so having only one copy of each gene might actually be
advantageous when producing culture which should remain alive and
replicating inside of our device for some time.

We are well on the way to verifying the functionality of our project
design. The time is however playing against us and we were thus unable to
perform the optimization and improvements needed. We are looking forward to
maybe finishing our project in the future.

![](https://static.igem.org/mediawiki/2021/3/39/T--Brno_Czech_Republic--Test-Icon.svg "engineering-pic-1")![](https://static.igem.org/mediawiki/2021/9/9f/T--Brno_Czech_Republic--A-Icon-bubliny.svg "engineering-pic-2")![](https://static.igem.org/mediawiki/2021/9/9c/T--Brno_Czech_Republic--A-Icon-geneconstruct.svg "engineering-pic-3")![](https://static.igem.org/mediawiki/2021/e/e1/T--Brno_Czech_Republic--A-Icon-klic.svg "engineering-pic-9")![](https://static.igem.org/mediawiki/2021/7/77/T--Brno_Czech_Republic--A-Icon-graf.svg "engineering-pic-13")![](https://static.igem.org/mediawiki/2021/f/f5/T--Brno_Czech_Republic--A-Icon-hvezdicka.svg "engineering-pic-26")

Igem Team Brno, Czech Republic 2021

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# Team:Brno Czech Republic/Experiments

Experiments

![](https://static.igem.org/mediawiki/2021/a/a9/T--Brno_Czech_Republic--uvodka-WetLab.jpg "wet-lab-uvodka")

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# WET LAB

This page will give you detailed information
about the experiments we have performed. You can read about
the reasons that led us to design and then execute the
experiment, its theoretical background, used materials and
methods, and we also give a brief description of our
workflow. The protocols we followed are a part of this
chapter. All of them can be found in the [Protocols](https://2021.igem.org/Team%3ABrno_Czech_Republic/Protocols)
section. For each experiment we add a hyperlink to the
protocol used.

At Masaryk University, we were allowed to work
in a laboratory that is usually used for teaching practical
courses during the semester. There, we had at our disposal
any equipment we need - flow boxes,
cyclers, centrifuges, pipettes and many others. We also had
the support of PhD students and technicians from the
Experimental biology department, to whom we are very
grateful for their advice.

The results of each experiment can be found in
the [Results](https://2021.igem.org/Team%3ABrno_Czech_Republic/Results)
section.

![](https://static.igem.org/mediawiki/2021/2/28/T--Brno_Czech_Republic--Experiments-01.jpeg "wet-lab-01")

![](https://static.igem.org/mediawiki/2021/5/56/T--Brno_Czech_Republic--Experiments-02.jpeg "wet-lab-02")

# Basic experiments

## Preparation of growth media

The protocols for media preparation can be
found here: [PROTOCOL:
Preparation of growth media](https://2021.igem.org/Team%3ABrno_Czech_Republic/Protocols).

## Preparation of vectors with our synthetic constructs

### 1. Preparation of commercial vector with synthetic constructs

### Motivation

For the transformation of *B. subtilis*,
we had 4 synthetic constructs prepared by company IDT
within the offer mediated by iGEM contest.

These synthetic modules were named A, B, C and
D and are further described in the [Project
design](https://2021.igem.org/Team%3ABrno_Czech_Republic/Design) section. Unlike last year, when we worked with
linearized construct segments, this year we received
synthetic constructs incorporated inside pUCIDT
plasmids.

### Short description

Synthetic genes were shortly centrifuged in
short spin mode and then resuspended in appropriate volume
of nuclease free water in a flowbox. Resuspended genes are
less stable, so they had to be stored on ice or put in a
freezer at -20 °C. We worked with them only inside a
flowbox to minimize the risk of DNA contamination. For gene
stocks, it is better to use pipette tips with filters as
pipettes might contain aerosols from previous pipetting.
Any contamination could devalue the whole stock.

### 2. Preparation of primers

### Motivation

Within the PHOSCAGE project we worked with
many primers. We mainly used the primers we received last
year. Their detailed description can be found [here](https://static.igem.org/mediawiki/2021/3/38/T--Brno_Czech_Republic--Table_of_primers.pdf).

### Short description

Our primers were also synthesised by the IDT
company. They had to be resuspended in nuclease free H2O
according to the instructions provided. When working with
the primers, we always used a 10 nM solution that was
prepared from 100 nM stock solution. The stock solution was
made by resuspending the delivered powder. We assigned a
number to each primer so that we could easily distinguish
them.

## Preparation of glycerol stocks and destination vectors for cloning

Protocols are available here ([PROTOCOL:
Preparation of glycerol stocks](https://static.igem.org/mediawiki/2021/4/41/T--Brno_Czech_Republic--PROTOCOL-_preparationglycerolstock.pdf) ) and here ([PROTOCOL:
Isolation of plasmids](https://static.igem.org/mediawiki/2021/d/d5/T--Brno_Czech_Republic--PROTOCOL-_Isolation_of_plasmids.pdf) ).

Last year, we received our strain of *B.
subtilis* and the vectors for our project from Dr. Libor
Krásný, the head of the Laboratory of Microbial Genetics
and Gene Expression at the Academy of Sciences of Czech
Republic. For this year's project, we used the same strains
of *B. subtilis* from the glycerol stocks we had
prepared a year ago. We also isolated the same destination
vectors from last year’s stocks of *E.coli.* The
preparation of these glycerol stocks is mentioned
in [our
wiki from last year.](https://2020.igem.org/Team%3ABrno_Czech_Republic/Experiments#bas-exp)

## Plasmid isolation

During our project we often needed to isolate
plasmids with the desired vectors from our bacteria. For
this purpose, the [PureYield™
Plasmid Miniprep System](https://worldwide.promega.com/-/media/files/resources/protcards/pureyield-plasmid-miniprep-system-quick-protocol.pdf?rev=592591947f9c47efb5a576173f4f77f6&sc_lang=en) by Promega Corporation was
used, where the plasmid DNA was obtained through column
purification. For some useful notes or advice visit
the [protocol
Isolation of plasmids](https://static.igem.org/mediawiki/2021/d/d5/T--Brno_Czech_Republic--PROTOCOL-_Isolation_of_plasmids.pdf).

## Restriction digestion

Restriction digestion was needed several times
in our wet lab experiments. It was used when our constructs
had to be separated from a particular plasmid so that it
could be separated and inserted into another vector, and
when the length of the restriction fragments needed to be
adjusted so that they would be further apart on the gel
after electrophoresis. This method was further intended,
also, for moving individual constructs between each other
or for identifying DNA using typical restriction sites and
restriction fragment sizes.

For digestion of plasmids isolated by the
method described above, only enzymes with permitted
restriction sites were used, namely BamHI, HindIII and
BsaI. The table describes use of enzymes and their
combination with buffers. Restriction digestion was carried
out according to the procedures indicated by the
manufacturer.

For some useful notes or advice visit
the [PROTOCOL:
Proof of transformation of *Escherichia
coli*](https://static.igem.org/mediawiki/2021/4/41/T--Brno_Czech_Republic--PROTOCOL-_proofEColitrans.pdf).

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **Commercial vector with construct** | **RE** | **Buffer** | **Digestion time (min)** | **Inactivation time (min) (80 °C)** |
| pUCIDT\_B | HindIII, BamHI  (Thermo Fisher Scientific) | Fast digest buffer | 25 | 15 |
| pUCIDT\_C | HindIII, BamHI  (Thermo Fisher Scientific) | Fast digest buffer | 25 | 15 |
| pUCIDT\_D | HindIII, BamHI, BsaI (NEB) | CutSmart | 15 | 20 |

Table 1 : Restriction digestion
according to the digested vector.

## Ligation

T4 ligase was used to link sequences with
complementary sticky ends cleaved by restriction enzymes.
The ligation is mentioned in [PROTOCOL:
Preparation of genetic modules in *Escherichia coli*
cloning/expression host](https://static.igem.org/mediawiki/2021/1/15/T--Brno_Czech_Republic--PROTOCOL-_Subcloning_and_transformation_of_Escherichia_col_host.pdf).

We had the best experience with overnight
ligation.

## PCR

Here we describe two modifications of the PCR
we used.

Colony PCR was applied only to *E. coli*
(in the case of *B. subtilis the* *E. coli*
chromosome must first be isolated). GoTaq Master Mix by
Promega was used for colony PCR. The temperature and
duration of each phase was related to the length of the
sequences and primers.

LongPCR was used to verify integration into
the chromosome in *B. subtilis*. Chromosome isolation
had to occur first (described above).

When performing Long-Range PCR, we followed a
protocol that came with the GoTaq® Long PCR Master Mix by
the company PROMEGA [here](https://worldwide.promega.com/-/media/files/resources/protocols/technical-manuals/101/gotaq-long-pcr-master-mix-protocol.pdf?la=en)).
However after a while we ran out of the Master Mix, so
after testing it first we continued using the GoTaq G2 DNA
polymerase from PROMEGA. For additional information, visit
[PROTOCOL: Long-Range PCR](https://static.igem.org/mediawiki/2021/2/2d/T--Brno_Czech_Republic--PROTOCOL-_ILong_range_pcr.pdf). All designed primers we used
in PCR reactions are described [here](https://static.igem.org/mediawiki/2021/3/38/T--Brno_Czech_Republic--Table_of_primers.pdf).

## Purification from gel

Electrophoresis is an effective separation
method. The separated DNA needs to be purified from the
agarose gel. The protocol used for this was
the [Wizard®
SV Gel and PCR Clean-Up System](https://worldwide.promega.com/-/media/files/resources/protcards/wizard-sv-gel-and-pcr-clean-up-system-quick-protocol.pdf?rev=29aa4610719c4f96afd213c345bc76d3&sc_lang=en). To obtain higher
concentration of your DNA, you can elute it using a smaller
amount of nuclease free water.

![](https://static.igem.org/mediawiki/2021/5/57/T--Brno_Czech_Republic--led.jpg "wet-lab-03")

# Preparation of Escherichia coli cloning host

## 1. E coli JM109

## Testing of competent cells E. coli JM109

### Motivation

This experiment was performed in order to test
effectiveness of the purchased competent cells. This was
accomplished by transforming the empty vectors pDG3661 and
pDG1664 into these competent cells.

### Short description

Competent cells - *E. coli* JM109
purchased from Promega company - were used for testing.
These competent cells were not prepared by us. They were
handled according to this protocol: *E. coli*
Competent Cells Quick Protocol #9FB035, which is available
on the website [here](https://se.promega.com/-/media/files/resources/protcards/e-coli-competent-cells-quick-protocol.pdf?la=en).
For testing competent cells, we used empty vectors pDG1664
and pDG3661. The transformation mixture was carefully
plated on LB medium plates containing the corresponding
antibiotic - in both cases ampicillin (final conc. 150
µg/ml).
A more specific description of the transformation of these
*E. coli* JM109 cells will be presented in the
following subsections.

## Transformation of JM109 with pUCIDT plasmids with constructs B, C and D

### Motivation

Transformation of *E. coli* JM109 with
our constructs incorporated inside pUCIDT plasmids with the
aim of amplifying these plasmids and making glycerol
stocks. Then use the amplified IDT plasmids containing
constructs B, C and D for subcloning.

### Materials

##### Bacterial strains

For this experiment, we used the commercial
strain of competent cells of *E. coli* JM109 from
Promega, which we stored at -80°C before use.

##### Chemicals and Media

We used Lysogeny Broth (LB) medium, LB plates
with corresponding antibiotic - ampicillin (final conc. 150
µg/ml). In addition, for transformation we used pUCIDT
plasmids with our constructs B, C and D.

### Short description

For this experiment, we followed the *E.
coli* Competent Cells Quick Protocol #9FB035, which can
be accessed at this website: [here](https://se.promega.com/-/media/files/resources/protcards/e-coli-competent-cells-quick-protocol.pdf?la=en).
To briefly introduce, competent JM109 cells stored at -80°C
were placed on ice. After thawing, 100 µl of competent
cells (volume of one transformation reaction) were
transferred to eppendorf tubes. Subsequently, 1-50 ng of
DNA pUCIDT plasmid with constructs B, C, and D (in a volume
of no more than 10 µl) were added to the cells. Both
positive and negative controls were also prepared to
confirm that the transformation was successful and the
antibiotic prevented cells without our desired antibiotic
resistance from growing. Immediately after the addition of
DNA, the eppendorf tubes were returned to the ice and after
10 minutes underwent a heath shock with heat in a water
bath at exactly 42°C. Immediately after that, they were put
back on ice again. Subsequently, LB medium was added to the
transformation mixture and a 60 min incubation was
performed at 37°C on a shaker at approximately 225 rpm.
After one hour incubation, competent cells were diluted to
1:10 and 1:100, and plated on ampicillin-containing Petri
dishes. Finally, the plates were transferred to a
thermostat and incubated overnight at 37°C. The next day,
the transformation was evaluated.

These cells serve as an endless source of our
synthetic constructs in the form of glycerol
stocks.

