iGEM 2025 — Team chalmers-gothenburg

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}This serves as a chronological record of your team's progress throughout the season. It documents your
daily activities, experiments, discussions, and decisions.{% endblock %}
{% block page\_content %}

![hyphae](https://static.igem.wiki/teams/5796/custom/hyphae/hyphae-tiling5.webp)

Below are the wetlab notes for our project, from the design process in June to the final results in September.

### June

---

Weeks 24-26

We designed and ordered our parts in june, and they didn't arrive until the end of the month. Since we
couldn't
make any progress on the wet-lab during this month, more focus was placed on research into expressing
more
chitin in *S. cerevisiae* as well as the dry-lab.

### July

---

Week 27

**1/7**
The ordered fragments arrived in powder form and were prepared to reach a concentration of 20ng/µL.
The *E. coli* strain containing our entry vector, pYTK095\*, was inoculated in LB media with
supplemented
chloramphenicol in preparation for extraction.

**2/7**
MoClo assembly was done on the ordered fragments in accordance with  [protocol](./experiments#MoClo). The two constructs we
had designed were assembled in parallel, one tube for the CHI92 gene and one for the SP1 gene. The
resulting plasmids
were transformed into competent *E. coli* using
[heat shock](./experiments#heat-ecoli), which were then plated on agar plates with LB and
chloramphenicol. The entry vector was extracted from the liquid culture using the
[GeneJET protocol](./experiments#extraction-GeneJET).

**3/7**
The plates from the previous day were green-white screened and were shown to only contain white
colonies.
This could suggest that the MoClo and transformation were both entirely successful, but it is more
likely that
something went wrong. 3 colonies were harvested and placed in liquid culture to confirm.

**4/7**
*Plasmid extraction*
Plasmids from the inoculated cells were extracted using the GeneJET miniprep Kit with the
 [plasmid extraction protocol](./experiments#extraction-GeneJET). Only 1.5mL of the liquid was used, which will be changed to all 5mL in the future to obtain
higher plasmid concentration.
The plasmid concentration was analyzed with a NanoDrop and one specimen was discarded due to low
concentration. The remaining were combined with a primer provided by a supervisor and sent for
sequencing.
Four new colonies from the same CHI92 and SP1 plates were inoculated and will be extracted after the
weekend.

Week 28

This week, we adjusted some of our methods

* The restriction enzymes BsmBI and BcuI were added to the MoClo reaction. Both the ordered
  vector and pYTK095\* have the same resistance gene. By letting the enzymes cut into the
  ordered vector we are reducing the likelihood that cells without our gene of interest
  survive
* Instead of extracting plasmids outright, we do colony PCR.
* We scaled up the MoClo reactions to 30µL

**7/7**
A new MoClo assembly was done according tho  [protocol](./experiments#MoClo). Both of the genes were done in parallel once
again. The assembled plasmids were transformed into *E. coli* which were plated and set to
incubate overnight.

**8/7**
Colony PCR was done on the *E. coli* colonies from yesterday using [colony PCR](./experiments#PCR-colony). A 1% agarose gel was casted in preparation for [gel electrophoresis](./experiments#Gel-electrophoresis). The gel ran clear, indicating
an error in our design.
After investigation, it appears like there was a BcuI cut site in the finished construct.
Another error might be the primer design. This left us with three options, and we decided to move
ahead with all three.

* Order everything again, but this time as a complete construct ready to put into the
  backbone
* Inoculate colonies, extract the plasmids and hope for the best
* Re-do the MoClo without BcuI, but with BsmBI and BsaI

**9/7**
*MoClo assembly and transformation*
A new MoClo assembly was done according to  [protocol](./experiments#MoClo). Both of the genes were done in parallel once
again. The assembled plasmids were transformed into *E. coli* which were set to incubate over
night.
After the thermocycler finished the plasmids were transformed into *E. coli* using
[heat shock](./experiments#heat-ecoli). Two plates for each construct
were made, one for a lower concentration of cells and one for
a higher concentrations. The plates were left to incubate overnight.

*Plasmid extraction*
The tubes that were inoculated yesterday were collected and their plasmids extracted in accordance
with the [GeneJET protocol](./experiments#extraction-GeneJET).
The resulting concentrations were high enough for five of the
tubes, which were diluted to more usable concentrations and placed in the freezer for storage.

**10/7**
*Result of yesterday's MoClo*
The colonies were screened with UV light. From the high concentration plate, one colony containing
SP1 was white and 7 containing CHI92 were white. Of the colonies on the low concentration plates,
none were visible.

*Colony PCR*
Colony PCR was done on the white *E. coli* colonies using  [colony PCR protocol](./experiments#PCR-colony) and Saphire polymerase. A
template-negative control was included.
*Gel electrophoresis*
The product from the colony PCR was run through a
[gel](./experiments#Gel-electrophoresis) which resulted in no bands, indicating an
unsuccessful PCR. The PCR will be redone with other primers.

**11/7**
*Colony PCR*
A new round of colony PCR was done with colonies both from the day before and from this day.
The [protocol](./experiments#PCR-colony) was modified in the following ways:

* Used a new master mix called "Phire Master Mix"
* The template DNA was boiled with the CTREAT program

*Gel electrophoresis*
The [gel electrophoresis](./experiments#Gel-electrophoresis) showed
bands for two of the tested colonies. One colony with CHI92 showed a
band at 1500bp, which suggested a successful transformation. One colony with SP1 also showed a band,
but at around 4000bp which was not expected. The remaining cells from both of these colonies were
inoculated for further testing.

Week 29

**14/7**

*Result of last week's inoculation of Chi92*
The inoculation of colonies from Friday showed bacterial growth of the Sp1 protein, but not the
Chi92. It is likely that the remaining colony was too small to survive. As a last resort, the area
of the colony was swabbed and inoculated. The remaining liquid media with cells transformed with
CHI92
were given fresh media and inoculated further. If this fails the MoClo will have to be repeated.
*Investigation of the SP1 transformation*
We did a [plasmid extraction](./experiments#extraction-GeneJET) and
a [GeneJet plasmid purification](./experiments#purification-GeneJET)
of the colony that had a band at 4000bp yesterday. A
restriction enzyme digestion was run according to [protocol](./experiments#FastDigest)
with NotI which should result in two fragments. The
[gel electrophoresis protocol](./experiments#Gel-electrophoresis)
resulted in a single band at 4000bp again. This made it evident that the transformation had failed
and that the MoClo would have to be repeated for SP1 as well.

**15/7**
*B. subtilis transformation media*
A new culture medium for future transformation of plasmids into *B. subtilis* was prepared
using the
 [Schaeffer's medium protocol](./experiments#Medium-Schaeffer).
*Inoculation result*
The result from yesterday's inoculation of CHI92 cells showed zero growth, so the E.coli culture was
discarded.

**16/7**
Another round of Moclo Assemblies were done for both proteins individually using the
[MoClo protocol](./experiments#MoClo).

**17/7**
*B. subtilis* cells were inoculated in 5mL LB media and placed in 37°C over night in
preparation
for making a glycerol stock for future use.

**16/7**
The inoculated *B. subtilis* from yesterday were combined with glycerol diluted with MQ and
placed
in -80°C for long term storage.

Week 30

**25/7**
We received the packages, but the CHI92 construct had a very low concentration of ≤ 0.5ng.
To combat this, it was diluted with 10nL MQ amplified with PCR and then transformed into
*E. coli*. The PCR was done in accordance with the [PCR protocol](./experiments#PCR-Sapphire)
with a template-negative control and the transformation into *E. coli* was done using
[heat shock](./experiments#heat-ecoli).

**26/7**
The *E. coli* transformed with CHI92 had not grown, so the procedure was redone with
electroshock instead of heat shock according to the [electroporation
protocol](./experiments#electro-ecoli).

Week 31

**28/7**
*PCR of CHI92*
The previous PCRs have been done using [Sapphire](./experiments#PCR-Sapphire).
Since they have been unsuccessful we switched to
Phusion polymerase. A PCR with this polymerase was done on the CHI92 construct, trying both GC buffer
and HF buffer. The product was run through a
[gel](./experiments#Gel-electrophoresis) together with the Sapphire product from last week.
The result was positive, indicating a successful amplification of the plasmid.

*Transformation of CHI92*
The CHI92 plasmid was transformed into *E. coli* using [heat shock](./experiments#heat-ecoli).

**29/7**
*PCR of yeast genome*
The PCR of CHS3 from the yeast genome was redone today with 1min 45s extension time (calculated from
30s
per kilobases) and an annealing temperature of 62°C. The PCR product will be run on a gel
tomorrow.

*Plasmid extraction*
Plasmid extraction on the inoculated *E. coli* from yesterday was done according to the
[GeneJET protocol](./experiments#extraction-GeneJET).
The protocol was modified in the following ways:

* Incubated at room temperature for 10min after transferring the solution to the GeneJET spin
  column to increase plasmid yield.
* Used 30μL of MQ instead of 50μL Elution Buffer for eluting the plasmid, also to
  increase yield.

The plasmid concentrations were measured with a NanoDrop and ranged from 330ng/μL to
380ng/μL.