## Subcloning of synthetic constructs to destination shuttle vector pDG3661 and pDG1664

Before transformation of JM109 cells with the
destination plasmids pDG3661 and pDG1664 with our
constructs, it was necessary to perform several
experiments. Detailed information about these experiments
can be found in the section [Basic
experiments](https://2021.igem.org/Team%3ABrno_Czech_Republic/Protocols#basicExperiments) and in [Protocol:
Isolation of plasmids](https://static.igem.org/mediawiki/2021/d/d5/T--Brno_Czech_Republic--PROTOCOL-_Isolation_of_plasmids.pdf).

### Chronological scheme of subcloning experiments:

1. isolation of vectors pUCIDT with our
   constructs
2. in case of B and D construct:

   (in case of C we had to perform PCR
   amplification of construct from pUCIDT\_C template - we
   were not able to digest this template successfully)

   1. restriction digestion with
      corresponding restriction enzymes
   2. visualization using gel
      electrophoresis
   3. purification
   4. ligation with appropriate destination
      shuttle vector

3. Transformation of JM109 cells
4. Proof of successful transformation
5. Glycerol stocks

![](https://static.igem.org/mediawiki/2021/a/ad/T--Brno_Czech_Republic--Experiments-04.jpeg "wet-lab-04")

## Transformation of JM109 with destination shuttle vectors pDG3661 and pDG1664 with constructs B, C and D

### Motivation

The motivation was to successfully perform
several intermediate subcloning steps mentioned above and
to transform *E. coli* JM109 with our constructs B, C
and D in shuttle vectors pDG3661 and pDG1664 with the aim
of amplifying these plasmids and making glycerol stocks. In
addition, we want to isolate these amplified vectors in
sufficient concentration to transform *B. subtilis*
168. The design of pDG3661 and pDG1664 vectors allows it to
be subsequently integrated into a specific location of the
*B. subtilis* 168 chromosome.

### Materials

##### Bacterial strains

For this experiment, we used the commercial
strain of competent cells of *E. coli* JM109 from
Promega, which we stored at -80°C before use.

##### Chemicals and Media

As for the chemicals, we used Lysogeny Broth
(LB) medium, LB plates with corresponding antibiotic -
ampicillin (final. conc. = 150 µg/ml). In addition, for
transformation we used shuttle vectors pDG3661 and pDG1664
with our constructs B, C and D.

### Short description

Several intermediate steps that were mentioned
earlier had to be taken before the actual transformation
was initiated.
For transformation *E. coli* with pDG3661 and pDG1664
containing constructs B, C and D, we again followed the
*E. coli* Competent Cells Quick Protocol #9FB035,
which can be accessed [here](https://se.promega.com/-/media/files/resources/protcards/e-coli-competent-cells-quick-protocol.pdf?la=en).
The same procedure was followed as for the transformation
experiment mentioned above - Transformation of JM109 with
pUCIDT plasmids of our constructs B, C and D, but instead
of using 1-50 ng of DNA, 10 µl of ligation mixture was
used. Subsequently, the success of the transformation was
confirmed by the steps presented in the following
section.

## Proof of transformation of Escherichia coli (JM109)

Protocol available here: [Protocol:
Proof of transformation of *Escherichia
coli*](https://static.igem.org/mediawiki/2021/4/41/T--Brno_Czech_Republic--PROTOCOL-_proofEColitrans.pdf)

### Motivation

To be sure of our success, we had to prove the
presence of destination vectors pDG3661 and pDG1664 with
our constructs. For this purpose, we used different methods
- selection on antibiotic-containing plates, colony PCR,
but also restriction digestion.

### Materials

##### Bacterial strains

We used transformed *E. coli* JM109
cells.

##### Chemicals and Media

As for the selection, we used Lysogeny Broth
(LB) medium, LB plates with corresponding antibiotic -
ampicillin (conc. 150 µg/ml). For the restriction
digestion, we used appropriate restriction enzymes and
buffer. And sequence-specific primers, listed in the
section [Primers](https://static.igem.org/mediawiki/2021/3/38/T--Brno_Czech_Republic--Table_of_primers.pdf),
were used to validate the colony PCR.

### Short description

Firstly, the presence of the destination
vectors pDG3661 and pDG1664 with constructs B, C and D in
*E. coli* JM109 was proved by growing the cells on LB
agar plates with ampicillin. We also performed better proof
of transformation using restriction digestion and
electrophoresis with isolated vectors from colonies of
transformants to check if the sizes of the fragments were
correct. Before the restriction digestion, we had to
isolate plasmids from overnight culture and isolate them.
You can find the detailed process of these experiments
here: [Basic
experiments](https://2021.igem.org/Team%3ABrno_Czech_Republic/Protocols#basicExperiments) and [Protocol:
Proof of transformation of *Escherichia coli*](https://static.igem.org/mediawiki/2021/4/41/T--Brno_Czech_Republic--PROTOCOL-_proofEColitrans.pdf).

Another strategy to confirm the success of the
transformation is to use sequence-specific primers. The
colonyPCR method can be used to amplify a specific segment
of the sequence of our constructs in the destination
vectors. The amplified sequence of DNA was thereafter used
for various purposes including electrophoresis or storage.
Further details can be found above in [Basic
experiments](https://2021.igem.org/Team%3ABrno_Czech_Republic/Protocols#basicExperiments).

![](https://static.igem.org/mediawiki/2021/f/f3/T--Brno_Czech_Republic--DM.jpg "wet-lab-05")

## 2. E. coli BL21(DE3)

## Preparation of competent cells E. coli BL21(DE3)

Protocol is available here: [Protocol:
Preparation of competent cells of *Escherichia coli*
BL21(DE3)](https://static.igem.org/mediawiki/2021/f/f6/T--Brno_Czech_Republic--PROTOCOL-_preparationBL21.pdf)

### Motivation

To prepare competent *E. coli* BL21(DE3)
cells to efficiently accept pUCIDT plasmids with our
synthetic constructs B and D. We need to use this
expression strain containing the gene for LacI in its
genome to verify the functionality of our synthetic
constructs B and D before and after IPTG induction.

### Materials

#### Bacterial strains

For this experiment, we used the expression
strain of competent cells of *E. coli* BL21(DE3),
which we stored at -80°C before use.

#### Chemicals and Media

We used Lysogeny Broth (LB) medium, LB plates
with corresponding antibiotic - ampicillin (conc. 150
µg/ml). In addition, for transformation we used pUCIDT
plasmids with our constructs B and D.

### Short description

After cultivation at 37 °C and subsequent
inoculation, the culture was allowed to grow to an OD of
0.27-0.33 and then placed on ice for a period of time. It
was now worked at 4 °C - centrifuged and a 0.05 M CaCl2
solution was added (this step was performed twice).
Glycerol was added to the cell suspension. *E. coli*
competent cells prepared in this way can be functionally
stored at -70°C for approximately one year.

## Testing of competent cells *E. coli* BL21(DE3)

### Motivation

We need to verify the viability of our
competent cells, which will then be transformed by plasmids
with our constructs.

### Materials

#### Bacterial strains

Expression strain of competent cells of *E.
coli* BL21(DE3).

#### Chemicals and Media

We used Lysogeny Broth (LB) medium, LB plates
with corresponding antibiotic - ampicillin (conc. 150
µg/ml). In addition, for transformation we used empty
shuttle vectors pDG3661 and pDG1664.

### Short description

*E. coli* BL21(DE3) cells were
transformed with empty shuttle vectors pDG3661 and pDG1664
(1 µg of DNA per 100 ul of competent cells). They were
plated on LB agar plates with the appropriate antibiotic,
ampicillin (150 µg/ml). Untransformed competent cells
plated on a plate with LB agar and ampicillin of the same
concentration were used as negative control.

![](https://static.igem.org/mediawiki/2021/6/64/T--Brno_Czech_Republic--Experiments-06.jpeg "wet-lab-06")

## Transformation of competent cells E. coli BL21(DE3) with IDT plasmids (containing constructs B and D)

Protocol is available here: [Protocol:
Subcloning and transformation of *Escherichia coli*
host](https://static.igem.org/mediawiki/2021/1/15/T--Brno_Czech_Republic--PROTOCOL-_Subcloning_and_transformation_of_Escherichia_col_host.pdf)

### Motivation

We performed this experiment to deliver pUCIDT
plasmids with synthetic constructs B and D into *E.
coli* BL21(DE3) cells.

### Materials

#### Bacterial strains

Expression strain of competent cells of *E.
coli* BL21(DE3).

#### Chemicals and Media

We used Lysogeny Broth (LB) medium, LB plates
with corresponding antibiotic - ampicillin (conc. 150
µg/ml). In addition, for transformation we used commercial
pUCIDT plasmids containing our synthetic constructs B and
D.

### Short description

After thawing 200 µl of competent *E.
coli* BL21(DE3) cells on ice, 1 µg of plasmid DNA was
added. They were then left on ice for 30 min, placed in a
water bath that was heated to 42 °C (heat shock) for 50 s
and placed back on ice for two minutes. 1 ml of LB medium
was added and left at 390 rpm for one hour at 37 °C,
centrifuged and the supernatant was discarded. To the
pellet of cells, 100 µl of LB medium was added and this
suspension was seeded on a plate with agar and ampicillin
(150 µg/ml). The dishes were retained to culture until the
next day in an incubator at 37°C.

## Proof of transformation of E. coli BL21(DE3)

### Motivation

We need to verify which colonies grown on the
antibiotic plates have pUCIDT plasmids with B and D
constructs (there may be some mutant colonies on the plates
that have developed resistance to the antibiotic and do not
actually have pUCIDT plasmids with synthetic constructs).
The verified colonies will be made into glycerol preserves
and later used in further experiments.

### Materials

#### Bacterial strains

We used transformed *E. coli* BL21(DE3)
cells.

#### Chemicals and Media

As for the selection, we used Lysogeny Broth
(LB) medium, LB plates with corresponding antibiotic -
ampicillin (conc. 150 µg/ml). For colony PCR we used
appropriate chemicals and sequence-specific primers, listed
in the section [Primers](https://static.igem.org/mediawiki/2021/3/38/T--Brno_Czech_Republic--Table_of_primers.pdf)
.

### Short description

In our experiments, we used to verify the
presence of plasmids with constructs in transformants in
two ways. The first way is restriction analysis and another
strategy to confirm the success of the transformation is to
use sequence-specific primers. The colony PCR method can be
used to amplify a specific segment of the sequence of our
constructs in the destination vectors. The amplified
sequence of DNA was thereafter used for various purposes
including electrophoresis or storage. Further details can
be found above in the part [Basic
experiments](https://2021.igem.org/Team%3ABrno_Czech_Republic/Protocols#basicExperiments) and [Protocols](https://static.igem.org/mediawiki/2021/4/41/T--Brno_Czech_Republic--PROTOCOL-_BStransformation.pdf).

![](https://static.igem.org/mediawiki/2021/1/1d/T--Brno_Czech_Republic--miska.jpg "wet-lab-07")

![](https://static.igem.org/mediawiki/2021/4/42/T--Brno_Czech_Republic--FEB.jpg "wet-lab-08")

# Integration of construct B, C and D into the chromosome of Bacillus subtilis

## Transformation of Bacillus subtilis

Our protocol for transformation of *Bacillus
subtilis* is available here: [PROTOCOL:
Transformation of *Bacillus subtilis*](https://static.igem.org/mediawiki/2021/4/41/T--Brno_Czech_Republic--PROTOCOL-_BStransformation.pdf)

### Motivation

Transformation of competent *B. subtilis*
cells was used, because we needed to insert various vectors
into our *B. subtilis* in order to integrate
our [constructs
B, C and D.](https://2021.igem.org/Team%3ABrno_Czech_Republic/Design) The first plan also included
integrating [construct
A](https://2021.igem.org/Team%3ABrno_Czech_Republic/Design), sadly due to a lack of time we did not have the
chance to try doing so.

### Materials

We used our own naturally competent cells
prepared from *B. subtilis* 168. The protocol
for [preparing
*B. subtilis* competent cells](https://static.igem.org/mediawiki/2021/0/09/T--Brno_Czech_Republic--PROTOCOL-_preparationBS.pdf) can be found far
ahead in the experiment section. We used vectors pDG1664
with construct B and pDG3661 with constructs C and D
isolated from our *Escherichia coli*. They were
carefully plated with LB medium and the corresponding
antibiotic.

### Short experimental description

We first had to isolate plasmids with
corresponding constructs from E. coli. The naturally
competent *B. subtilis* cells were preserved in the
form of glycerol stocks. We added 1 µg of plasmid DNA to
100 µl of naturally competent cells. We also prepared
positive and negative controls. The positive control
contained empty supercoiled plasmid carrying antibiotic
resistance. The competent cells without plasmid were used
as the negative control. These controls confirmed that the
transformation was performed correctly and that the
antibiotic prevented cells without our desired plasmid with
antibiotic resistance from growing. We incubated the tubes
containing competent cells and plasmids at 200 rpm and 37
°C for 1 hour. Afterwards, we plated the competent cells on
agar plates with a selection medium containing the
corresponding antibiotic. The cells grew overnight at 37
°C. Next day we interpreted the results. ​​We performed the
same set of experiments with pDG1664\_B, pDG3661\_C and
pDG3661\_D.

## Proof of successful integration

## 1. AmyE test (Starch hydrolysis test)

Protocol is available here: [PROTOCOL:
AmyE test (Starch hydrolysis test)](https://static.igem.org/mediawiki/2021/f/fa/T--Brno_Czech_Republic--PROTOCOL-_AmyE_test_%28Starch_hydrolysis_test%29.pdf)

Original publication:

Guérout-Fleury, A. M., Frandsen, N., &
Stragier, P. (1996). Plasmids for ectopic integration in
*Bacillus subtilis*. Gene, 180(1-2), 57–61.
 [https://doi.org/10.1016/s0378-1119(96)00404-0](https://doi.org/10.1016/s0378-1119%2896%2900404-0)

### Motivation

AmyE test is a simple method that can
demonstrate ectopic integration into the chromosome of
*B. subtilis*. 𝛼-amylase, witch degrades starch in the
medium is encoded in the integration site of our plasmides.
If this integration site remains intact, 𝛼-amylase is able
to function normally. After the iodine treatment, this can
be observed as a clear halo effect around the colonies,
created by the digestion of the starch. If the plasmid with
our construct is integrated successfully, 𝛼-amylase is
unable to function properly and therefore cannot degrade
the starch. While doing our experiments, the AmyE test was
used to prove the successful integration of our inserts by
comparing them with unmodified *B.subtilis*
168.

### Materials

We used *B. subtilis* 168 as well as agar
and starch to make the two layered agar for the plates. We
also used iodine to make the digestion of starch
visible.

### Short description

This method utilised a two layer agar: the
first is a regular agar that contains the corresponding
antibiotic and the second is a thinner layer that contains
starch supplement. Subsequently a few iodine crystals are
added and the starch will start slowly turning dark blue.
Iodine fumes are highly toxic so do not forget to work in a
laboratory hood.

![](https://static.igem.org/mediawiki/2021/c/cd/T--Brno_Czech_Republic--Notebook-figure7.jpeg "wet-lab-09")

## 2. Isolation of chromosomal DNA

### Motivation

Isolation of chromosomal DNA is used to
confirm that our plasmids with our constructs have been
successfully integrated into the DNA of *B. subtilis*.
The isolated DNA is later used for a long PCR, where the
part that we want to confirm can be amplified.

### Materials

We used our modified *B. subtilis* 168.
The full protocol can be found here: [Protocol:
Proof of the successful transformation of *Bacillus
subtilis*](https://static.igem.org/mediawiki/2021/7/71/T--Brno_Czech_Republic--PROTOCOL-_proofBStrans.pdf)

### Short experimental description

Using an overnight culture, we used what we
call a physical method which uses high temperatures to
isolate the chromosomal DNA.

## 3. PCR

### Motivation

PCR methods can amplify a certain part of DNA
using the appropriate primers. We used PCR to amplify our
constructs in plasmids from *E. coli* as well as in
the chromosomal DNA of *B. subtilis*. Long-range PCR
was routinely used in our experiment for the amplification
of our longer constructs. For amplifying DNA from *B.
subtilis,* we used colony PCR. The amplified DNA was
thereafter used for various purposes including
electrophoresis, DNA sequencing or storage. Further details
can be found above in basic experiments.

## 4. Sequencing of Bacillus subtilis - amplicons of chromosomal DNA

### Motivation

We needed to verify the sequence which was
integrated into the chromosome of *B. subtilis*. This
would demonstrate the success of our transformation and
chromosomal integration. This experiment further
demonstrates the success of our subcloning activities in
*B. subtilis*. Few tries were needed before we
achieved the final data, but the results were worth
it.

### Short description

We used the services of the
company [Eurofins Genomics](https://eurofinsgenomics.eu/) for
sequencing of our *B. subtilis*. The sequencing was
performed twice. Since our targeted sequences were too
long, we had to divide them into two parts. First sequenced
part targeted the end of plasmid and the beginning of a
construct. The second targeted part included the end of the
targeted chromosome and the beginning of plasmid. We
used   [primers
44, 46, 51 and 52](https://static.igem.org/mediawiki/2021/3/38/T--Brno_Czech_Republic--Table_of_primers.pdf).

As far as the exact steps and methods used to
prepare our cells for sequencing, we followed instructions
supplied to us by the company [Eurofin genomics](https://eurofinsgenomics.eu/), which
can be found on their website. It is important to point out
that with all three constructs: B, C and D we tried to
sequence both strands of DNA using forward and reverse
sequencing.

![](https://static.igem.org/mediawiki/2021/e/e3/T--Brno_Czech_Republic--Experiments-09.jpeg "wet-lab-10")

# Characterization of construct B and D

## Measurement of expression of fluorescent proteins

### Motivation

In these experiments, we tried to demonstrate
the functionalities of our project design. In our approach,
we designed a reporter system to help us verify the
functionality of each part of the system.

For the purpose of this experiment, we
determined the growth curve based on the OD600 of our
cultures. In particular, we tested the response of
promoters to the presence of IPTG in the expression strain
of E.coli BL21(DE3) cells. In *B. subtilis* cells, we
were interested in constitutive expression.

We decided to verify the presence of the
reporter proteins GFP and mScarlet-I in several ways. We
used fluorescence measurements simultaneously with OD600
measurement of cultures on a Tecan plate fluorometer. We
also used fluorescence spectrum measurements in cuvettes on
a Fluoromax instrument from Horiba Scientific. Finally, we
also verified the results on a fluorescence
microscope.

These three experiments were performed with
the same cultures. The experimental design was very complex
- the cultures used, the methods, their processing and
other details can be found in this table: [Table
of samples for measurement](https://static.igem.org/mediawiki/2021/5/5b/T--Brno_Czech_Republic--PROTOCOL-_table1measurments.pdf). In the following sections,
you can read about more detailed specifications of the
experiments in each facility.

### 1. Measurement of fluorescence and OD of culture in time - Tecan device

This device allows the measurement of
fluorescence of two fluorescent proteins to be measured
simultaneously. At the same time, the instrument also
measures the OD of the culture, so the two types of data
can be analyzed simultaneously. The instrument measures in
a microtiter plate. Therefore, its use was convenient for
us as it can automatically analyze a large number of
samples simultaneously.

### Materials

See [Table
of samples for measurement](https://static.igem.org/mediawiki/2021/5/5b/T--Brno_Czech_Republic--PROTOCOL-_table1measurments.pdf) and other specifications.
The Tecan device settings can be found in this
document: [Tecan
settings](https://static.igem.org/mediawiki/2021/9/9c/T--Brno_Czech_Republic--PROTOCOL-_table1tescan.pdf). For the measurement, we used a black
microtiter plate with 96 wells with a lid.

Fluorometer device type: Tecan Infinite 200
Pro

![](https://static.igem.org/mediawiki/2021/3/3f/T--Brno_Czech_Republic--tecan.jpg "wet-lab-11")

### Method

This machine allows you to measure samples in
a microtiter plate. The instrument maintains the culture
temperature at 37 °C and also provides shaking of the
microtiter plate. Measurements of OD, GFP intensity and
mScarlet-I were performed automatically at 15 min intervals
over a 24 h period. A bottom beam was used to measure
fluorescence intensity and OD. This method of measuring
from underneath is well proven and more accurate in the
laboratories where we made the measurements. For higher
reproducibility of the results, we worked in technical
duplicates.

### Preparation of microtiter plate lid

We used a black microtiter plate with a lid
for 96 samples. 0.05% Triton in ethanol was poured onto the
lid. Care must be taken to ensure that the entire surface
of the cap is coated with an even thin layer. Then the lid
is left inside up, the flowbox is closed and the plate and
lid are illuminated with UV light. This ensures the
sterility and cleanliness of the plate. The plate should
also be sterilized with UV light before each subsequent
use. For subsequent use, the sample can only be placed in
the unused wells only.

### Sample preparation for measurement

Overnight cultures in liquid LB were prepared
the previous evening (culture at 110 RPM shaking, 37 °C).
The following morning their OD was measured. Subsequently,
the cultures were diluted appropriately so that their OD
was 0.05. 200 ul of each culture was pipetted into a well
in the plate. Once the OD of the samples reached 0.6, the
BL21(DE3)\_B IPTG 0.6, BL21(DE3)\_D IPTG 0.6 and BL21(DE3)
IPTG 0.6 samples were induced with IPTG to a final
concentration of 100 uM. The *B. subtilis* positive
control GFP in chromosome sample was also induced at OD =
0.6, but by adding xylose to a final concentration of
0.5%. [See
the supplementary chart](https://static.igem.org/mediawiki/2021/5/5b/T--Brno_Czech_Republic--PROTOCOL-_table1measurments.pdf).

## 2. Measurement of fluorescence in device FluoroMax Plus

The spectrofluorometric measurement is an
essential confirmation that the correct fluorescent
proteins are formed in our constructs. It can very
accurately measure the excitation and emission spectra of
any fluorescent protein. However, this instrument is very
computationally expensive, so we are grateful to the
management of Loschmidt Laboratories for allowing us to
make measurements on it.

### Material

For this experiment, we used a similar set of
samples as for the previous measurements in the Tecan
device. The list of samples used can be found in this
document: [Table
of samples for measurement](https://static.igem.org/mediawiki/2021/5/5b/T--Brno_Czech_Republic--PROTOCOL-_table1measurments.pdf). Cultures were not diluted
before measurement. Each sample was measured in a
polystyrene disposable 4 sided cuvette.

### Preparation of samples for measurement

Overnight cultures of all samples in LB medium
were inoculated the previous day (shaking culture at 110
RPM, 37 °C). Measurements were performed 20 hours after
inoculation of overnight cultures. Certain cultures were
induced with IPTG immediately after inoculation. Some
samples were diluted after 15 hours of cultivation to OD =
0.8. Some samples were induced at OD = 0.8 by IPTG to a
final concentration = 100 uM and these were returned to the
shaker. For other samples, a double concentration of IPTG
was added.

### Method:

The type of device: FluoroMax Plus TCSPC,
Horiba Scientific

Other device specifications:

* xenon lamp,
* monochromatic excitation = diffraction
  grating,
* the reference detector = photodiode,
* the emission monochromator = diffraction
  grating,
* the main detector = PMT,
* slit = 1 nm for excitation and
  emission

![](https://static.igem.org/mediawiki/2021/f/f1/T--Brno_Czech_Republic--Fluoromax.jpg "wet-lab-12")

We used the Horiba Scientific FluoroMax Plus
instrument provided by Loschmidt Laboratories for this
experiment, to whom we are very grateful. Spectral analysis
of the emission spectra of each sample was performed. There
was a range of wavelengths for mScarlet-I and GFP which you
can see in the following table:

|  |  |
| --- | --- |
| Measured fluorescent protein | Setting the range of spectra |
| GFP | 400 - 600 nm |
| mScarlet-I | 500 - 700 nm |

Table 2 : Measurement specifications of
FluoroMax Plus

## Fluorescence microscopy

We aimed to verify the measurements made on
the previous instruments using a fluorescence microscope.
Selected cultures were photographed in a brightfield and
using fluorescence filters.

### Material:

We used some of the cultures prepared for the
previous two experiments - which exactly is indicated in
the [Table
of samples for measurement](https://static.igem.org/mediawiki/2021/5/5b/T--Brno_Czech_Republic--PROTOCOL-_table1measurments.pdf). For less light refraction,
immersion oil was used.

### Method:

For this experiment, we used this type of
fluorescence microscope: Imager Z2, Zeiss.

The used microscope and other specific device
settings can be seen at the two following pictures:

![](https://static.igem.org/mediawiki/2021/4/40/T--Brno_Czech_Republic--mikroskop.jpg "wet-lab-13")

![](https://static.igem.org/mediawiki/2021/2/2a/T--Brno_Czech_Republic--nastavenimickroskopu.jpg "wet-lab-14")

15 ul of culture were taken and mixed with 15
ul of immersion oil. The sample prepared was thus ready for
photography. A drop of immersion oil was added to the top
of the coverslip before photographing. The appropriate
filters and magnification were set on the microscope. Each
specimen was attempted to be captured with either
brightfield, GFP and mScarlet-I filters.