*Plasmid verification*
Restriction digestion was used to cut the plasmids using the
[FastDigest protocol](./experiments#FastDigest).
Two different restriction enzymes
were used: BsmBI (Eco31I) which should cut twice, and BcuI which should cut once.
A gel electrophoresis was done according to the
[gel electrophoresis protocol](./experiments#Gel-electrophoresis)
and the correct bands seemed to appear.
However they were pretty slanted and there were some extra fragments.
The gel will be rerun tomorrow at 80V for 45 minutes.

**30/7**
A new gel electrophoresis of the CHI92 plasmids was done at 80V for 45min. The result was much the
same as the previous day; BsmBI cut at two sites and BcuI cut at one site, which was expected. This
implies that the colonies we picked not only took up the antibiotic gene from the plasmid, but the
CHI92 gene as well.
![](https://static.igem.wiki/teams/5796/test/gel-v31-wednesday-5148.webp)

The PCR product from yesterday was collected and run through a
[gel](./experiments#Gel-electrophoresis). Negative (-Template) controls
were included.
All specimens, including the controls, seemed to have the same extremely short fragments. The
genomic CHS3 will have to be amplified again.

![](https://static.igem.wiki/teams/5796/test/gel-v31-wednesday-5153.webp)

**31/7**
*PCR pf CHS3 and verification*
The PCR of CHS3 from the yeast was redone with both Saphire, Phusion, and Phire polymerases.
Template-negative
controls were included for each polymerase. A [gel](./experiments#Gel-electrophoresis) was cast in preparation for electrophoresis. The
gel showed
a single band for the Saphire mix at about 3500bp which was the expected length. All of the other
columns were empty.

*SP1 transformation*
Plasmids containing SP1 were transformed into *E. coli* using
[heat shock](./experiments#heat-ecoli) which were left to grow
overnight in 37°C on selective plates.

**1/8**
*SP1 transformation*
The *E. coli* containing SP1 that were plated yesterday were placed in the fridge.

*CHS3 PCR purification*
The PCR product from the Saphire tube that resulted in a band yesterday was purified using the
[GeneJet purification protocol](./experiments#purification-GeneJET).
It was checked with [gel electrophoresis](./experiments#Gel-electrophoresis)
again and the same band at 3500bp appeared.

*CHS3 plasmid transformation*
The plasmid from the purification was inserted into the pYTK001 backbone using
[MoClo](./experiments#MoClo) (PART) and the
finished plasmid was transformed into *E. coli* using
[heat shock](./experiments#heat-ecoli). The cells were plated and left to grow in
room temperature
over the weekend.

### August

---

Week 32

**4/8**
We began the week by doing a colony PCR for the CHS3 gene both with the high and the low
concentrations using the  [colony PCR protocol](./experiments#PCR-colony).
It was realized later that the extension time we used was incorrect.
We still got bands in the
[gel electrophoresis](./experiments#Gel-electrophoresis)
at the length of our gene of interest for the high
concentration group. We still wanted to redo the PCR with the correct PROGRAM to enhance results.
![](https://static.igem.wiki/teams/5796/wetlab/chs3-4-8.webp)
Today we also picked colonies transformed with the SP1 plasmid from the plates that were made on
Thursday last week. They were inoculated in 5mL LB medium+kanamycin and incubated in a 37°C
shaking incubator overnight.

**5/8**
The failed colony PCR from yesterday will not be redone since the big size of the fragment may
cause problems. Instead, the colonies were inoculated in the hopes that they would work.
Tomorrow they will be checked with restriction digestion.
Plasmid extraction was done on the SP1 colonies using the
[GeneJET protocol](./experiments#extraction-GeneJET). These will be mutated to include
a stop codon since it had been lost when the gene was produced.

**6/8**
Plasmids carrying CHS3 were isolated from E. coli colonies using the
[GeneJET protocol](./experiments#extraction-GeneJET) and quantified
by NanoDrop (264.6-495.1 ng/μL). For construct verification, the plasmids were digested using
the [FastDigest protocol](./experiments#FastDigest) with
Green buffer. The Eco31I (BsaI) enzyme was predicted to yield three fragments and the SacI was
expected to only linearize the plasmid.
Samples (with negative controls for each clone) were run on a
[gel](./experiments#Gel-electrophoresis). As anticipated, BsaI produced three bands
and SacI a single band. Although slight lane waviness reduced readability, banding patterns were
consistent with the in-silico map.
![](https://static.igem.wiki/teams/5796/wetlab/chs3-6-8.webp)
After confirmation, we performed
[Mutation PCR](./experiments#PCR-mutation) to template DNA to remove
internal restriction sites using primers 003 and 004.

**7/8**
*CHS3 mutation*
A gel was casted according to
[protocol](./experiments#Gel-electrophoresis) and was used to
run the CHS3 plasmids that were mutated yesterday. It resulted in
bands at about 5kb as hoped, but the bands were hard to read. We proceeded with DpnI
[digestion](./experiments#FastDigest)
to remove the unmutated template plasmids and ran the product on another gel. This unfortunately resulted
in bands at 3kb instead of 5kb. The result of the NanoDrop was strange, and ranged from
-14.1ng/μL to 7.7ng/μL.
![](https://static.igem.wiki/teams/5796/wetlab/chs3-7-8.webp)
After some investigating, it was discovered that the wrong primers had been used to do the mutation.
They did not overlap and also lacked the desired mutation, leading to the 3kb bands in the gel.

*CHS3 update*
Despite the delay, we confirmed today that the CHS3 gene that we ordered a few weeks ago would
be delivered tomorrow. This means that MoClo can be done tomorrow before transforming it into
*E. coli* and no more mutation PCR has to be done on it.

*SP1 and CHI92 update*
On this day, it was also discovered that the SP1 and CHI92 constructs we had ordered lacked
stop codons. SP1 requires a point mutation, but CHI92 requires the insertion of an entire stop codon
which will impact the temperatures for the mutation PCR. We will probably do a gradient PCR for
CHI92
to determine the optimal temperature.

**8/8**
We received our package with the CHS3 gene, so we proceeded by doing a Moclo assembly according to the
[MoClo protocol](./experiments#MoClo). The parts can be viewed in the Engineering page.
This was followed by preparing plates for the plasmid
transformation into E.coli, one with
amplicilin and two with kanamycin resistance (high and low concentration) and heating them to
37°C.
To create the high concentration plate we simply span down the cell mixture and got rid of excess
liquid.
These plates were then inoculated during the weekend in room temperature.

Deviations from transformation protocol

Took two tubes with competent cells.
2. Pipetted 5μL of ligation reaction from the MoClo into the first tube (meant for MoClo
product and to be selected for with ampicillin). In the second tube, we pipetted 1μL
ligation reaction meant to be selected for with kanamycin.
6. Added 900μL LB-media at room temperature into each tube.
10. From the tube with ampicillin selection, we spread 50μL on an LB plate with ampicillin.
From the tube with kanamycin selection, we spread 50μL onto an LB plate with kanamycin.
11. To make plates with a higher cell concentration, the tubes were centrifuged at 10'000g for
30s and most of the supernatant was discarded. The pellet was resuspended in the remaining
supernatant and plated on the corresponding selection plates.

Week 33

**11/8**
Nothing had grown on the CHS3 plates from Friday and they were placed in the 37°C incubator for
a while.
To be safe, the MoClo assembly was redone using the same parts and
[protocol](./experiments#MoClo). as last time. The finished construct
was transformed into *E. coli* using [heat shock](./experiments#heat-ecoli)
and plated on LB+Amp plates and placed in the incubator overnight. Both a plate with a high and
a low cell concentration were made. The plates from Friday never grew anything.

**12/8**
*CHS3*
Colonies had grown on the plates from yesterday, both white and green ones. 4 colonies from each
of the two plates were picked and inoculated in liquid LB and ampicillin overnight.

*Stop codon mutation PCR*
[Mutation PCR](./experiments#PCR-mutation) was started in order
to re-introduce the lost stop codons in both CHI92 and SP1,
but two forward primers had accidentally been ordered instead of one forward and one reverse primer
for the SP1 gene. This was discovered only after the protocol was started, so it was continued
regardless. The primers were diluted to 1muM with sterile MQ and placed in the freezer for
future use.
Dilutions of the template DNA for CHI92 and SP1 were made to 1ng/muL and also placed in the
freezer.
A [gel electrophoresis](./experiments#Gel-electrophoresis)
was done to check the PCR product, with a template-negative control for
each of the genes. Both the ladder and the bands looked very odd and smudgy. There seemed to be
some primer dimers shorter than 250bp on the CHI92 wells and the control, which was expected.
There were none in the SP1 wells which is reasonable since the duplicate primers were not
expected to dimerize. This will be redone with the correct primers when they arrive.