![](https://static.igem.org/mediawiki/2021/6/6e/T--Brno_Czech_Republic--balime.jpg "wet-lab-15")

![](https://static.igem.org/mediawiki/2021/7/79/T--Brno_Czech_Republic--Experiments-figure15.jpeg "wet-lab-16")

![](https://static.igem.org/mediawiki/2021/d/d5/T--Brno_Czech_Republic--A-Icon-pipoeta.svg "wet-lab-ic-1")![](https://static.igem.org/mediawiki/2021/2/24/T--Brno_Czech_Republic--A-Icon-miska.svg "wet-lab-ic-2")![](https://static.igem.org/mediawiki/2021/4/40/T--Brno_Czech_Republic--A-Icon-primery.svg "wet-lab-ic-3")![](https://static.igem.org/mediawiki/2021/d/d4/T--Brno_Czech_Republic--A-Icon-ELFO.svg "wet-lab-ic-4")![](https://static.igem.org/mediawiki/2021/1/11/T--Brno_Czech_Republic--A-Icon-ecoli.svg "wet-lab-ic-5")![](https://static.igem.org/mediawiki/2021/2/28/T--Brno_Czech_Republic--A-Icon-plazmnid.svg "wet-lab-ic-6")![](https://static.igem.org/mediawiki/2021/1/13/T--Brno_Czech_Republic--A-Icon-Epinka.svg "wet-lab-ic-7")![](https://static.igem.org/mediawiki/2021/d/d5/T--Brno_Czech_Republic--A-Icon-pipoeta.svg "wet-lab-ic-13")![](https://static.igem.org/mediawiki/2021/c/cd/T--Brno_Czech_Republic--A-Icon-sinice.svg "wet-lab-ic-8")![](https://static.igem.org/mediawiki/2021/0/01/T--Brno_Czech_Republic--A-Icon-bacilus.svg "wet-lab-ic-9")![](https://static.igem.org/mediawiki/2021/2/26/T--Brno_Czech_Republic--A-Icon-falkona.svg "wet-lab-ic-10")![](https://static.igem.org/mediawiki/2021/5/56/T--Brno_Czech_Republic--A-Icon-prednaska.svg "wet-lab-ic-11")![](https://static.igem.org/mediawiki/2021/7/77/T--Brno_Czech_Republic--A-Icon-graf.svg "wet-lab-ic-12")![](https://static.igem.org/mediawiki/2021/c/cd/T--Brno_Czech_Republic--A-Icon-sinice.svg "wet-lab-ic-14")

Igem Team Brno, Czech Republic 2021

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# Team:Brno Czech Republic/Results

Results

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# Results

## Introduction

The synthetic sequences as well as regulatory
regions used in our project PHOSCAGE were designed using
the Benchling software. *In silico*, 4 gene constructs
were designed, called as constructs A, B, C and D.

Construct A was designed to test BMC
production. However, this construct could not be
synthesized by IDT and hence will not be mentioned in the
following sections.

Construct B contains a gene for *sfGFP*
production with a PduP tag that should ensure *sfGFP*
packing to BMCs.

Construct C contains genes for the cI
repressor and mScarlet-I downstream of the PPho
promoter. This construct is designed to test the response
of PPho to changes in phosphate levels.

Construct D is responsible for the formation
of cI repressor and mScarlet-I with a degradation tag upon
IPTG induction. In the absence of IPTG, GFP with a
degradation tag is in turn expressed.

More detailed information about our constructs
can be found in  [Parts
and Project design](https://2021.igem.org/Team%3ABrno_Czech_Republic/Design).

Each construct contains BamHI and HindIII
restriction sites at the 5' and 3' end respectively.
Corresponding restriction enzymes were used to clone each
construct into its destination integration plasmid from
which it can be integrated into the specific locus of *B.
subtilis* 168 chromosome.

Synthetic sequences ([Parts](https://2021.igem.org/Team%3ABrno_Czech_Republic/Parts))
received from the company IDT were delivered in pUCIDT
plasmids. For this reason, we used a technique known as
subcloning. This technique is used for transferring a
specific DNA sequence from a parent vector, in our case the
commercial plasmid pUCIDT, to a target vector. Target
vectors used in our project were plasmids pDG3661 and
pDG1664. Both of them are “shuttle vectors”, which means
that they can be used in both *E. coli* and *B.
subtilis*.

After successful transformation of *E.
coli* JM109 with pUCIDT\_B, pUCIDT\_C, and pUCIDT\_D, these
plasmids were isolated from overnight cultures, digested by
corresponding restriction enzymes, and ligated to the
shuttle vector pDG3661 (in the case of constructs C and D)
or pDG1664 (in the case of construct B). The destination
vectors thus ligated with constructs B, C and D (pDG3661\_C,
pDG3661\_D, pDG1664\_B) were again used for the
transformation of *E. coli* JM109 which was then
utilised for their replication. All thusly prepared vectors
were isolated from overnight cultures. *B. subtilis*
168 was transformed with these vectors and our constructs
and parts of plasmids were integrated into its chromosome.
The functionality of synthetic systems B and D was
subsequently tested.

After the first testing measurements, the
*E. coli* strain BL21(DE3) was also transformed with
pUCIDT\_B and pUCIDT\_D and the functionality of the
synthetic constructs was tested in this expression strain
with *LacI* repressor gene in its genome (Schema of
our experimental approach is shown in Figure 1).

In the following section you will find
detailed information about results of all the experiments
performed.

# Preparation of Escherichia coli cloning host

## Experimental design

![](https://static.igem.org/mediawiki/2021/b/b0/T--Brno_Czech_Republic--Experiment-schema.svg "results-img-1")
**Figure 1:Scheme of our experimental
approach.**

Construct B was cloned into the destination
pDG1664 vector by restriction digestion and ligation -
creating pDG1664\_B which was then used to transform *E.
coli* JM109. Transfromants were selected by their
ampicillin resistance. Constructs C and D were cloned into
the destination vector pDG3661 - creating pDG3661\_C and
pDG3661\_D. After transformation of *E. coli* JM109,
ampicillin was also used for selection. We were able to
clone constructs B and D into the destination vectors
without too much trouble. However, in the case of construct
C, this was more complicated and therefore a PCR cloning
method was used instead of subcloning.

In order to perform complex measurements,
which are described in the last section of “Wet lab and
Results” , the constructs were cloned not only into cloning
strain *E. coli* JM109, but also into expression
strain *E. coli* BL21(DE3).

At the end of the section “Preparation of
*Escherichia coli* cloning host” , we have isolated
the vectors pDG3661\_D, pDG3661\_C and pDG1664\_B in high
concentrations. The success of cloning and subsequent
transformation of JM109 and BL21(DE3) was demonstrated by
plasmid isolation, colony PCR or control restriction
digestion.

### Transformation of E. coli JM109 with pUCIDT\_B, pUCIDT\_C and pUCIDT\_D

All of our constructs were synthesized by the
IDT company and delivered in pUCIDT vectors. pUCIDT\_B,
pUCIDT\_C and pU CIDT\_D were successfully used for
transformation of *E. coli* JM109 competent cells
(Figure 2 and 3) from which cell glycerol stocks were
prepared. The transformation is described in the
 [PROTOCOL:
Subcloning and transformation of *Escherichia coli*
host](https://static.igem.org/mediawiki/2021/1/15/T--Brno_Czech_Republic--PROTOCOL-_Subcloning_and_transformation_of_Escherichia_col_host.pdf). The pink coloration of some colonies caused by
mScarlet-I production in the Figure 3 is a strong
indication of successful transformation of *E. coli*
JM109 with pUCIDT\_C. The colonies transformed with pUCIDT\_D
were more orange in colour, probably resulting from the
combination of red and green fluorescent proteins. This
therefore suggests that expression of our constructs is
taking place in *E. coli* in spite of the fact that
the constructs are designed and optimized for *B.
subtilis*.

![](https://static.igem.org/mediawiki/2021/0/09/T--Brno_Czech_Republic--Figure2.jpg "results-img-2")**Figure 2: Transformation of *E.
coli* JM109 competent cells with pUCIDT\_B.** Plate
number 2 is vital control for competent cells. Plate
number 3 contains transformed cells with vector pUCIT\_B
and number 4 contains negative control. Plate number 1
is a test of our antibiotic stock, which is not
significant for this experiment.

![](https://static.igem.org/mediawiki/2021/c/c9/T--Brno_Czech_Republic--Figure3.jpg "results-img-3")**Figure 3: Transformation of *E.
coli* JM109 with pUCIDT\_C and pUCIDT\_D.** Plate 1
and 2 are vital controls for competent cells, plate 3,
and 5 are cells transformed with pUCIDT\_C and 4 and 6
are cells transformed with pUCIT\_D. Plate number 7 is
negative control.

A problem arose in the synthesis of our
construct A, which required a design change. pUCIDT\_A was
therefore not delivered to us until late summer and thus we
were not able to start working with this construct before
the end of the competition.

### Proof of transformation of E. coli JM109 by pUCIDT\_B, pUCIDT\_C and pUCIDT\_D

Successful transformation of the vector
pUCIDT\_B was confirmed by selection on plates containing
ampicillin. Transformation was also verified by restriction
digestion with the enzymes HindIII and BamHI. Size of the
cleaved construct (1128 bp) and vector (2770 bp) was
determined using gel electrophoresis and a gene marker see
Figure 4. Fragments of construct B (marked by arrow) were
extracted from gel and also used for ligation with digested
vector pDG1664 described in the next section.

![](https://static.igem.org/mediawiki/2021/d/db/T--Brno_Czech_Republic--Figure4.jpg "results-img-4")**Figure 4:****Proof of
transformation of *E.coli* JM109 - restriction
analysis.**DNA fragments from restriction digestion
of pUCIDT\_B isolated from *E.coli*JM109
demonstrating successful transformation. M is marker -
GeneRuler 1 kb (ThermoFisher Scientific). Samples 1 and
2 are pUCIDT\_B digested with BamHI and HindIII -
fragments labeled by arrow had the length of digested
construct B - 1128 bp and vector 2770 bp.
Electrophoresis conditions: 0,8% agarose gel, 120V, 45
min.

![](https://static.igem.org/mediawiki/2021/8/85/T--Brno_Czech_Republic--Figure5.jpg "results-img-5") **Figure 5: Restriction digestion of
pUCIDT\_C and pUCIDT\_D plasmid as a proof of successful
transformation.**DNA fragments obtained by
restriction digestion of pUCIDT\_C and pUCIDT\_D proved
successful transformation of JM109 *E.coli* cells.
M is marker - GeneRuler 1 kb (ThermoFisher Scientific),
First sample to the right is digested pUCIDT\_C 2295 bp
long. Next sample is the vector pDG3661 (which was used
for subsequent ligation) digested with BamHI and
HindIII. The last sample is pUCIDT\_D digested with
BamHI, HindIII and BsaI where the size of the digested
construct was 2891 bp. Electrophoresis conditions: 0,8%
agarose gel, 120V, 45 min.

Successful transformation of *E. coli*
JM109 with pUCIDT\_C and pUCIDT\_D was verified in the same
manner. After restriction digestion, construct C had 2295
bp and construct D 2891 bp, see Figure 5.

Construct D was similar in size to the
digested empty pUCIDT, so pUCIDT\_D had to be additionally
digested with enzyme BsaI, resulting in two plasmid
fragments of sizes 1355 bp and 1415 bp (which appear as a
single band on the gel). This allows for nice separation of
digested construct D and pUCIDT plasmid and thus improves
gel extraction.

### Cloning of pDG1664\_B, pDG3661\_C and pDG3661\_D and transformation of E. coli JM109

After previously described restriction
digestion, gel electrophoresis and gel extraction,
construct D was successfully ligated with shuttle vector
pDG3661 and used for the transformation of *E. coli*
JM109.

Attempts at ligating construct C with this
vector resulted in repeated contamination with the original
pUCIDT vector. pUCIDT\_C was likely to have a significantly
better transformation rate or better ability to replicate
in competent cells. This problem was most likely the result
of choosing the same selection marker, i.e. ampicillin
resistance, for both vectors. This problem was eventually
solved by amplifying construct C with PCR (primers marked
as 55 and 56 can be found [here](https://static.igem.org/mediawiki/2021/3/38/T--Brno_Czech_Republic--Table_of_primers.pdf), the size of the amplicon
was 2471 bp) and then ligating the amplicons with vector
pDG3661. Products of this ligation reaction were then
successfully used for the transformation of E. coli
JM109.

After subcloning our constructs into shuttle
vectors pDG3661 (C and D) and pDG1664 (B) the cloning and
transformation was verified as described below (see Figure
6, Figure 7, Figure 8 and Figure 9).

![](https://static.igem.org/mediawiki/2021/3/33/T--Brno_Czech_Republic--Figure6.jpg "results-img-6")**Figure 6:Transformants of *E.
coli* JM109 containing pDG1664\_B on plates with
ampicillin.**Plate 1 contains negative control. Plate
2 contains positive control. Plates 3 and 4 were
inoculated with cells transformed by pDG1664\_B from a
ligation mix marked as B2, these two plates differ by
the amount of inoculum. Plates 5 and 6 contain cells
transformed by pDG1664\_B from a ligation mix marked as
B1, these two plates also differ by the amount of
inoculum.

![](https://static.igem.org/mediawiki/2021/e/ee/T--Brno_Czech_Republic--Figure7.jpg "results-img-7")**Figure 7 : Transformants of *E.
coli* JM109 containing pDG3661\_C on plates with
ampicillin.**Plates 1 and 3 contain cells transformed
with a ligation mix marked as C1. The difference
between them is in the amount of inoculum (400 µl for
the plate 1 and 110 µl for the plate 3). Plates 2 and 4
contain cells transformed with ligation mix marked as
C6. The difference between them is in the amount of
inoculum (400 µl for the plate 2 and 110 µl for the
plate 4). Plate 5 is positive control containing cells
transformed with plasmid pDG3661 and plate 6 is
negative control.

![](https://static.igem.org/mediawiki/2021/4/4d/T--Brno_Czech_Republic--Figure8.jpg "results-img-8")**Figure 8: Transformation of *E.
coli* JM109 containing pDG3661\_D on plates with
ampicillin.**Plate 1 is vital control of our
competent cells on medium without ampicillin. Plate 2
is positive control and plate 3 is negative control.
Plate 4 contains transformants with pDG3661\_D from the
ligation mix marked as D2 (colony marked with an
arrow). Plate 5 contains transformants with pDG3661\_D
from the ligation mix marked as D1 (colony marked with
an arrow).