**13/8**
The liquid E. coli culture with CHS3 which was inoculated yesterday was used today. Plasmid
extraction using the [GeneJET protocol](./experiments#extraction-GeneJET)
was done for each colony sample, 8 in total. To verify them,
restriction digestion was done with either PacI or Esp3I for all the samples. The digestion
was done with FastDigest enzymes using the
[FastDigest protocol](./experiments#FastDigest).
PacI should linearize the plasmids while Esp3I should cut twice, resulting in two fragments.
A [gel electrophoresis](./experiments#Gel-electrophoresis)
was run with the 16 samples and ladders on either side. The gel showed a
lot more cuts than initially expected. It was discovered that this was due to the use of the
wrong primers for gene amplification. The amplification was redone with the correct primers to
be checked the next day.
 ![](https://static.igem.wiki/teams/5796/wetlab/chs3-13-8.webp)

**14/8**
After testing a few different combinations of agarose concentration, voltage and time we landed
on 1% agarose run at 90V for 40 minutes for the CHS3 assembly. The gel resulted in the predicted
bands for some of the samples, and these were assembled into the backbone PLE\_YTK007 using
[MoClo](./experiments#MoClo).
The finished product was placed in the freezer.

**15/8**
*CHS3*
The MoClo products from the day before were transformed into *E. coli* and the cells were
plated on LB+Kanamycin plates after an inoculation period of 1h in liquid LB. They were placed
in the incubator overnight.

*Stop codon mutation PCR*
A [Mutation PCR](./experiments#PCR-mutation)
was done on CHI92 and SP1 to introduce the stop codons using Phusion polymerase
and an HF buffer with two template-negative controls, one for each gene. For CHI92 an entire
codon needed to be inserted, making the optimal annealing temperature unknown. Due to this, a
gradient PCR was done ranging from 53°C to 63°C with one tube of CHI92 for each temperature.
Since SP1 only required a single substitution it was run at 59.1°C annealing temperature,
which was the closest temperature we could get to the optimal using the gradient PCR. The
extension time was calculated as 25s/kb. The success of the PCR will be checked next week.

Week 34

**18/8**
Green-White screening was done on the plated *E. coli* transformed with CHS3 on
Friday and nearly all colonies were white. 2 white colonies from each plate were picked and
transferred into liquid LB+Kanamycin. They were placed to incubate overnight.

*Colony PCR*
More white colonies were picked from the same plates (2 from each). A colony PCR was done following
the  [colony PCR protocol](./experiments#PCR-colony).
The PCR products were left in the thermocycler overnight.

*Mutation PCR*
We checked the mutation PCR product from yesterday using the
[gel electrophoresis protocol](./experiments#Gel-electrophoresis).
There was
very little liquid left in the tubes, likely due to a gap between the lids and the tubes.
Because of this, only 1μL of product was used for the gel electrophoresis, diluted with 4μL
MQ and mixed with 1μL loading dye. There were no bands apart from a lot of short fragments.
![](https://static.igem.wiki/teams/5796/wetlab/mutpcr-18-8.webp)

**19/8**
The colony PCR from yesterday was run through a gel according to the
[gel electrophoresis protocol](./experiments#Gel-electrophoresis)
which showed only short fragments, meaning that it has to be redone.
A new [Mutation PCR](./experiments#PCR-mutation)
of CHI92 and SP1 was done, since last week yielded no bands. The same gradient of
temperatures was used and the tube with SP1 was again placed on the row closest to the optimal
temperature. The PCR product was run through a gel, which was pretty hard to read and will be redone
tomorrow.

**20/8**
The gel from yesterday was redone, and indicated that the mutation PCR was unsuccessful and will
need to be redone. Next time we will increase the annealing temperature of the gradient PCR and
increase the annealing temperature for SP1.
It was discovered that the wrong primers were used for the colony PCR yesterday and there weren't
enough of the colonies on the plates to inoculate them again. Plasmid extraction was done again on 6
remaining liquid cultures using the [GeneJET protocol](./experiments#extraction-GeneJET)
and their concentrations measured with a NanoDrop. The
concentrations ranged between 178 and 235 ng/muL, which was quite low but expected since we used
less liquid culture than usual in case we needed to repeat it again.
The plasmids were linearized with the EcoRI restriction enzyme using the
[FastDigest](./experiments#FastDigest) protocol at 37°C
for 30min. A control was done containing the original backbone pYTK\_007.
After digestion they were run through a 0.5% agarose gel. Each well showed three bands, which was
unexpected since they were only supposed to be cut once. The only exception was the original
backbone, which was only linearized. A decision on how to proceed will be done tomorrow with a
supervisor.

**21/8**
A gradient PCR was done for the mutation of CHI92 and SP1 with annealing temperatures ranging from
60C to 70C. The extension time was recalculated to 30s/kb as well to hopefully give better results.
A 1% agarose gel was casted for the PCR product which was run as usual. The controls were in a
different kind of PCR tube, where the lids are attached in strips. There must have been a gap
between the lids and the tubes because they were empty when taken from the thermocycler, likely due
to evaporation.
The gel showed a lot of bands, some of which could have been our mutated plasmid but we decided to
redo the mutation PCR to hopefully get a better result.
![](https://static.igem.wiki/teams/5796/wetlab/chi92-sp1-21-8.webp)
A larger volume was used along with the
temperature that yielded the best result from this run: CHI92 at 68C and SP1 at 60C. These will be
run through a gel tomorrow.
Despite the strange bands from yesterday it was decided to send the CHS3 plasmid with the prettiest
bands for sequencing. The plasmid was combined with a primer and MQ and sent to Eurofins for
sequencing.

**22/8**
A gel was run with the mutation PCR product from yesterday. There were no bands in the gels, so
mutation PCR has to be redone again.
This time, extra care was taken to follow the protocol as closely as possible as well as keeping the
tubes on ice at all times and foregoing the mastermix. Additionally, the tubes were placed on a
pre-warmed PCR machine. SP1 was run at 60C and CHI92 at 68C.
After the PCR the samples were run through a gel. This time, good bands at the right size appeared
along with some that were assumed to come from primer dimerization. The DNA from the bands were
extracted using the [GeneJET protocol](./experiments#extraction-GeneJET) [plasmid extraction](./experiments#extraction-GeneJET)
and the purified samples were stored in preparation for Gibson assembly and later transformation into *E. coli*.

Week 35

**25/8**
The GenArt [Gibson assembly protocol](./experiments#Gibson)
was followed to make the plasmids from Friday circular again.
After incubation the plasmids were transformed into competent *E. coli* using using [heat shock](./experiments#heat-ecoli) which were then
plated onto LB+Chloramphenicol plates and left to incubate overnight.

**26/8**
The plates did not grow as expected and the transformation and plating from yesterday was redone,
this time with a higher concentration of the plasmids.

**27/8**
Colonies grew on the plates from yesterday, so three colonies from each CHI92-containing and
SP1-containing plate were picked and inoculated in liquid LB+Chloranphenicol.
In preparation for the binding affinity test with chitin beads, a buffer was prepared using:

* 220mL MQ
* 7.3g NaCl
* 0.788g Tris-HCL
* 0.09g EDTA
* 250muL Tween 20

**28/8**
Plasmids from yesterday’s inoculated *E. coli* were extracted using
[GeneJET protocol](./experiments#extraction-GeneJET) and placed in the
freezer.
*B. subtilis* cells were inoculated in liquid transformation media in preparation
for transformation.

**29/8**
The *B. subtilis* from yesterday were harvested and their OD450 measured. It was fairly low and they
were left to grow further with 1mL fresh transformation media added. After measuring again a while
later the OD was still very low and they were left to incubate for even longer.
After a discussion with some lab colleagues regarding handling of *B. subtilis* the inoculation
procedure was tweaked. The following procedure will be followed in the future:
Plate cells on LB using Quadrant streaking with an inoculation loop and grow over night at 37C
Pick a big, well isolated colony and inoculate in 10mL transformation medium overnight at 37C. After
growth the OD450 should be 0.5-1
Continue with the transformation protocol.
With this in mind, Quadrant streaking was done and placed to incubate.

### September

---

Week 36

**2/9**
*B. subtilis* cells from the glycerol stock were inoculated in LB media in preparation for
transformation.

**3/9**
The *B. subtilis* culture from yesterday were placed in the fridge to prevent overgrowth.

**4/9**
The culture had an OD450 of 0.1, so we spun it down at 3000g for 5min. We calculated
that we should remove media until we reached a final volume of 1.4mL to reach a better OD450
of 0.7 using the following:
OD1×V1=OD2×V2
→0.1×9900 = 0.7×V2
→V2 = 9900/0.7 = 1.4mL
We ended up with an OD450 of 0.43 but our supervisor deemed it close enough to proceed
with the [1-step transformation protocol](./experiments#transform-bsubtilis-1step).
We used the CHI92 plasmid with the highest concentration
at 334.4ng/μL. No SP1 plasmids were transformed since there weren't enough cell culture for that.
The transformed cells were plated on LB+chloramphenicol plates and left to grow in the 37°C
incubator.

**5/9**
The plates from yesterday were checked several times over the course of the day, but nothing grew
unfortunately.

Week 37

**8/9**
*B. subtilis* was inoculated in LB medium, 2 from the glycerol stock (stored at -80°C) and 2
from the previous colonies. Additional  *B. subtilis* cells were also plated on LB plates in case
the liquid cultures don’t grow.