![](https://static.igem.org/mediawiki/2021/7/77/T--Brno_Czech_Republic--Figure9.jpg "results-img-9")**Figure 9: Transformation of *E.
coli* JM109 containing pDG3661\_D on plates with
ampicillin.**Figure 9 is a close up of the figure 8
for better visibility of our colonies (marked with
arrows).

### Proof of transformation of E. coli JM109 by pDG1664\_B, pDG3661\_C and pDG3661\_D

Successful transformation of *E. coli*
with pDG1664\_B was verified by restriction digestion with
BamHI and HindIII (Figure 10). Fragment size of the
construct remains 1128 bp, the size of digested pDG1664
plasmid is 6042 bp.

![](https://static.igem.org/mediawiki/2021/9/96/T--Brno_Czech_Republic--Figure10.jpg "results-img-10")**Figure 10: Proof of transformation of
*E.coli* JM109 by pDG1664\_B - restriction
analysis.**This figure shows DNA fragments from
restriction digestion of pDG1664\_B which were isolated from
*E.coli* JM109 demonstrating successful
transformation. M is marker - GeneRuler 1 kb (ThermoFisher
Scientific). First three samples (1 - 3) are pUCIDT\_B
digested with BamHI and HindIII fragments had the length of
digested construct B - 1128 bp and vector - 2770 bp.
Samples 4-6 are pDG1664\_B digested with BamHI and HindIII
fragments had the length of digested construct B - 1128 bp
and vector - 6042 bp. Electrophoresis conditions: 0,8%
agarose gel, 120V, 40 min.

Successful transformation of pDG3661\_C into
*E. coli* was confirmed by colony PCR (primers 1 and 2
were complementary to pDG3661\_C, primers 55 and 56 were
complementary to pUCIDT\_C and were used to detect potential
contamination by this plasmid - they can all be found
[here](https://static.igem.org/mediawiki/2021/3/38/T--Brno_Czech_Republic--Table_of_primers.pdf)). As can be seen on the gel in Figure 11, some pUCIDT
contamination was still present. The colony which contained
only the plasmid pDG3661\_C (amplicon of the size 2570 bp)
was marked as number 1 and was used for subsequent
experiments. Shuttle vector containing construct C was
isolated from colony 1 and could finally be used for the
transformation of *B. subtilis* , where it was
integrated into its chromosome (described in Transformation
of *Bacillus* subtilis below).

![](https://static.igem.org/mediawiki/2021/f/fd/T--Brno_Czech_Republic--Figure11.jpg "results-img-11")**Figure 11: Colony PCR proving successful
transformation of *E. coli* with pDG3661\_C. M is
marker - GeneRuler 1 kb (ThermoFisher Scientific).**The
numbers (1, 2, 3, 4) indicate the number of the colony
which was sampled for the cPCR. The letters (a and b)
indicate the combination of used primers (a = primers 1 and
2 - to identify pDG3661\_C, b = primers 55 and 56 - to
identify pUCIDT\_C). In the sample 1a was confirmed
pDG3661\_C (size of the amplicon was 2570 bp, marked by an
arrow). Sample 1b contains a non-specific product, sample
2a contains a non-specific product (pDG3661\_C not present),
2b shows the presence of pUCIDT\_C and non-specific
products. In 3a, no products were detected. 3b shows the
presence of pUCIDT\_C and a non-specific product.
Electrophoresis conditions: 1% agarose gel, 120V, 40
min.

Successful transformation of *E. coli*
with pDG3661\_D was demonstrated by restriction digestion.
Fragment size of construct D was 2955 bp and 7271 bp for
plasmid pDG3661, see Figure 12.

![](https://static.igem.org/mediawiki/2021/2/22/T--Brno_Czech_Republic--Figure12.jpg "results-img-12")
**Figure 12: Proof of cloning construct D into the
shuttle vector pDG3661 andof successful transformation of
*E. coli* : M is marker - GeneRuler 1 kb
(ThermoFisher Scientific).**Based on the size of the
fragments (2295 bp for construct C and 2768 bp pUCIDT)
from restriction digestion were samples C1 and C2
identified as pUCIDT\_C. Samples D1 and D2 contain
pDG3661\_D (2955 bp for construct D and 7271 bp for
pDG3661). PC = positive control (pDG3661), NC = negative
control. Electrophoresis conditions: 1% agarose gel,
120V, 40 min.

### Transformation of competent E. coli BL21(DE3) with pUCIDT\_B, pUCIDT\_D

We have successfully integrated our constructs
B and D into the *B. subtilis* 168 chromosome
(described below), but after testing their exprethis was a
result of the absence of the LacI repressor gene in the
genome of B. subtilis 168. As there is nothing to repress
IPTG inducible promoters in the absence of IPTG, the
expression of genes under their control is
constitutive.

We therefore decided to also transform *E.
coli* BL21(DE3) expression strain, which contains the
gene for LacI in its genome, to demonstrate the response of
our system to IPTG induction.

Plasmids pUCIDT were used for the
transformation of *E. coli* BL21(DE3) as they are
smaller than the shuttle vectors pDG3661 and pDG1664 and
are present in *E. coli* cells in more copies. This
should ensure a high rate of production of target
proteins.

After testing competent *E. coli*
BL21(DE3) cells, we transformed them with pUCIDT\_B (see
Figure 14) and pUCIDT\_D (see Figure 13). Both
transformations were successful. As a negative control, we
used competent cells without plasmids plated on a Petri
dish with ampicillin (150 µg/ml). The verification of
successful transformation and its outcome will be described
in the following section.

![](https://lh5.googleusercontent.com/HpFdlGj81RZd0r95QjDdl_QAqfwe5BwvcOSMvj3s_1b_01dqAonR140Dr41VLJWg_cEykcrvc0unSFyMB6OcjNU9NmIxA_lNqcwegFFXUM9AsIK5-2E9OohsypV8La2R6-q74qRh=s1600 "results-img-13")
**Figure 13: Colonies of *E. coli* BL21(DE3)
competent cells transformed with pUCIDT\_D. Plate 1 - 1x
diluted, plate 2 - 10x diluted, plate 3 - 100x
diluted.**Plates contain ampicillin.

![](https://static.igem.org/mediawiki/2021/a/af/T--Brno_Czech_Republic--Figure14.jpg "results-img-14")
**Figure 14: Colonies of *E. coli* BL21(DE3)
competent cells transformed with pUCIDT\_B. Inoculate on
plate 1 was 10x diluted. Inoculate on plate 2 - 100x
diluted.**Plates contain ampicillin.

### Proof of transformation of E. coli BL21(DE3) with pUCIDT\_B and pUCIDT\_D

First preliminary indication of successful
transformation was the growth of colonies on our ampicillin
(150 µg/ml) plates. Colony PCR was also used for additional
verification of the transformants. Sequence-specific
primers 55 and 56 annealing to pUCIDT were designed to be
used with all constructs ligated to pUCIDT plasmids. All
primers can be found in the [Primers section](https://static.igem.org/mediawiki/2021/3/38/T--Brno_Czech_Republic--Table_of_primers.pdf). Successful
transformation of *E. coli* BL21(DE3) with pUCIDT
plasmids containing constructs B and D was thus confirmed.
pUCIDT plasmids with the respective constructs isolated
from our stocks of transformed E. coli JM109 were used as
positive controls. In negative control, 1 µl of PCR water
was added to the PCR reaction instead of DNA.
Electrophoresis was performed at 120 V for 33 min and the
products were analyzed on a 0.8% agarose gel.

The products of the PCR reactions had the
expected size in both cases. The gel in Figure 15
demonstrate successful transformation of 2 colonies with
pUCIDT\_B and two colonies with pUCIDT\_D. Later on, more
colonies were tested and glycerol stocks were made from the
cells of verified colonies.

![](https://static.igem.org/mediawiki/2021/6/6b/T--Brno_Czech_Republic--Figure15.jpg "results-img-15")
**Figure 15: PCR products proving the successful
transformation of *E. coli* BL21(DE3) with pUCIDT\_B
and pUCIDT\_D.**First sample from the left (NC) is the
negative control. Samples D1 and D2 are products of PCR
reaction which confirm the presence of pUCIDT\_D in tested
colonies (size of the amplicon is 3070 bp). Sample D2
also contains a non-specific product. PC-D is positive
control (isolated pUCIDT\_D from previous
experiments).Samples B1 and B2 are products of PCR
reaction which confirm the presence of pUCIDT\_B in tested
colonies (size of the amplicon is 1275 bp). (PC-B) is
positive control (isolated pUCIDT\_B verified in previous
experiment). M is marker - GeneRuler 1 kb (ThermoFisher
Scientific). Electrophoresis conditions: 1% agarose gel,
120V, 40 min.

# Integration of synthetic constructs into the chromosome of Bacillus subtilis

## Experimental design

In the next phase of our work in the wet lab,
we focused on transforming *Bacillus subtilis* strain
168 with plasmids pDG1664\_B, pDG3661\_C and pDG3661\_D.
Working with bacteria *B. subtilis* was a challenge,
since there was no one at our University with previous
experience with this bacterium except from our 2020 iGEM
team. Therefore this project was a challenge for us as well
as our PIs at times. Despite this, we have managed to
perform our experiment successfully and finish as planned
with the exception of a few time management flaws.

We are very thankful for the clever and
insightful advice we received from Dr. Krásný and
Laboratory of Microbial Genetics and Gene Expression in
Prague. Their help is greatly appreciated.

The vectors pDG1664 and pDG3661 were designed
for the integration into the chromosome of *B.
subtilis* . The part of pDG1664 designed for integration
additionally encodes resistance to erythromycin and the
combination of erythromycin and lincomycin was used for the
selection of transformants containing pDG1664\_B. The part
of pDG3661 designed for integration carries resistance to
chloramphenicol. AmyE test was used to demonstrate
successful transformation and integration of pDG3661\_C and
pDG3661\_D into the AmyE region of the chromosome *B.
subtilis* 168. Additionally, chromosomal DNA was
isolated and the region of insertion was amplified by PCR.
PCR products were then sent for sequencing.

## Testing competent cells of Bacillus subtilis

We worked with competent cells of *B.
subtilis* 168 which we obtained last year from Dr.
Krásný from Prague. Before using them, we first decided to
test them and see if they are still functional. To make
sure that they are still ready to be transformed, we
transformed them with empty vectors pDG3661 and verified
the integration into the chromosome by PCR. The PCR showed
that the cells were still competent.

## Transformation of Bacillus subtilis with pDG1664\_B, pDG3661\_C and pDG3661\_D

More details about our experiments can be
found in the section Wet lab.

As the synthesis of some of our composite
parts took longer than others and some trouble arose during
cloning and transformation of certain constructs, we were
not able to work with all of our constructs simultaneously,
though we tried to work in parallel when possible. The
first successful transformation and integration into the
chromosome of *B. subtilis* was performed with
Construct D. Then integration of construct B was close
second. Working with construct C however took substantially
longer due to the difficulties with subcloning into
pDG3661. Transformed strains of B. subtilis 168 were named
*B. subtilis\_B* , *B. subtilis\_C* and *B.
subtilis\_D* , according to the construct which was
integrated into their chromosome.

Transformed cells were streaked onto plates
with the appropriate antibiotic and incubated overnight at
37°C. In the figure 16 below you can see plates with *B.
subtilis* transformants carrying construct C.

![](https://static.igem.org/mediawiki/2021/d/dd/T--Brno_Czech_Republic--Figure16.jpg "results-img-16")
**Figure 16: Transformants of *B. subtilis* 168
with pDG3661\_C integrated in its chromosome on plates
with chloramphenicol.**Plates 1, 2 and 3 contain
transformants with pDG3661\_C. In this case, positive
control did not grow. The transformation with pDG3661\_C
was however tested and confirmed by PCR and sequencing.
Plate 5 contains negative control. The fact that colonies
can be seen on negative control is probably due to
spontaneous mutations leading to resistance.

### Proof by PCR and agarose gel DNA electrophoresis

First step of verifying the integration of our
constructs into the chromosome of *B. subtilis* 168
was long PCR using primers which annealed either near the
integration site on chromosome of *B. subtilis* or to
our composite part. Chromosomes isolated from transformed
colonies were used as templates for these PCR reactions.
Chromosomes were isolated using a “physical method” of
isolation which utilizes the alternation of extreme
temperatures to break the cell wall. More details can be
found in the section [Wet
Lab](https://2021.igem.org/Team%3ABrno_Czech_Republic/Experiments).

For constructs B and C, only the region
encompassing the transition between the chromosome of *B.
subtilis* , the part of plasmids pDG1664 and pDG3661
designed for integration and the 5' end of our construct
were amplified. For chromosomes containing construct B,
primers 43 and 46 were used to amplify the transition
between chromosome and plasmid (685 bp) and primers 50 and
51 were used to obtain a PCR product encompassing the
transition between plasmid and our construct (935 bp). For
chromosomes containing construct C, primers 44 and 46 were
used to amplify the transition between chromosome, plasmid
and our construct (1117 bp).

For construct D, The entire inserted region
was amplified using primers 28 and 29, which anneal to the
chromosome of *B. subtilis* at each side of the
integration site, producing an amplicon of 6511 bp.

![](https://static.igem.org/mediawiki/2021/2/26/T--Brno_Czech_Republic--Figure17.jpg "results-img-17")
**Figure 17: PCR verification of successful
integration of construct B intochromosome.**For
sample 2 primers 43 and 46 were used and the amplicon
of 685 bp was produced. For sample 1 primers 50 and 51
were used and the size of the amplicon was 935 bp. The
sequence of sample 2 in the second well was also
verified by sequencing (see below). M is marker -
GeneRuler 1 kb (ThermoFisher Scientific).
Electrophoresis conditions: 1 % agarose gel, 120V, 40
min.

![](https://static.igem.org/mediawiki/2021/0/07/T--Brno_Czech_Republic--Figure18.jpg "results-img-18")
**Figure 18: PCR verification of successful
integration of construct D intochromosome of *B.
subtilis* 168. M is marker - GeneRuler 1 kb
(ThermoFisher Scientific).**Primers 28 and 29 were
used for samples D1 and D2. The size of the product
should be 6511 bp. Amplicon of this size is only
present in sample D1. Positive control (PC) is a sample
of *B. subtilis* with plasmid pDG1664 inserted in
its chromosome. NC is a negative control.
Electrophoresis conditions: 1 % agarose gel, 120V, 40
min.

![](https://static.igem.org/mediawiki/2021/0/0a/T--Brno_Czech_Republic--Figure19.jpg "results-img-19")
**Figure 19: PCR verification of successful
integration of construct C intothe chromosome of *B.
subtilis* 168. PCR products amplified from the B.
subtilis\_C chromosome were loaded on the
gel.**Primers 44 and 46 were used to amplify the
region of transition between the chromosome and the
insert. Vector pDG3661\_C was used as a positive control
with primers 52 and 53. The size of the amplicons
(1117bp) confirms the presence of the insert in the
chromosome of *B. subtilis* . The amplicon of the
positive control is also of the correct size - 900 bp.
The same samples were then extracted from the gel and
sent for sequencing (see below). NC = negative control.
M is marker - GeneRuler 1 kb (ThermoFisher Scientific).
Electrophoresis conditions: 1 % agarose gel, 120V, 40
min.

### Proof by AmyE test

Another proof of successful integration of
plasmid pDG3661 into the *amyE* region of the
chromosome of *B. subtilis* 168 is the AmyE test. In
the case of construct B, the insert is not integrated into
*amyE* region but into thrC so AmyE test would not
work. More details about this experiment can be found in
the section Wet Lab.

The ability of transformed cells to digest
starch is compared with cultures of unmodified *B.
subtilis* 168 strains. The cells are grown on plates
containing starch. After some time of incubation, iodine is
added onto the plates. If the cells are able to metabolize
starch, iodine will not stain the solid medium in their
proximity, forming a sort of halo. As the *amyE* gene
which is necessary for starch digestion is inactivated by
the insertion of pDG3661 into its sequence, no halo should
be present in cell cultures with successfully integrated
pDG3661\_C and pDG3661\_D. The AmyE test confirmed successful
integration in all tested colonies (see Figure 20).

![](https://static.igem.org/mediawiki/2021/8/8e/T--Brno_Czech_Republic--Figure20.jpg "results-img-20")
**Figure 20: AmyE tests. 1.: Unmodified *B. subtilis*
- negative control, 2: B. subtilis\_D 3: *B.
subtilis\_C* .**Typical halo around the lawn of
unmodified B. subtilis can be seen on starch plate 1,
demonstrating the functionality of *amyE* gene of our
negative control. On starch plates number 2 and 3, no halo
can be seen around the lawn of *B. subtilis* . This
demonstrates successful insertion of pDG3661\_C and
pDG3661\_D into *amyE* locus responsible for starch
digestion.

### Proof by sequencing

Firstly, the chromosomal DNA of *B.
subtilis\_B*, *B. subtilis\_C* and B. subtilis\_D had
to be isolated using the previously mentioned “Physical
method” which utilises extreme temperatures to break the
cell wall.

As our constructs are rather long, we decided
to only send the transition between the chromosome and the
part of the integrated plasmid and the transition between
the integrated part of the plasmid and our construct for
sequencing as these sequences were crucial for
demonstrating successful integration. The selected
sequences were first amplified by long PCR. The PCR product
was then separated from the rest of the chromosomal DNA on
agarose gel DNA. The correct amplicon was extracted from
the gel and the correct amount of DNA with a corresponding
primer (see [Table of primers](https://static.igem.org/mediawiki/2021/3/38/T--Brno_Czech_Republic--Table_of_primers.pdf)) was sent for sequencing.

In the case of B.subtilis\_D we used primers 44
(transition from chromosome to plasmid) and 52 (transition
from plasmid to construct D), for B we used 46 (transition
from chromosome to plasmid) and 51 (transition from B to
plasmid) and for C we used 47 and 44. We prepared samples
according to the instructions from [Eurofins
corporation](https://eurofinsgenomics.eu/media/1587670/web_tubeseq_samplesubmissionguide_dinlang_final.pdf).

The alignments of our sequences can be found
here:

[B.subtilis\_D\_Primer44](https://static.igem.org/mediawiki/2021/c/c7/T--Brno_Czech_Republic--Primers44.pdf) ,[B.subtilis\_D\_Primer52](https://static.igem.org/mediawiki/2021/5/5a/T--Brno_Czech_Republic--B.subtilis_D_Primer52-alignment.pdf)

[B.subtilis\_C\_Primer44](https://static.igem.org/mediawiki/2021/a/ae/T--Brno_Czech_Republic--B.subtilis_C_Primer44-alignment.pdf) ,[B.subtilis\_C\_Primer47](https://static.igem.org/mediawiki/2021/7/74/T--Brno_Czech_Republic--B.subtilis_C_primer47-alignment.pdf)

[B.subtilis\_B\_Primer46](https://static.igem.org/mediawiki/2021/c/c2/T--Brno_Czech_Republic--B.subtilis_B_primer46-alignment.pdf) ,[B.subtilis\_B\_Primer51](https://static.igem.org/mediawiki/2021/9/94/T--Brno_Czech_Republic--B.subtilis_B_primer51-alignment.pdf)

The integration of constructs B, C and D into
the chromosome of *B. subtilis* 168 was successfully
demonstrated by sequencing. The schemes below show
sequenced parts of integration regions of *B.
subtilis* chromosome - B. subtilis\_B (Figure 21), *B.
subtilis\_C* (Figure 22) and *B. subtilis\_D* (Figure
23).

![](https://static.igem.org/mediawiki/2021/2/26/T--Brno_Czech_Republic--sekvenace-konstrukt-B.png "results-img-21")
**Figure 21: Scheme of sequenced part of integration
region of *B.subtilis\_B* chromosome.**

![](https://static.igem.org/mediawiki/2021/8/8e/T--Brno_Czech_Republic--sekvenace-konstrukt-C.png "results-img-22")
**Figure 22: Scheme of sequenced part of integration
region of *B.subtilis\_C* chromosome.**

![](https://static.igem.org/mediawiki/2021/0/01/T--Brno_Czech_Republic--sekvenace-konstrukt-D.png "results-img-23")
**Figure 23: Scheme of sequenced part of integration
region of *B.subtilis\_D* chromosome.**

# Characterization of constructs B and D

## Measurement of expression of fluorescent proteins

In our PHOSCAGE project, four gene constructs
- A, B, C and D - were designed. Following experiments were
performed with constructs B and D.

Construct B contains a gene encoding
*sfGFP* (Superfold GFP) with a PduP tag that should
ensure its encapsulation into BMCs (production of which is
governed by construct A).

Construct D is responsible for the expression
of cI repressor and mScarlet-I with a degradation tag upon
IPTG induction. In the absence of IPTG, GFP with a
degradation tag is expressed in turn.

More detailed information about our constructs
can be found in Parts and Project design.

### Experimental design

After successful transformation of *E.
coli* and *B. subtilis* , we designed the following
experiments to demonstrate the functionality of the
different components in our project. We included a reporter
system in our design to more easily verify and visualize
the functionality of each component of the system.

First, we determined the growth curve for each
culture of *E. coli* and B. subtilis recombinants from
regular measurements of OD600. We tested how the system
responds to the presence of IPTG in the culture of the
expression strain *E.coli* BL21(DE3). In *B.
subtilis* cells, we presumed that IPTG inducible
promoters would behave as constitutive.

We used several different methods to detect
the presence of the reporter proteins GFP and mScarlet-I.
We used a Tecan Infinite 200Pro plate spectrophotometer for
simultaneous and continuous measurement of both OD600 and
fluorescence of the cultures. Additionally, we used
FluoroMax Plus from Horiba Scientific for more sensitive
measurement of fluorescence of B. subtilis and *E.
coli* cells in cuvettes in end point mode. We then
verified the results obtained using these two techniques by
fluorescence microscopy.

All the experiments were performed with the
same cultures. The experimental design was complex and more
details about the cultures used, courses of the cultivation
and more can be found in this table: Table of samples for
measurement. Following pictures (Figures 24, 25 and 26)
were taken during sample preparation.

![](https://static.igem.org/mediawiki/2021/4/4d/T--Brno_Czech_Republic--Results-figure24.jpeg "results-img-24")**Figure 24: Presence of fluorescent
protein in liquid cultures of strainscarrying
constructs B and D. Cultures of *E. coli*
BL21(DE3) transformed with constructs B (left flask)
and D (two flasks on the right).**The presence of
matured fluorescent protein is clearly visible after
prolonged cultivation even to the naked eye.

![](https://static.igem.org/mediawiki/2021/1/1a/T--Brno_Czech_Republic--Results-figure25.jpeg "results-img-25")**Figure 25 : Liquid cultures of
strains carrying constructs B and D under UV light.**
After prolonged cultivation, cultures containing our
constructs were placed under UV light. The culture of
*E. coli* *BL21(DE3)\_pUCIDT\_D* on the bottom
left shows bright pink fluorescence. The culture at the
bottom right contains *B. subtilis* strain from
Dr. Krásný which was transformed with the *gfp*
gene using plasmids. This culture glows with hints of
green fluorescence. We planned on using this as a
positive control, but ended up using a different strain
instead. The two cultures at the back are the negative
controls - *B. subtilis* 168 and *E. coli*
DH5𝛂 which do not carry any genes encoding fluorescent
proteins.

![](https://static.igem.org/mediawiki/2021/1/1f/T--Brno_Czech_Republic--Results-figure26a.jpeg "results-img-26")

![](https://static.igem.org/mediawiki/2021/b/b2/T--Brno_Czech_Republic--Results-figure26b.jpeg "results-img-26-b")

**Figure 26: BL21(DE3) colonies of strains carrying
constructs B and D onplates under UV
light.**BL21(DE3) colonies with strains transformed
with our constructs were streaked on LBA plates with
corresponding antibiotics. The colonies displaying
green fluorescence on the left are of the strain
*BL21(DE3)\_pUCIDT\_B* . The plate below is the
BL21(DE3) control which was not transformed with any of
our constructs. On the right are
*BL21(DE3)\_pUCIDT\_D* colonies, glowing bright pink
due to mScarlet-I expression. The plate below is the
control. Plates were incubated for 16 hours at 37
͒C.

**As demonstrated in figures 24 and 25, the
presence of fluorescent proteinsin the cells carrying our
constructs is visible to the naked eye both under UV light
and daylight after prolonged cultivation. This observation
corresponds to the results obtained from further
measurements.**

**BL21(DE3) cells depicted in figure 26 were
plated on petri dishes with LBmedium and appropriate
antibiotics. Unlike the controls (two plates at the
bottom), the colonies carrying construct B (on the left)
and D (on the right) showed fluorescence of corresponding
colour.**

### 1. Continuous measurement of OD and fluorescence in liquid cultures with recombinant Bacillus or E. coli using Tecan spectrophotometer.

This device is able to simultaneously measure
the fluorescence of two different fluorescent proteins as
well as the OD of the culture, so the data obtained can be
analyzed together. The parameters were measured in cultures
incubated in a microtiter plate.

For a more detailed description, see the
section Characterization of construct B and D on the
page [Wet
lab](https://2021.igem.org/Team%3ABrno_Czech_Republic/Experiments). Sample selection and preparation can be found in
the following table: Table of samples for measurement.

Following measurements were all conducted
during one run of the experiment in the Tecan
spectrophotometer. Figure 27 shows the growth curves of E.
coli BL21(DE3) strains carrying constructs B or D as well
as controls that were not transformed with our constructs.
Some of the cultures were induced with IPTG during
inoculation, some were induced at OD 0.6 and some were not
induced.

![](https://static.igem.org/mediawiki/2021/7/72/T--Brno_Czech_Republic--Figure27.jpg "results-img-27")
**Figure 27: Growth curves of *E. coli* BL21(DE3)
cultures used in following experiments.**The graph shows
the growth curves of selected strains of *Escherichia
coli* BL21(DE3). The different strains are labeled in
figure legend. The designation "\_pUCIDT\_B/D" signifies the
presence of our constructs in pUCIDT plasmid. The label
"IPTG" indicates the presence of the inducing agent and the
label "0.6 / overnight" indicates the point of induction.
The black curve is the measurement of the blank.The blue
dashed line indicates the time of induction at OD
0.6.