**9/9**
The plates from yesterday had grown colonies and were placed in the fridge.
OD450 measurements were taken of the *B. subtilis* cells from yesterday. The result
was as follows:
Cryo.L: 0.54
Cryo.K: 2.04
Colony.L: 0.64
Colony.L: 1.4
They were deemed high enough to proceed with the transformation. We chose one sample from each
of the 4 tubes from the OD measurement.
We followed the
[transform-bsubtilis-2step](./experiments#transform-bsubtilis-2step)
of *B.subtillis*. We took 500μL of each sample and placed it in 2mL of
[Schaeffer's medium](./experiments#Medium-Schaeffer) in inoculation
tubes that were placed in the
shaking incubator for 4.5h. After the time had passed, we used a Nanodrop for the three
colonies with the SP1 plasmids which had not been done previously. Colony two had the highest
concentration (339.7ng/μL) in comparison to C1 and C3 (160.4 ng/μL and 60.5 ng/μL respectively).
3μL of the SP1 plasmid from C2 was added to half of the tubes of cells. The other half of the
tubes were combined with 3μL of the CHI92 plasmid from last week (C3). The tubes were placed in
a shaking incubator at 37°C for 2.5h.
After 2.5h the cells were plated on selective plates and placed in 37C to grow overnight.
We also started the
[1-step transformation protocol](./experiments#transform-bsubtilis-1step)
again. This was done by taking one colony from each plate from yesterday and placing it in 10mL
transformation medium to grow overnight.

**10/9**
We checked plates from the 2-step transformation and there was unfortunately no growth. We moved
on to finishing the
[1-step transformation](./experiments#transform-bsubtilis-1step)
that was started yesterday.
We checked the OD for the inoculated *B. subtilis* cultures from yesterday:
1: 042
2: 0.8
Those values were close enough to the protocol so genes were introduced. 4 tubes were made, 2 for
CHI92 in each of the cultures and 2 for SP1 in each of the cultures. They were left to grow for an
hour according to the protocol and were tne plated on LB+chloramohenicol plates.

**11/9**
Nothing had grown on the plates for the 1step transformation from yesterday. At this point there
was no more time for another round of transformation. It was decided that we would send the CHI92
and SP1 plasmids that we extracted from *E. coli* for sequencing to see if they were assembled
correctly.

Week 39

**22/9**
Today, two tubes of plasmid DNA were sent to Eurofins for sequencing. One for the CHI92 plasmid and one for the
SP1 plasmid.

**19/8**
The sequencing results came in and were checked on Benchling.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one
iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

![hyphae](https://static.igem.wiki/teams/5796/custom/hyphae/hyphae-tiling5.webp)

##### On This Page

* [Introduction](#intro)
* [Design](#design)

+ [CHI92](#chi92)
+ [SP1](#sp1)
+ [CHS3](#CHS3)

* [Build/Test](#buildtest)
* [Learn](#learn)
* [References](#reference)

### Introduction

To make a strain of *Bacillus subtilis* express fungal adhesion proteins and assimilate on the
surface of
baker's yeast (*Saccharomyces cerevisiae*), we utilized the Modular Cloning Method (MoClo).
The idea was to assemble multiple plasmid parts simultaneously into a single vector by utilizing type
IIS restriction
enzymes (BsaI and BsmBI). These enzymes cut outside their recognition sites to generate four-base
overhangs, allowing
customizable vectors (Addgene: Modular Cloning Guide). For our project, a yeast MoClo toolkit found in
an article by Lee et al.[1](#ref1)
was used for all assemblies.

The engineering principles of synthetic biology have been a guide in our process. The first step of the
DBTL cycle,
Design, included brainstorming sessions to narrow down our problem, identifying tools and resources, and
planning.
The Build phase revolved around constructing the plasmids through Modular Cloning as described. In the
Test phase,
we relied on methods such as gel electrophoresis, colony PCR, and restriction
digestion to test if our design worked. Lastly, in the Learn phase, we checked the results from the Test
phase and made adjustments. Also
included in our project was the DBTL cycle of testing the organism that we engineered. We designed our
final plasmids in
the last DBTL cycle, then built our organism by transforming them with our plasmids. We intended to test
them against
each other (*S. cerevisiae* to *B. subtilis*) and in a chitin column, learning by doing
statistical analysis
of replicates.

![](https://static.igem.wiki/teams/5796/engineeringsuccess/igem-eng-cycle.webp)

Design-Build-Test-Learn cycle.

### Design

The two different adhesion proteins, Synthetic Peptide 1 (SP1) and Chitinase 92 (CHI92), were
investigated, as well
as native chitinase in the *S. cerevisiae* cell wall. The three final constructs are presented in
the figures below.
All plasmids were built in the software Benchling.

All final constructs utilized the pYTK095\* backbone from the toolkit presented in Lee et al.[1](#ref1) which
can be seen
in the figure below. The backbone
contains a resistance gene, CamR, that gives the carrier of the plasmid resistance against
chloramphenicol, used
for selection in case of successful integration. It also contains an origin of replication as well as
promoters/terminators,
an ori and the sfGFP gene. The sfGFP gene, as the name suggests, produces GFP. Outside the GFP gene and
its promoters
sit recognition sites for BsaI cut sites. All of these parts make it a good candidate to use as a
backbone for MoClo.
As described before, when we perform MoClo we cut our desired gene fragment and the backbone with BsaI.
This creates
overhangs that then connect together, effectively swapping out the sfGFP gene with the desired gene
fragment.
The reason for replacing sfGFP is that we can easily check which colonies have the right insert, as the
wrong ones will
fluoresce because of the GFP gene remaining.

![](https://static.igem.wiki/teams/5796/engineeringsuccess/eng-pmk3-tp.webp)

pYTK095\* plasmid by Lee et al.[1](#ref1)

A paper by Chamas et al. explored the display of adhesion proteins on *Escherichia coli* Nissle for
localisation
of *Candida albicans*[2](#ref2). This paper described two basic parts that were proven to bind to *Candida*.
We wanted to express them in *B. subtilis* as it is found on the human body and might be more
viable in a
clinical environment. But *E. coli* and *B. subtilis* have different cell walls, which led us
to seek a
different expression path. During our design stage we went through several options before landing on a
basic part
that Kim et al. presented [3](#ref3). They called the part Yuab, which produced a cell wall protein that
protrudes through
the cell wall. This expression pathway was native to *B. subtilis*. In the paper, they used a type
of linker
sequence together with the desired protein and Yuab, which made the protein be expressed and linked to
the membrane
of the bacterium. By adding our GOI to this display construct, we could express them on the *B.
subtilis* membrane.

The transcriptional units of each plasmid were designed to produce different outcomes and will be
discussed individually from this point.

#### Sp1

* pYTK095\* backbone (pmK3\_CamR\_GFP)
* P43 promoter
* Yuab and linker
* SP1
* T24 terminator

The transcriptional unit of SP1 included the following parts: P43 promoter, Yuab and linker, SP1, and
the T28
terminator. Sp1 originates from the Chamas et al. paper. They described SP1 as “synthetic protein 1”
and speculated
that it binds to non-specific carbohydrates. We included it in our project to find out if it binds
to chitin and as
a control to see if it binds to our engineered yeast. Chamas et al. concluded that SP1 may have
higher binding
capability to *Candida* than CHI92. The promoter was also found to be effective in the Kim et
al. paper.
The terminator is a common terminator that works in *B. subtilis*. The linker was also copied
from the Chamas et al.
paper.

The plasmid was then built in the Benchling software. When it was approved by our lab assistants we
ordered the
plasmid in two parts, separating the Yuab and Sp1 regions. We added BsaI recognition sites as
overhangs. We then
attempted MoClo for several weeks with poor results, testing different polymerases like Sapphire,
Phire, and Phusion.
Eventually, we ordered the entire construct instead of separate parts. With this change, we
succeeded in building
our first plasmid. Later, we found that the stop codon had been mutated away. By ordering mutation
primers and
performing the necessary steps, we restored the stop codon, finally completing the first
plasmid.

#### Chi92

* pYTK095\* backbone (pmK3\_CamR\_GFP)
* P43 promoter
* Yuab and linker
* CHI92
* T24 terminator

Similar to SP1, all parts for the CHI92 construct were found in the paper mentioned above. The only
key difference was that instead of
SP1 as the binding domain, we used CHI92 from Chamas et al. They described CHI92 as a protein that
binds to chitin,
which is abundant in *Candida* cell walls. We treated SP1 and CHI92 as sibling constructs in
ordering and testing.
The timeline was identical, so the steps are not repeated here.

#### CHS3

* pYTK095\* backbone
* pYTK002 connector 1
* pYTK011 Promoter
* CHS3 gene
* pYTK056 terminator
* pYTK072 connector 2

For the *S. cerevisiae* construct, the goal was to overexpress chitin in the cell wall. This
would allow for
better testing and for mimicking *C. albicans* more accurately. For this, we chose to use CHS3,
a native gene
in baker's yeast responsible for chitin expression in the cell wall. The backbone consisted of
different elements
from Lee et al.'s paper. We constructed it this way to be able to build in *E. coli* and later
cut the CHS3
gene out into a new backbone built for *S. cerevisiae*.

![](https://static.igem.wiki/teams/5796/engineeringsuccess/eng-p001-tp.webp)

p001 plasmid with ampicillin resistance and the CHS3 gene.