**The decrease in growth rate of cultures
after induction indicates a slighttoxic effect of IPTG.
Even the control culture which was not transformed with our
construct and was induced overnight ( *BL21(DE3) IPTG
overnight* ) was negatively affected by IPTG as it grew
slower compared to control BL21(DE3) without IPTG where
IPTG was not added.**

**Interestingly, the growth curve of strains
with construct B - *BL21(DE3)\_pUCIDT\_B IPTG overnight*
and *BL21(DE3)\_pUCIDT\_B IPTG 0.6* did not show
significant differences after induction compared to the
cultures of cells carrying construct B which were not
induced. The growth curve of these cultures was similar to
the controls. This could imply that the expression of
construct B does not pose a metabolic burden on the
BL21(DE3) cells.**

**The presence of construct D, on the other
hand, poses a great metabolicburden on the BL21(DE3) cells.
The lag phase of cultures transformed with construct D was
prolonged by more than 1 hour.**

Figure 28 shows the growth curve of *B.
subtilis* strains used in following experiments. We
analyzed strains containing constructs B or D, negative
controls which were not transformed with our constructs and
also a positive control with GFP gene integrated in its
chromosome. The positive control with integrated GFP gene
was induced at the same time as BL21(DE3) cultures as both
experiments were performed simultaneously.

![](https://static.igem.org/mediawiki/2021/6/69/T--Brno_Czech_Republic--Figure285.jpg "results-img-28")
**Figure 28: Growth curves of *B. subtilis* 168
cultures used in following experiments.**The graph shows
the growth curves of selected strains of *Bacillus
subtilis*. The different strains are labeled in the
annotation of the graph. The label "B/D" signifies the
presence of our construct. The black curve is the
measurement of the blank (LB medium). The blue dashed line
indicates the time of xylose induction of *B. subtilis
positive control GFP in chromosome*.

**These results are in accordance with
predicted behaviour of selected B. subtilis cultures.
*Bacillus* with constructs B or D integrated into
their chromosome as well as cultures where only a part of
the empty plasmid was integrated grew more slowly in the
late growth phase.**

Simultaneously with OD, relative fluorescence
of these cultures was also measured. Figure 29 shows the
measurement of mScarlet-I fluorescence of E. coli BL21(DE3)
cultures in time. Measured were strains with construct D
inserted into their chromosome as well as control strains
with no integration - *BL21(DE3) without IPTG* and
*BL21(DE3) IPTG overnight* . Some samples were induced
with IPTG already at inoculation and one was induced at OD
0.6. The exact instrument settings for this type of
measurement can be found in the section: Wet lab,
Characterization of construct B and D.

![](https://static.igem.org/mediawiki/2021/f/f0/T--Brno_Czech_Republic--Figure28.jpg "results-img-29")
**Figure 29: Fluorescence of mScarlet-I in cultures of
*E.coli* BL21(DE3) strains carrying construct D and
negative controls measured in time.**The graph displays
the fluorescence values in Relative Fluorescence Unit
(RFU) of mScarlet-I of selected strains of *Escherichia
coli* BL21(DE3) measured in time. The different
strains are labeled in the annotation of the graph. The
label "IPTG" indicates the presence of the inducing agent
and the label "0.6/overnight" indicates the time of
induction. The black curve is the measurement of the
blank.The blue dashed line indicates the time of
induction.

**No increase in fluorescence was detected in
the blank nor in the negativecontrols. The results show an
increase in fluorescence of strains with integrated
construct D when compared to the controls. Sharp increase
in fluorescence was however recorded only after a longer
period of time. IPTG induction at OD 0.6 had no effect on
the intensity of fluorescence. The increase in intensity of
fluorescence of the *BL21(DE3)\_pUCIDT\_D IPTG
overnight* was delayed.**

**Unfortunately, we did not have the time to
optimize the settings of GFPfluorescence measurement in the
Tecan fluorometer and thus the results we obtained so far
were inconclusive and are not presented here.**

### 2. Fluorescence measurements in FluoroMax Plus and fluorescence microscopy of Bacillus or E. coli recombinants

Spectrofluorimetry was used to confirm correct
folding and expression of fluorescent proteins in
recombinant strains cultured in liquid media. Horiba
Scientific FluoroMax Plus can very accurately measure the
emission spectra of our fluorescent proteins. Range of
measured spectra displayed in the figures below cover the
emission peaks of GFP and mScarlet-I.

Other device specifications are listed on the
page [Wet
lab](https://2021.igem.org/Team%3ABrno_Czech_Republic/Experiments) in the section Characterization of project design
in case of construct B and D.

#### a) The effects of IPTG induction on fluorescence intensity in E.coli BL21(DE3)

As mentioned above, we decided to insert our
plasmid constructs into E. coli expression cells to
demonstrate the effect of IPTG induction on the expression
of proteins from IPTG inducible promoters in constructs B
and D.

We used two strains of *E. coli*
BL21(DE3) which were not transformed with our constructs as
negative controls. One of the negative controls and strains
carrying plasmids with our constructs were induced at OD =
0.8 immediately after diluting overnight cultures to reach
this OD. Fluorescence was measured 4 hours after induction
with IPTG.

Samples from cultures of strains containing
our constructs were also analyzed using fluorescence
microscopy. Further details of the work with the
fluorescence microscope can be found here: [Wet
lab](https://2021.igem.org/Team%3ABrno_Czech_Republic/Experiments), Characterization of construct B and D.

##### Analysis of GFP emission spectra of E.coli BL21(DE3)\_pUCIDT\_B

![](https://static.igem.org/mediawiki/2021/0/09/T--Brno_Czech_Republic--Results-figure30.png "results-img-30")
**Figure 30: GFP emission spectra measured in cultures
of *E.coli* BL21(DE3) strains carrying construct B
and negative controls.**This graph shows the emission
spectra of GFP in *E.coli* BL21(DE3) strains.
Different samples analyzed in the experiment are labeled
in the annotation of the graph. Two samples are negative
controls (one of which was induced by IPTG at OD 0.8).
The other two samples come from cultures of strains
containing our construct B (one of which was induced by
IPTG at OD 0.8). The scattered peaks in the left portion
of the graph which do not correspond to the emission
spectrum of GFP are the effects of fluorescence signal
scattering on insoluble structures and overall
heterogeneity of the measured sample. This scattering may
occur during the work with cell suspensions. The green
line indicates the emission maximum of GFP at 510
nm.

**The presence of a typical GFP emission
profile and the increase influorescence intensity compared
to the negative controls confirm the presence of construct
B in *E.coli* BL21(DE3) as well as the expression and
correct function of GFP encoded by this construct.**

**There is however no significant difference
between the fluorescenceintensity of
*BL21(DE3)\_pUCIDT\_B* cultures with and without IPTG
induction. This suggests that IPTG induction has no effect
on the expression of target proteins.**

##### Fluorescence microscopy of induced cultures:

##### Fluorescence microscopy of not-induced cultures:

![](https://static.igem.org/mediawiki/2021/1/19/T--Brno_Czech_Republic--Results-figure31.png "results-img-31")**Figure 31: Induced *E. coli*
BL21(DE3) cells transformed with construct B under
fluorescence microscope.**Images of native unfixed
samples of *E. coli*cultures carrying construct B
taken with a fluorescence microscope. This image shows
*BL21(DE3)\_pUCIDT\_B 2xIPTG* which was induced with
double concentration of IPTG. The image on the left is
a brightfield image, the image in the middle was taken
with a GFP filter and on the right is the overlay of
the two previous images. Immersion oil was used. M =
52x

![](https://static.igem.org/mediawiki/2021/4/48/T--Brno_Czech_Republic--Results-figure32.png "results-img-32")**Figure 32: Uninduced *E. coli*
BL21(DE3) cells transformed with construct B under
fluorescence microscope.**Images of native unfixed
samples of *E. coli*cultures carrying construct B
taken with a fluorescence microscope. This image shows
*BL21(DE3)\_pUCIDT\_B* 2xIPTG without IPTG
induction. The image on the left is a brightfield
image, the image in the middle was taken with a GFP
filter and on the right is the overlay of the two
previous images. M = 52x

**Observations made using fluorescence
microscopy correspond to the findingsfrom fluorescence
spectra measurements and confirm the expression and correct
folding of GFP in *E. coli* cultures transformed with
construct B. GFP was detected in similar quantities in both
induced and uninduced cultures of *BL21(DE3)\_pUCIDT\_B*
. Induction does not affect the expression of our target
protein.**

*The same experimental approach was used for
BL21(DE3)\_pUCIDT\_D cells.*

##### Analysis of GFP emission spectra of E.coli BL21(DE3)\_pUCIDT\_D

![](https://static.igem.org/mediawiki/2021/2/20/T--Brno_Czech_Republic--Results-figure33.png "results-img-33")**Figure 33: GFP emission spectra
measured in cultures of *E.coli* BL21(DE3) strains
carrying construct D and negative controls.**This
graph shows the emission spectra of GFP in
*E.coli* BL21(DE3) strains. Different samples
analyzed in the experiment are labeled in the
annotation of the graph. Two samples are negative
controls (one of which was induced by IPTG at OD 0.8).
The other two samples come from cultures of strains
containing our construct D (one of which was induced by
IPTG at OD 0.8). The scattered peaks in the left
portion of the graph which do not correspond to the
emission spectrum of GFP are the effects of
fluorescence signal scattering on insoluble structures
and overall heterogeneity of the measured sample. This
scattering may occur during the work with cell
suspensions. The green line indicates the emission
maximum of GFP at 510 nm.

##### Analysis of mScarlet-I emission spectra of E.coli BL21(DE3)\_pUCIDT\_D

![](https://static.igem.org/mediawiki/2021/a/a4/T--Brno_Czech_Republic--Results-figure34.png "results-img-34")**Figure 34: mScarlet-I emission
spectra measured in cultures of *E.coli*BL21(DE3)
strains carrying construct D and negative
controls.**This graph shows the emission spectra of
mScarlet-I in *E.coli* BL21(DE3) strains.
Different samples analyzed in the experiment are
labeled in the annotation of the graph. Two samples are
negative controls (one of which was induced by IPTG at
OD 0.8). The other two samples come from cultures of
strains containing our construct D (one of which was
induced by IPTG at OD 0.8). The scattered peaks in the
left portion of the graph which do not correspond to
the emission spectrum of mScarlet-I are the effects of
fluorescence signal scattering on insoluble structures
and overall heterogeneity of the measured sample. This
scattering may occur during the work with cell
suspensions. The green line indicates the emission
maximum of mScarlet-I at 510 nm.

**The presence of a typical GFP and
mScarlet-I emission profiles and theincrease in
fluorescence intensity compared to the controls confirmed
the presence of construct D in *E.coli BL21(DE3)* as
well as the expression and correct function of both
fluorescent proteins encoded in this construct.**

**The issue observed when measuring GFP
emission spectra of construct B ishowever also present
here. There is no significant difference between the
fluorescence intensity of *BL21(DE3)\_pUCIDT\_D*
cultures with and without IPTG induction. This suggests
that IPTG induction has no effect on the expression of
target proteins.**

**These findings indicate that mScarlet-I and
therefore also repressor cI areexpressed constitutively.
Repressor cI should bind to our modified promotor P
Grac-OcI and inhibit the expression of genes
downstream this promoter - namely *sfGFP* .
Significantly lower intensity of *sfGFP* emission
signal in samples of cultures of strains bearing construct
D compared to mScarlet-I and *sfGFP* in samples with
construct B might therefore suggest that our modified
promoter can be repressed by the cI repressor although some
leakage is present.**

**Further experiments would be helpful for
confirming these conclusions. Wewere unfortunately unable
to perform additional experiments due to the time
constraints.**

##### Fluorescence microscopy of uninduced cultures:

![](https://static.igem.org/mediawiki/2021/a/a1/T--Brno_Czech_Republic--Results-figure35.png "results-img-35")**Figure 35: Uninduced *E. coli*
BL21(DE3) cells transformed with construct D under
fluorescence microscope.**Images of native unfixed
samples of *E. coli* cultures carrying construct D
taken with a fluorescence microscope. This image shows
*BL21(DE3)\_pUCIDT\_D* without IPTG induction. The image
on the left is a brightfield image, the following image was
taken with a GFP filter, the next image to the right was
taken with mScarlet-I filter and on the right is the
overlay of the three previous images. Immersion oil was
used. M = 52x![](https://static.igem.org/mediawiki/2021/1/14/T--Brno_Czech_Republic--Results-figure36.png "results-img-36")**Figure 36: Uninduced *E. coli*
BL21(DE3) cells transformed with construct D under
fluorescence microscope.**Images of native unfixed
samples of *E. coli* cultures carrying construct D
taken with a fluorescence microscope. This image shows
*BL21(DE3)\_pUCIDT\_D* without IPTG induction. The image
on the left is a brightfield image, the following image was
taken with a GFP filter, the next image to the right was
taken with mScarlet-I filter and on the right is the
overlay of the three previous pictures. Immersion oil was
used. M = 88.2x

**These images from fluorescence microscope
confirm the expression andcorrect function of both GFP and
mScarlet-I even in the absence of IPTG in
*BL21(DE3)\_pUCIDT\_D* .**

**Observations made using fluorescence
microscopy correspond to the findingsfrom fluorescence
spectra measurements and confirm the expression and correct
function of both GFP and mScarlet-I in *E. coli*
cultures transformed with plasmid bearing construct D. Both
GFP and mScarlet-I were detected in similar quantities in
samples from induced and uninduced cultures of
*BL21(DE3)\_pUCIDT\_D* . Induction does not have any
effect on the expression of our target proteins.**

#### b) Optimization of IPTG induction in E.coli strain BL21(DE3)

As we did not observe any difference in the
expression of our target proteins in induced cultures
compared to cultures which were not induced, we decided to
try to optimize the IPTG concentration used for induction.
The final concentration used in previous experiments was
100 μM IPTG. We decided to also two-fold higher
concentration (200 μM IPTG). The emission spectra of
samples from cultures induced with this increased
concentration of IPTG were then analyzed using FluoroMax
Plus.

We prepared two negative controls from
cultures of strains which were not transformed with our
constructs. One of the negative controls was induced with
IPTG during inoculation. Samples *BL21(DE3)\_pUCIDT\_B/D
IPTG* were induced during inoculation. Samples
*BL21(DE3)\_pUCIDT\_B/D 2xIPTG* were firstly induced
with 100 μM IPTG during inoculation and a second dose of
IPTG was added 15 hours later. The emission spectra were
measured 4 hours after the second dose of IPTG.

##### Analysis of GFP emission spectra of E.coli BL21(DE3)\_pUCIDT\_B:

![](https://static.igem.org/mediawiki/2021/2/28/T--Brno_Czech_Republic--Results-figure37.png "results-img-37")**Figure 37: Comparison of GFP
emission spectra measured in cultures of *E.coli*
BL21(DE3) strains carrying construct B induced with
different concentrations of IPTG and negative
controls.**This graph shows the emission spectra of
GFP in *E.coli* BL21(DE3). Different samples
analyzed in the experiment are labeled in the
annotation of the graph. Two samples are negative
controls (one of which was induced by IPTG at
inoculation). The other two samples are cultures
containing construct B. Both of them were induced by
IPTG already at the beginning of cultivation. An
additional dose of IPTG was added to one of them after
15 hours. The scattered peaks in the left portion of
the graph which do not correspond to the emission
spectrum of GFP are the effects of fluorescence signal
scattering on insoluble structures and overall
heterogeneity of the measured sample. This scattering
is expected when working with cell suspensions. The
green line indicates the emission maximum of
GFP.

##### Analysis of GFP emission spectra of E.coli BL21(DE3)\_pUCIDT\_D:

![](https://static.igem.org/mediawiki/2021/3/3a/T--Brno_Czech_Republic--Results-figure38.png "results-img-38")**Figure 38: Comparison of GFP
emission spectra measured in cultures of *E.coli*
BL21(DE3) strains carrying construct D induced with
different concentrations of IPTG and negative
controls.**This graph shows the emission spectra of
GFP in *E.coli* BL21(DE3). Different samples
analyzed in the experiment are labeled in the
annotation of the graph. Two samples are negative
controls (one of which was induced by IPTG at
inoculation). The other two samples are cultures
containing construct D. Both of them were induced by
IPTG already at the beginning of cultivation. An
additional dose of IPTG was added to one of them after
15 hours. The scattered peaks in the left portion of
the graph which do not correspond to the emission
spectrum of GFP are the effects of fluorescence signal
scattering on insoluble structures and overall
heterogeneity of the measured sample. This scattering
is expected when working with cell suspensions. The
green line indicates the emission maximum of
GFP.

##### Analysis of mScarlet-I emission spectra of E.coli BL21(DE3)\_pUCIDT\_D:

![](https://static.igem.org/mediawiki/2021/c/c9/T--Brno_Czech_Republic--Results-figure39.png "results-img-39")
**Figure 39: Comparison of mScarlet-I emission spectra
measured in cultures of *E.coli* BL21(DE3) strains
carrying construct D induced with different
concentrations of IPTG and negative controls.**This
graph shows the emission spectra of mScarlet-I in
*E.coli* BL21(DE3). Different samples analyzed in
the experiment are labeled in the annotation of the
graph. Two samples are negative controls (one of which
was induced by IPTG at inoculation). The other two
samples are cultures containing construct D. Both of them
were induced by IPTG at inoculation. An additional dose
of IPTG was added to one of them after 15 hours. The
scattered peaks in the left portion of the graph which do
not correspond to the emission spectrum of mScarlet-I are
the effects of fluorescence signal scattering on
insoluble structures and overall heterogeneity of the
measured sample. This scattering is expected when working
with cell suspensions. The red line indicates the
emission maximum of mScarlet-I.

**These measurements demonstrate that in both
construct B and D, theexpression of both GFP and mScarlet-I
is not influenced by the increase in IPTG
concentration.**

#### c) Measurement of fluorescence spectra in Bacillus subtilis

The emission spectra of our target fluorescent
proteins were also measured in samples of cultures with
*B. subtilis* strains bearing constructs B or D
integrated into their chromosome. In the case of
chromosomal integration, only one copy of the construct is
present and therefore lower intensity of fluorescent signal
then in *E. coli* , transformed with multicopy
plasmids carrying our construct, can be expected. We used a
positive control kindly provided by Dr. Krásný's research
group - *B. subtilis* with GFP integrated in its
chromosome downstream of a xylose-inducible promoter.

Unfortunately, we were unable to obtain
positive *B. subtilis* control containing mScarlet-I
integrated in chromosome.

B.subtilis\_B and B.subtilis\_D cultures were
also analyzed using fluorescent microscopy.

##### Analysis of GFP emission spectra of B. subtilis\_B:

![](https://static.igem.org/mediawiki/2021/0/0d/T--Brno_Czech_Republic--Figure40.jpg "results-img-40")**Figure 40: GFP emission spectra
measured in cultures of *B. subtilis* 168 strains
carrying construct B and negative controls.**This
graph shows the emission spectra of GFP produced in
B.subtilis. Different samples analyzed in the
experiment are labeled in the annotation of the graph.
The positive control has GFP gene integrated in its
chromosome and was kindly provided by Dr. Kásný. The
negative control is a strain of B.subtilis 168 without
construct B. The last sample is a culture with
construct B integrated into the chromosome. The
scattered peaks in the left portion of the graph which
do not correspond to the emission spectrum of GFP are
the effects of fluorescence signal scattering on
insoluble structures and overall heterogeneity of the
measured sample. This scattering is expected when
working with cell suspensions. The green line indicates
the emission maximum of GFP.

##### Analysis of GFP emission spectra of B. subtilis\_D:

![](https://static.igem.org/mediawiki/2021/1/1c/T--Brno_Czech_Republic--Figure41.jpg "results-img-41")**Figure 41: GFP emission spectra
measured in cultures of *B. subtilis* 168 strains
carrying construct D and negative controls.**This
graph shows the emission spectra of GFP in B.subtilis.
Different samples analyzed in the experiment are
labeled in the annotation of the graph. The positive
control has GFP gene integrated in its chromosome and
was kindly provided by Dr. Kásný. The negative control
is a strain of B.subtilis 168 without construct D. The
last sample is a culture with construct D integrated
into the chromosome. The scattered peaks in the left
portion of the graph which do not correspond to the
emission spectrum of GFP are the effects of
fluorescence signal scattering on insoluble structures
and overall heterogeneity of the measured sample. This
scattering is expected when working with cell
suspensions. The green line indicates the emission
maximum of GFP.

##### Analysis of mScarlet-I emission spectra of B. subtilis\_D:

![](https://static.igem.org/mediawiki/2021/3/31/T--Brno_Czech_Republic--A5.jpg "results-img-42")
**Figure 42: mScarlet-I emission spectra measured in
cultures of *B. subtilis*168 strains carrying
construct D and negative controls.**This graph shows
the emission spectra of mScarlet-I in B.subtilis.
Different samples analyzed in the experiment are
labeled in the annotation of the graph. The negative
control is a strain of B.subtilis 168 without construct
D. The last sample is a culture with construct D
integrated into the chromosome. The scattered peaks in
the left portion of the graph which do not correspond
to the emission spectrum of mScarlet-I are the effects
of fluorescence signal scattering on insoluble
structures and overall heterogeneity of the measured
sample. This scattering is expected when working with
cell suspensions. The red line indicates the emission
maximum of mScarlet-I.

##### Fluorescence microscopy of B. subtilis\_B and B. subtilis\_D cultures:

![](https://static.igem.org/mediawiki/2021/d/da/T--Brno_Czech_Republic--Results-figure43.png "results-img-43")
**Figure 43: Negative control - *B. subtilis* 168
cells with no insert in its chromosome - under
fluorescence microscope.**Images of native unfixed
samples of B. subtilis taken with a fluorescence
microscope. This image shows a culture of *B.
subtilis* 168 with no insertion in it's chromosome.
The image on the left is a brightfield image, the image
in the middle was taken with a GFP filter and on the
right is the overlay of the two. Immersion oil was
used. M = 52x

![](https://static.igem.org/mediawiki/2021/a/a5/T--Brno_Czech_Republic--Results-figure44.png "results-img-44")**Figure 44: *B. subtilis* 168
cells transformed with construct B under fluorescence
microscope.**Images of native unfixed samples of
*B. subtilis* taken with a fluorescence
microscope. This image shows *B. subtilis\_B* . The
image on the left is a brightfield image, the image in
the middle was taken with a GFP filter and on the right
is the overlay of the two. Immersion oil was used. M =
52x

![](https://static.igem.org/mediawiki/2021/4/42/T--Brno_Czech_Republic--Results-figure45.png "results-img-45")**Figure 45: *B. subtilis* 168
cells transformed with construct D under fluorescence
microscope.**Images of native unfixed samples of
*B. subtilis* taken with a fluorescence
microscope. This image shows *B. subtilis\_D* . The
image on the left is a brightfield image, the following
image was taken with a GFP filter, the next image to
the right was taken with a mScarlet-I filter and on the
right is the overlay of the three. Immersion oil was
used. M = 52x

**Analysis of GFP emission spectra of *B.
subtilis\_B*using FluoroMax Plus confirmed the expression
and correct function of *sfGFP* . The emission profile
is comparable to the positive control provided by Dr.
Krásný, which also has a GFP gene inserted into its
chromosome.**

**Analysis of emission spectra of both of our
target fluorescent proteins in *B. subtilis\_D* using
FluoroMax Plus show the absence of a typical emission
profile or significant increase in fluorescence intensity
for both GFP and mScarlet-I compared to the negative
control. This indicates that there are no or very little
fluorescent proteins produced by *B. subtilis\_D*
cells.**

**Observations made using fluorescence
microscopy however indicate a presenceof small fluorescent
signals of GFP in *B. subtilis\_B* and GFP and
mScarlet-I in *B. subtilis\_D* . There was no
fluorescence detected in the negative control, so the
signals from *B. subtilis\_B* and *B. subtilis\_D*
should be the result of integration of our constructs in
bacterial chromosome.**

**To confirm expression of *sfGFP* and
mScarlet-I genes integrated in the B. subtilis\_D
chromosome, the spectrum analysis would need to be
optimized.**

# Conclusion

To conclude everything, we would like to
briefly summarize the successes achieved in the wet lab. We
have managed to transform *E. coli* strains JM109 and
BL21(DE3) with three out of four constructs (namely
constructs B, C and D) and we achieved the same success
with *B. subtilis* . We have verified the cloning and
transformation of our constructs using restriction
digestion, PCR and sequencing. We were able to demonstrate
the expression of fluorescence proteins from constructs B
and D, both of which proved to be functional under the
fluorescence microscope in *B. subtilis* . Last but
not least, we were able to figure out how to bipas the
issues with the synthesis of our construct A by using
Golden Gate assembly (which is not included in the Result
section - due to lack of time to perform this experiment
until wiki freeze).

# What could be improved?

Because we know failures are part of every
successful experiment, and our project was no exception, we
want to talk about them and share the lessons we learned
from our mistakes. In order to correct one's failings, one
must first recognise them. When subcloning our constructs
from the original pUCIDT plasmids into the pDG destination
vectors, we naively choose the same selection marker for
both vectors (resistance to ampicillin). This flaw in our
experimental design made successful subcloning into the
shuttle vector very difficult, especially in the case of
construct C, as we were unable to distinguish colonies
containing successfully ligated pDG vectors with our
construct from colonies contaminated with pUCIDT.

Another important lesson was learned at the
end of our experimental work. After verifying the
expression of functional fluorescence proteins from our
constructs, we still need to perform more experiments as
well optimize some which did not work as expected to prove
the function of our system as a whole. Unfortunately, we
did not manage to do this due to time constraints. For
future reference, we will know that a longer period of time
should be set aside for measurements and optimization of
characterization of each construct.

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Igem Team Brno, Czech Republic 2021