The main focus with the different backbones was to create recognition sites for BsaI and BsmBI,
allowing us to use
BsaI for the construction of plasmids in *E. coli*. This was done to produce more CHS3. Then we
would use BsmBI
to transfer it into a pre-made backbone prepared by our lab supervisor, called pLE\_Ytk007. It also
has its origin
in Lee et al. and contains a yeast ori, which was the main reason for swapping the backbones. We
also used a kanamycin
resistance gene in this construction.

![](https://static.igem.wiki/teams/5796/engineeringsuccess/eng-p002-tp.webp)

p002 plasmid, with the URA3 and KAN genes for selection and the CHS3 gene.

### Build/Test

Following the Design phase, the Build phase began and took place in the wet lab. The bulk part of the
build phase
is the assemblies from level 1 plasmids to level 2. Naturally, tests must be run simultaneously as they
are built
to confirm progress, and therefore the “Test” phase will be discussed in the following section as well.
All the
experiential methods that were used will be presented, but the protocols can be found under the tab
[Experiments](/experiments).
The build phase started with ordering the Sp1 and Chi92 parts. As mentioned before we started ordering
the
constructs in two parts. With the linker being the separation point. When the order arrived we
started doing Moclo to connect the parts with our pYTK095\* backbone. After every MoClo experiment we
transformed the
ligation mixture into *E.coli*. We used heat shock the
majority of the time and every time we plated the cells with low and high concentration separately to
ensure a high
likelihood of getting colonies. Plasmid extraction was
done with Thermo Fisher's plasmid miniprep kits. The testing of these plasmids varied during the
project. In the
first week we encountered a problem with the first test. The green white screening was hard to do as not
many
colonies had grown and none were green which was unusual. We put the plates in the incubator longer and
more
colonies grew. After that we decided to send the plasmids in for sequencing, instead of doing a test in
the lab.
The result came back and after checking in Benchling we noticed that a lot was wrong. Thus sending us
back to the
design stage. The parts that we ordered arrived in shuttle vectors with the same resistance as our final
backbone.
This complicated the transformation, as some bacteria might take up the original plasmid instead of our
new one
and still survive the antibiotics. To try to fix the issue we went back into the lab and added more
enzymes
that only cut in the shuttle vector. The goal was to create a new incentive to use our correct plasmid
by cutting up
the shuttle vector, thus hopefully rendering it useless and unable to give resistance.
In addition we also ordered primers that extend our GOI with the cut sites, so we can do PCR and just
add the
resulting fragment in future MoClo. In case our first strategy did not work. We chose this approach to
try to mimic
real lab work where ordering new constructs that are not sponsored by iGEM, and primers are a lot
cheaper than whole plasmids.
To test the MoClo assembly with the extra enzymes we performed a colony PCR and ran the results on a 1%
agarose
gel. The result came back wrong as we had more bands than we expected and no band was the correct size.
We checked
Benchling again and when we added the extra enzymes to cut the shuttle vector we had accidentally added
an enzyme
that had a recognition site in our GOI. That recognition site had been mutated in when Twist made our
order. So we
decided to make three new strategies moving forward.
The first was ordering the whole construct in one piece to then cut into our backbone. The other was to
inoculate and transform the vectors we had
and hope for the best. The third was redoing the MoClo with the first order but remove the enzyme that
had several
cut sites.
We redid the MoClo several times and checked the transformed cells with colony PCR which often resulted
in no bands in
the gel. We initially thought that our issues had to do with the PCR reaction. Initially we used
Sapphire Star polymerase and buffer, then
we tried Phyre and later we used Phusion polymerase and buffer. Phyre and Phusion are more precise than
Sapphire, meaning
that they cause fewer mutations when replicating the DNA, however they require longer run times. The
Phusion polymerase
resulted in successful bands of the CHI92 plasmid but not of SP1.
When we transformed these promising plasmids into *E.coli* again, the CHI92 plate did not grow but
SP1 did. So we
did a restriction digestion to further check if it was correct. Sadly it still showed too large of a
band thus
proving the MoClo was the problem.
This erased the option of redoing but without the extra enzymes. We then switched to our most promising
plan which
was to try to order the whole construct in one piece.
At the same time we started working on the *S. cerevisiae* construct. As we mentioned above, we
wanted to over express CHS3 by
placing it in an expression vector. For this we needed CHS3 with cut sites flanking the coding region.
As CHS3 is a native gene
in *S. cerevisiae* we
designed primers that had recognition sites on the CHS3 gene but with overhangs that included cut sites
for BsaI and
BsmbI. One problem we encountered was that CHS3 has a BsaI cut site in the middle of it. We designed a
mutation primer to
mutate away this cut site, so it would not cause issues later on. Then we used these flaking primers and
with yeast
DNA as template we performed PCR to acquire our GOI. Additionally, as insurance we ordered the whole
CHS3 gene without the
cut site and with the flanking regions in case we encountered more issues with the PCR. In the end,
after many PCR trials with different
polymerases, we got a correct band on a gel and proceeded to use this result to create
our *E .coli* construct (p001) using MoClo.
Instead of using colony PCR to check the results of the MoClo we did restriction digestion. Colony PCR
can be quite
tricky with large fragments which we indeed had with our CHS3 gene
(~3500 bs). We did amplification PCR before mutation PCR to get more of our gene. The plan was that
after mutation PCR we would digest it
with Dnp1 to remove the original template DNA, check the results on a gel, then amplify with PCR, and
purify the PCR results.
Finally we would perform gibson assembly, and
later transform into *E .coli*. This plasmid would then be used to create our final construct,
ready to transform
into yeast.
However, when we checked the results after Dnp1 digestion we noticed that the mutation primer had been
ordered wrong: it missed the actual base pair mutation. We also realized that the whole CHS3 gene
ordered in the
beginning would arrive the next day. Therefore, we pivoted to work with our ordered CHS3 gene which we
removed the
cut site from. MoClo was then performed with the backbone and our ordered CHS3 gene, which worked much
better.
We got the correct bands with restriction digestion and the plasmid containing CHS3 could then be
transformed into
E.Coli using heat shock and from a green white screening the colonies looked to be successful. A colony
PCR however
gave vague results showing faint bands at the right site but to many bands. We sent the plasmid in for
sequencing
which was inconclusive, the DNA sequenced was not our gene insert but not the backbone either. Due to
lack of time
we decided to extract and transform it into yeast anyway since the green white screening was successful
and there
looked to be bands where we expected them. After transformation, *S. cerevisiae* grew which also
indicated successful uptake of the plasmid.
Separately, when the SP1 and CHI92 orders arrived which contained the
whole constructs, we changed the plan again. As time was running out we noticed that we could use the
shuttle
vector instead of our backbone as it already had the correct ori. By changing the resistance it became a
sufficient backbone. But one problem remained, which was that the stop codons for the constructs were
either lost
in the manufacturing phase. Thus, we needed to order mutation primers to mutate back the stop codons.
This was
discovered in Benchling after double-checking the sequence of our order. What followed was many weeks of
mutation
PCR, including a second order of mutation primers since we ordered two of the same primers for one of
the genes,
instead of a forward and a reverse.
After many different settings on the
thermocycler and different polymerases were tested, we succeeded by following every protocol very
carefully, keeping
the reaction on ice at all times, adding the polymerase last and dNTPs second to last, and preheating
the PCR machine.
After we checked that the correct length was found on a gel and extracted the DNA from the gel, and
annealed the
amplification strand. From there we moved on to attempting to transform the two constructs int *B.
subtilis*.
Several methods were tested for transforming into *B. subtilis* but
nothing was successful. The problem seemed to be getting them competent as the growth was very
slow. In the end the transformation was never successful, so we could never test it on our *S.
cerevisiae* cells.
We decided to send our CHI92 and SP1 plasmids in for sequencing. We discovered that both the promoter
(though slightly
mutated), the RBS and YuaB was present in our construct which told us that it was very likely that CHI92
and SP1 were too.

### Learn

Through the project a lack of effective communication and inexperience affected the wetlab work and
results. Everyone had
different levels of independence in the lab which we should have taken into consideration when making
the
schedule, pairing people who had more experience with those who felt less sure.
The project could have benefited from a more consistent approach in labeling, documenting and following
protocols as small mix-ups and misinterpretations of protocols sometimes caused us to have to repeat the
same
protocol several times, losing valuable time.
We believe that more time would change our results drastically as it would allow us to redo the
transformation of *B. subtilis* and the transformation of CHS3 if it turned out to be faulty.
Because of the
lack of time we had to assume that the CHS3 had been implemented which turned out not to matter since
the
*B. subtilis* transformation didn't work anyway.
The project planning phase in the beginning of the projects faced many issues. Our first project leader
left which
set us back a couple of weeks. With a more concise plan and defined roles and ambition, the project
could
have started earlier, which in turn may have enabled us to start the lab work earlier.
Moreover because of the lack of time we did not do the rest of the planned project. The plan was to
successfully
transform *B. subtilis* with our CHI92 and SP1 constructs, make them express the binding proteins
and test them.
We aimed to test *B. subtilis* against our transformed yeast. We would grow *B. subtilis* and
*S. cerevisiae* together and then check if *B. subtilis* is bound to *S. cerevisiae*
through microscope imaging. This test
was to see if SP1 was better at binding as it binds to carbohydrates instead of chitin, which CHI92
binds to.
We would not get data out of this test, but still confirmation that the constructs worked as intended in
the real
world. The other test was to use an affinity column with chitin beads. By counting the cells before
adding them to
the affinity column and after, we would have been able to assess how well SP1 and CHI92 bind to chitin.
A
negative control would also have been performed using wild type *B. subtilis*. The
counting of the cells would have been performed using a haemocytometer. The amount of cells that would
have stuck to the
column over several replicates would have allowed us to test if the modified cells bound to chitin more
than the wild type
cells with statistical significance.

### References

1. Lee ME, DeLoache WC, Cervantes B, Dueber JE. A highly characterized yeast toolkit for modular,
   multipart
   assembly. ACS Synthetic Biology. 2015;4(9):975-986. doi:10.1021/sb500366v
2. Chamas A, Aranda-Díaz A, Harousseau G, Zulauf KE, Torrealba J, Cruz C, et al. Engineering adhesion
   of the
   probiotic strain Escherichia coli Nissle to the fungal pathogen Candida albicans. mBio.
   2024;15(3):e0063924.
   PMID: 39265099; PMCID: PMC11669158.
3. Kim D, Kim W, Kim J. New bacterial surface display system development and application based on
   Bacillus subtilis
   YuaB biofilm component as an anchoring motif. Biotechnology and Bioprocess Engineering.
   2021;26(1):39-46.
   doi:10.1007/s12257-020-0397-7

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide
sufficient information for other teams to replicate our work.{% endblock %}
{% block page\_content %}

![hyphae](https://static.igem.wiki/teams/5796/custom/hyphae/hyphae-tiling5.webp)

#### Transformation

[Transformation of *E. coli* - heat shock](#protocol-1-1)
[Transformation of *E. coli* - electroporation](#protocol-1-2)
[Transformation of *B. subtilis* - 1-step](#protocol-1-3)
[Transformation of *B. subtilis* - 2-step](#protocol-1-4)
[Transformation of *S. cerevisiae*](#protocol-1-5)

#### PCR

[PCR - Phusion](#protocol-2-1)
[PCR - PHIRE](#protocol-2-2)
[PCR - Sapphire](#protocol-2-3)
[Colony PCR](#protocol-2-4)
[Mutation PCR](#protocol-2-5)

#### Other DNA Techniques

[MoClo](#protocol-3-1)
[DNA digestion - FastDigest](#protocol-3-2)
[Gibson Assembly](#protocol-3-3)
[Plasmid extraction - GeneJET](#protocol-3-4)
[Gel Extraction - GeneJET](#protocol-3-5)
[PCR purification - GeneJET](#protocol-3-6)
[DNA concentration measurement](#protocol-3-7)
[Gel Electrophoresis](#protocol-3-8)

#### Medium & Buffer Preparation

[LB Medium](#protocol-4-1)
[Schaeffer's one-step *B. subtilis* transformation medium](#protocol-4-2)
[Two-step *B. subtilis* transformation media (MGI&II)](#protocol-4-3)

#### Inoculation and Plating

[Plating of cells](#protocol-5-1)
[Inoculation of cells](#protocol-5-2)

**Click an experiment to view it here!**

**Transformation of *E.coli* - heat shock**

1. Thaw a tube of competent cells from the -80°C freezer on ice for 10 minutes.
2. Add 10μl of DNA solution to the tube and mix gently by flicking.
3. Leave the tube on ice for 30 minutes.
4. Place the tube in a water bath or heating block at 42°C for exactly 45 seconds.
5. Place on ice for 5 minutes.
6. Add 900μl of LB medium at 37°C or room temperature.
7. Incubate at 37°C for 60 minutes in a shaking incubator.

**Transformation of *E. coli* - electroporation**

**Preparing competent cells**1. Pour the bacterial culture into the prechilled 15-ml glass centrifuge tube on ice.
2. Centrifuge at 7000 rpm at 4 °C, for 5 min.
3. Discard the supernatant.- Resuspend the pellet in 1 ml of sterile, ice-cold ddH2O and transfer into a chilled 1.5-ml microcentrifuge tube.
   - Spin in a microcentrifuge at 7000 rpm at 4 °C, for 5 min.
   - Discard the supernatant.
   - Resuspend the pellet in 1 ml of ice-cold ddH2O.
   - Repeat steps 1.5–1.7 two more times for a total of three washes.
   - Spin a final time at 7000 rpm at 4 °C, for 5 min, discard the supernatant, resuspend the pellet in 50 μl of ice-cold ddH2O, and put the cells on ice.
   **Electroporation**- Add 50–100 ng of supercoiled plasmid to the electro-competent E. coli and mix gently (do not pipette up and down).
   - Transfer the bacteria to a chilled electroporation cuvette. Be careful to pipette straight in between the metal plates and avoid introducing any bubbles.
   - Cap the cuvette and tap it lightly on the bench to settle the bacteria/DNA mix.
   - Put the cuvette back on ice and carry it to the electroporator.
   - Turn on the electroporator and set it to 1.8 kV, 25 μF, 200 Ω. This is a standard setting for most E. coli strains. Other bacterial strains may require an adjustment of the electroporation conditions.
   - Wipe the cuvette briefly with a Kimwipe to remove any residual water or ice, and then place it in the electroporation chamber.
   - Push the pulse button. The time constant displayed should be around 4 ms.
   - Immediately after the pulse has been delivered, add 1 ml of LB (or other growth medium, e.g., SOC) to the cuvette and pipette quickly but gently up and down. Be aware that the transformation efficiency decreases proportionally to the lag time between the electric pulse and the addition of media.
   - Transfer the mixture to a fresh 1.5-ml microcentrifuge tube.
   - Incubate at 37 °C for 1 h with shaking.
   - Evenly spread 100 μl of your transformation onto a selective plate.

**Transformation of B. subtilis - 1-step**

1. Streak the B. subtilis on LB agar plates, incubate overnight at 37 ℃.
2. Pick a single colony, inoculate it in 10 mL of one-step transformation medium and incubate overnight at 37 ℃ in a shaking incubator (180 rpm).
3. After ~18 h, measure the OD450 (typically reach 0.5-1.0), if lower, grow longer time or centrifuge down and resuspend in lower volume of culture supernatant.
4. Transform 1.0 mL of the culture into a sterile Eppendorf tube containing transforming DNA and incubate 30 min at 37 ℃ in a shaking incubator.
5. Plate the medium on a selective agar plate.
6. Incubate the plate overnight at 37 ℃.

**Transformation of B. subtilis - 2-step**

1. Transformation of B. subtilis
2. Grow the recipient strain(s) overnight in MGI medium, supplemented with antibiotics if necessary.
3. In the morning, take 500 μl of the overnight culture and inoculate in 2 ml of pre-heated medium MGI.
4. Grow for 4.5 h with shaking.
5. Transfer 250 μl of culture to 1 ml of pre-heated medium MG II and add approximately 1 μg of με plasmid DNA.
6. Grow for 2.5 h with shaking.
7. Plate the cells on selective plates.

**S. cerevisiae transformation**

2-3 days before the transformation: 1. Grow yeast strain of interest on (YEPhD) agar plate (~2 days). For strains that grow poorly on glucose other carbon sources can be used.
2. Scrape cells from the plate and inoculate ~10 ml YEPhD with the cells.
3. Incubate the cells o/n at 30°C and shaking at 280rpm.
Transformation:4. Prepare a YEPhD culture (5 ml/transformation) with an OD600 of 0.4.
5. Grow the cells to an OD600 of 1.0-1.5 (2 to 6 h) at 280 rpm at 30°C.
6. Spin down 3 min at 3,000 rpm.
7. Wash the cells twice with 25 ml sterile water.
8. Resuspend the pellet in sterile 0.1 M LiAc (100 µl/transformation) and transport to an eppendorf tube.
9. Spin at 1,300g for 1 min.
10. Resuspend the pellet in 0.1 M LiAc (use 100 µl/transformation) and fill out eppendorf tubes with approximately 100 µl/transformation.
11. Spin at 1,300g for 1 min.
12. Remove the supernatant and add to the dry pellet in this order:

* 240 μl 50% PEG4000
* 35 μl 1 M LiAc
* 75 μl DNA mix (up to 5 μg of DNA + 50 μg [5 μl] of salmon sperm [carrier DNA], with sterile water added to a final volume of 75 μl).

13. Vortex until the pellet is completely resuspended.
14. Incubate 30 min at 30°C.
15. Heat shock 20min at 42°C.
16. Centrifuge tubes at 1,300g for 1 min and discard the supernatant.
17. If an antibiotic resistance marker is introduced, carefully resuspend the cells in 0.5 ml YEPhD and incubate shaking at 30ºC for the time specified in Table 1. If not, continue with step 18.
18. Resuspend the cells carefully in 1 ml sterile water and plate 50 μl onto one selection plate (1/10).
19. Spin down the remaining cells for 1 min at 1,300 rpm.
20. Discard supernatant and resuspend the pellet in 100 μl sterile water and plate onto a second selection plate (9/10).
21. Incubate 2-5 days at 30°C until transformants appear.

**PCR - Phusion**

1. Prepare sufficient master mix for 10µL reactions (note: scaleable) following this scheme:

| Volume | Reagent |
| --- | --- |
| 2.0 µl | 5X Phusion HF buffer |
| 0.8 µl | 2.5 mM dNTPs |
| 0.5 µl | Forward primer (10 µM stock) |
| 0.5 µl | Reverse primer (10 µM stock) |
| 0.1 µl | Phusion polymerase |
| 5.1 µl | Purified water |
| 1.0 µl | Template DNA |

2. Pipette 10 µl of the mastermix to PCR reaction tubes
3. Perform the following PCR protocol on a thermocycler (note: varies with template/primers):

| Step | Temp. | Time |
| --- | --- | --- |
| Initial denaturation | 98°C | 60 sec |
| Denaturation | 98°C | 10 sec x 32 |
| Annealing | ...°C | 30 sec x 32 |
| Elongation | 72°C | 30 sec/kb x 32 |
| Final elongation | 72°C | 300 sec |
| Hold | 4°C | Indefinitely |

**PCR - PHIRE**

1. Prepare sufficient master mix for 20µL reactions (note: scaleable) following this scheme:

| Volume | Reagent |
| --- | --- |
| 10.0 µl | 2x PHIRE Green Hot Start II DNA Polymerase Mix |
| 0.15 µl | Primer Forward 1 (100μM stock) |
| 0.15 µl | Primer Reverse 2 (100μM stock) |
| 9.70 µl | MQ-H2O |

2. Pipette 20 µl of the mastermix to PCR reaction tubes
3. Pipette 1 µl of the templates to their respective tubes
4. Perform the following PCR protocol on a thermocycler (note: varies with template/primers):

| Step | Temp. | Time |
| --- | --- | --- |
| Initial denaturation | 95°C | 5 min |
| Denaturation | 95°C | 10 sec |
| Annealing | ...°C | 10 sec |
| Elongation | 72°C | 30 sec + 20 sec/kb |
| Repeat from Denaturation for 30 cycles |
| Final elongation | 72°C | 10 min |
| Hold | 12°C | Indefinitely |

**PCR - Sapphire**

1. Create the following reaction mixture (50 μl; scale to desired volume):

| Amount | Reactant |
| --- | --- |
| 25 μl | SapphireAmp Fast PCR Master Mix (2X Premix) |
| To 0.2 μM (final conc.) | Forward Primer |
| To 0.2 μM (final conc.) | Reverse Primer |
| 0.1-10 ng | Plasmid DNA |
| To 50 μl | Purified water |

2. Perform the following PCR protocol on a thermocycler (note: varies with template/primers):

| Temp. | Time |
| --- | --- |
| 94°C | 1 min |
| 98°C | 5 sec x 30 |
| ..°C | 5 sec x 30 |
| 72°C | 10 sec/kb x 30 |
| 12? | Hold? |

**Colony PCR**

1. Add 15 μL of MQ-water to a PCR tube.
The following steps are done in a LAF bench.2. Choose a colony from the agar plate and mark the chosen colony on the backside of the plate and label it the same as on the PCR tube.
3. Take a small pipette tip and gently take up some of the colony (not the entire colony) and transfer it into the MQ water in the PCR tube. Slowly pipette up and down to mix.
4. Incubate the PCR tubes in the thermocycler at 98 °C for 10 min and then store at 12 °C.
While waiting → Prepare the components for the PCR and proceed following a regular PCR protocol.

**Mutation PCR**

Use a regular PCR protocol (preferably Phusion) with the mutagenic primers!

**MoClo assembly**

1. Mix all of the reagents in the table in a tube

| Volume | Reagent |
| --- | --- |
| 0.5 µl of each insert | DNA insert |
| 1.0 µl | T4 DNA ligase buffer |
| 0.5 µl | T4 DNA ligase (enzyme) |
| 0.5 µl | BSAI restriction enzyme |
| 0.5 µl | BSMBI restriction enzyme |
| 6.5 µl | Purified water (pipette this first) |

2. Incubate the mixture in a thermocycler according to the following table:

| Step | Temp. | Time (Regular enzymes) | Time (FastDigest) |
| --- | --- | --- | --- |
| Initial restriction | 37°C | 4 min | 4 min |
| Restriction (cycling) | 37°C | 2 min x 50 | 1 min x 20-50 |
| Ligation (cycling) | 16°C | 5 min x 50 | 2 min x 20-50 |
| Final restriction | 37°C | 4 min | 4 min |
| Heat inactivation | 65°C | 10 min | 10 min |
| Hold | 12°C | Indefinitely |

**DNA digestion (FastDigest enzymes)**

1. In a PCR tube, mix:

| Volume | Reagent |
| --- | --- |
| 7 μl | Purified water |
| 1 μl | FastDigest Green |
| 1 μl | Template |
| 1 μl | EcoRI |

2. Incubate at 37°C for 30 min.

**Gibson Assembly - GeneArt**

1. Thaw GeneArtTM Gibson Assembly® HiFi Master Mix on ice.
2. Vortex GeneArtTM Gibson Assembly® HiFi Master Mix immediately before use.
3. In a microcentrifuge tube on ice, set up the GeneArtTM Gibson Assembly® cloning reaction as described in the table below:

|  | 1-3 inserts | 4-5 inserts | Positive control |
| --- | --- | --- | --- |
| Amount of each fragment | 0.08 pmol vector, 0.08 pmol each fragment, up to 10 μL combined |  | 10 μL |
| GeneArt Gibson Assembly HiFi Master Mix | 10 μL |  | 10 μL |
| Nuclease-free water | Fill to 20 μL |  | - |
| Incubation time at 50°C | 15 min | 60 min | 15 min |

4. Mix the reactions by vortexing, spin down and incubate at 50°C for the recommended time.
5. After incubation, place the reaction mix on ice.

**Plasmid extraction - GeneJET MiniPrep**

All centrifugations are recommended to be done at 12000g for 1 minute unless specified otherwise.1. Resuspend pelleted cells in 250μl resuspension solution, mix by pipetting up and down (or vortexing) and transfer to a microcentrifuge tube.
2. Add 250μl lysis solution and mix by inverting 4-6 times (do not vortex). Incubate for 2-3 minutes (not more than 5 minutes).
3. Add 350μl neutralization solution and mix by inverting 4-6 times.
4. Centrifuge for 5 minutes and transfer the **supernatant** to a spin column.
5. Centrifuge the spin column and discard the flow-through.
6. Add 500μl wash solution (with ethanol).
7. Centrifuge and discard the flow-through.
8. Wash again and centrifuge an additional time.
9. Transfer the spin column to a microcentrifuge tube.
10. Add 50μl elution solution to the center of the spin column membrane without touching it.
11. Incubate for 2 minutes.
12. Centrifuge for 2 minutes and **discard the spin column (keep the tube)**.
13. Make sure the tube is labelled and measure the concentration by Nanodrop.

**Gel Extraction - GeneJET**

All centrifugations are recommended to be done at 12000g for 1 minute.1. Weigh a microcentrifuge tube.
2. Using a scalpel, excise an as small as possible gel slice containing the DNA (avoid UV overexposure). Put the gel slice in the microcentrifuge tube.
3. Weigh (the tube with) the gel.
4. For every 1 mg of gel, add 1 μl of binding buffer to the tube with the gel slice.
5. Incubate the mixture at 60°C until completely dissolved (around 10 minutes). Mix every few minutes by inverting.
6. When the gel is completely dissolved, if it is not yellow, add 10 μl pH 5.2 3 M sodium acetate solution.
7. Vortex briefly and transfer up to 800 μl to a purification column.
8. Centrifuge at 12000g for 1 minute and discard the flow-through.
9. Optional if the DNA will be used for sequencing: Add 100 μl binding buffer, centrifuge and discard the flow-through.
10. Add 700 μl wash buffer (with ethanol), centrifuge and discard the flow-through.
11. Centrifuge again.
12. Transfer the column to a microcentrifuge tube.
13. Add 50μl elution solution to the center of the column membrane without touching it.
14. Centrifuge and **discard the column (keep the tube)**.
15. Make sure the tube is labelled and measure the concentration by Nanodrop.

**PCR Purification - GeneJET**

All centrifugations are recommended to be done at 12000g for 1 minute.1. To the PCR product, add an equal volume of Binding Buffer and vortex.
2. If the color is not yellow, add 10μl pH 5.2 3M sodium acetate solution.
3. Transfer up to 800μl of the solution to the purification column
4. Centrifuge and discard the flow-through.
5. Add 700μl wash buffer (with ethanol), centrifuge and discard the flow-through.
6. Centrifuge again.
7. Transfer the column to a microcentrifuge tube.
8. Add 50μl elution buffer to the column membrane without touching it.
9. Centrifuge and **discard the column (keep the tube)**.
10. Make sure the tube is labelled and measure the concentration by Nanodrop.

**DNA concentration measurement (Nanodrop)**

1. Blank the machine by placing 1μL purified water in the notch and pressing “blank”.
2. Wipe the notch dry.
3. Run every sample, wiping after each one.
4. Read the measured concentrations and write them on the tubes.

**Gel Electrophoresis**

**Preparing agarose solution**1. If not prepared, make an agarose solution by mixing agarose into 1x TAE buffer to a w/w concentration of 0.5% or 1% and microwaving until completely dissolved. Store the solution in 60°C when not in use.
**Casting the gel**2. Cast a gel by pouring the solution into a tray of the desired size with a comb of the desired number of wells (including 2 for ladders). The gel should be around 0.5cm in depth.
3. Add stain to the solution (we use GelRed, 1.5μL for 8cm gel) and mix while removing eventual bubbles. Leave the gel to solidify for 30 minutes.
**Running the gel**4. Remove the comb and transfer the tray with the gel into an electrophoresis chamber. Make sure the gel is covered in TAE buffer (add more if necessary).
5. Separately mix 1μL of each sample with 4μL purified water and 1μL 6x loading dye.
6. Transfer 3μL of each sample mix into their respective wells, taking care not to touch the gel.
7. Transfer 1.5μL ladder (GeneRuler 1kb) into wells on both sides of the wells with the samples.
8. Close the lid, connect the cables (make sure the polarity is correct) and turn on the machine.
9. Set the machine to the desired voltage and time (our standard: 100V / 30min for 8cm well) and press Run. Look for tiny bubbles near the edges to make sure it’s running.
10. When the run is done, transfer the gel off the tray and onto the machine's gel plate (wipe the gel plate with ethanol first). Place it in the machine and click analyse in the computer program. Edit the contrast etc. for visibility, save the image, discard the gel in the special gel bin and wipe the gel plate with ethanol.

**LB medium preparation**

Create a mixture of the desired volume with the following composition (w/w):* 1% Tryptone
* 1% NaCl
* 0.5% Yeast extract
* Purified water (to desired volume; recommended 500 ml)
Stir the mixture until everything has dissolved and autoclave it (remember not to close the lid too tight).

**Schaeffer's medium preparation**

1. Medium Preparation (500 mL):
2. Dissolve K2HPO4 (9.54 g), KH2PO4 (3.0 g), trisodium citrate dihydrate (0.57 g), MgSO4·7H2O (0.1 g), and Na2SO4 (1.0 g) in ~300 mL dH2O.
3. Adjust to pH 7.0 at room temperature using HCl.
4. Add glucose (2.0 g), sodium L-glutamic acid potassium salt monohydrate (1.38 g), L-tryptophan (20.0 mg), FeCl3 (4.055 mg) and MnSO4·H2O (0.169 mg), mix until fully dissolved.
5. Bring volume to 500 mL with dH2O.
6. Sterilize by sterile filtration and store at room temperature.

**MGI & MGII media preparation for two-step *B. subtilis* transformation**

* Media composition:
* MGI:

|  |  |
| --- | --- |
| Ammonium sulfate | 2 g/l |
| Na-citrate | 1 g/l |
| Potassium phosphate dibasic trihydrate | 14 g/l |
| Potassium dihydrogen phosphate | 6 g/l |
| Magnesium sulfate | 1.6 mM |
| Caseine hydrolysate | 0.25 g/l |
| Glucose (filter) | 5 g/l |
| Tryptophane (filter) | 0.25 g/l |

* MGII:

|  |  |
| --- | --- |
| Ammonium sulfate | 2 g/l |
| Na-citrate | 1 g/l |
| Potassium phosphate dibasic trihydrate | 14 g/l |
| Potassium dihydrogen phosphate | 6 g/l |
| Magnesium sulfate | 4 mM |
| Yeast extract | 1 g/l |
| Caseine hydrolysate | 0.1 g/l |
| Calcium nitrate | 2.5 mM |
| Glucose (filter) | 5 g/l |
| Tryptophane (filter) | 0.25 g/l |

**Plating of cells**

Media should only be exposed to air inside the LAF bench. Components vary with organism.1. Add 100 μl cell solution to the middle of a plate.
2. Rub around the solution on the plate with a T-spreader until it stops feeling wet.
3. Close the plate (keep plates bottom-up when not in use) and label it (LC) if not done already.
4. Pellet the cells by centrifugation and discard half (or other amount depending on desired concentration) of the supernatant.
5. Repeat steps 1-3 for the new “high concentration” (HC) cell solution.
6. Seal the plates with parafilm and incubate at 37°C shaking overnight.
Plates are stored in the refrigerator when not in use. Tape plates together with colored tape for organization.

**Inoculation of cells**

Media should only be exposed to air inside the LAF bench. Components vary with organism.1. Identify the colony (or colonies) to inoculate, preferably isolated colonies.
2. Using the electric pipette, add 3ml (or 5ml) LB medium to a tube.
3. Add the appropriate antibiotic to the tube (amount depends on type).
4. With an inoculation loop, touch the identified colony lightly to pick some (not all) of it up. Hold the loop in the medium and shake it around a bit.
5. Incubate at 37°C shaking overnight.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their
significance. Also outline future plans and reflections on the impact of your project.{% endblock %}
{% block page\_content %}

![hyphae](https://static.igem.wiki/teams/5796/custom/hyphae/hyphae-tiling5.webp)

##### On This Page

* [Wetlab results](#wetlab)

+ [Mutation PCR](#PCR)
+ [Transformation of plasmids](#plasmids)

* [Drylab results](#drylab)
* [Project outlook](#outlook)
* [References](#references)

## Wetlab results

Below follows the results for the wetlab section of our project.

### Mutation PCR Results

After several attempts at mutating stop codons into the CHI92 and SP1 genes, the mutation PCR gave a
band of
the
expected size: 3044bp. This can be seen in the figure below.

![CHI92 mutation PCR](https://static.igem.wiki/teams/5796/wetlab/chi92-mut.webp)

Mutation PCR result of the CHI92 plasmid.

![SP1 mutation PCR](https://static.igem.wiki/teams/5796/wetlab/sp1-mut.webp)

Mutation PCR result of the SP1 plasmid.

These gel images suggest that we obtained functioning PCR settings for both a single nucleotide
substitution and for
a full codon insertion. These settings can be seen in the table below.

| Step | Temperature (°C) | Time (s) | Other |
| --- | --- | --- | --- |
| Initial denaturation | 98 | 60 |  |
| Denaturation | 98 | 10 |  |
| Annealing for substitution | 60 | 30 | 32 cycles |
| Annealing for codon insertion | 68 | 30 | 32 cycles |
| Extension | 72 | 90 | 30s/kbp |
| Final extension | 72 | 300 |  |
| Hold | 4 | inf |  |

It would be interesting to further improve these settings to obtain a better result with more of the
correct plasmid and less undesirable bands like those in the figures above, but that was not
possible
within the timeframe of this project.

### Transformation of Plasmids

After the transformation of the mutated plasmids into *E. coli*, cell colonies grew. This
suggests
that at least the antibiotic resistance part of our plasmid was taken up by them. The transformation
of the
CHS3 plasmid into *S. cerevisiae* also yielded colonies, suggesting that at the very least
the URA3
gene of the backbone was taken up.

To verify this, all three plasmids were extracted from the colonies and sent for sequencing. This
showed a
nearly 90% match between the designed promoter-RBS-Yuab section of the plasmid and the corresponding
part of
the sequencing result. This highly suggests that our entire construct is intact in the *E.
coli*. As
for the CHS3 plasmid, the received sequence had more mutations in it. This is not surprising since
the CHS3
plasmid seemed to be contaminated with several additional sequences prior to transformation. Due to
a lack
of time it was transformed into the yeast regardless.

These results signify the effectiveness of the protocols used, as well as the construction of the
plasmids.
However they also highlight the need for further investigation into the CHS3 plasmid.

The final transformation into *B. subtilis* did not yield any colonies. There are a number of
factors
which might have caused this. Since the sequencing result of that plasmid was good, it is assumed
that there
is nothing inherently wrong with the plasmid. One reason for the failure might simply be that the
transformation protocol either wasn’t very effective or that it was not followed properly, either in
the
media preparation or in the actual transformation steps. Missteps in either of these could have
caused the
high cell mortality. Another reason could be that *B. subtilis* does not take up plasmids as
readily
as *E. coli* does. *E. coli* has been genetically modified in several published
studies, but it’s
possible
that it has a higher learning curve than model organisms. A final reason might have been a simple
lack of
luck. At times, it is hard to weed out the specific reason that a method did not work.

## Drylab results

The docking simulations showed that both chitinase-92 and SP1 are capable of binding chitin-related
ligands with comparable binding energies, particularly for the closed-ring structures. The lowest free
binding energy was observed for the chitinase-92 NAG trimer complex, with a free binding energy of -5.23
kcal/mol, indicating a strong and stable interaction. The open-ring ligand docks with chitinase-92 were
roughly 1.5 kcal/mol higher in energy, suggesting slightly weaker but still favorable binding. SP1
demonstrated stronger predicted binding to chitobiose, consistent with previously reported experimental
adhesion results, while chitinase-92 showed stable interactions across multiple ligands.

However, due to the low-confidence structural model of SP1, these results should be interpreted with
caution. The predicted binding sites were mainly located within structured regions of the proteins, with
chitinase-92 favoring the interface between its two dense domains. Although the planar β-sheet region of
SP1 was not accessible under the rigid docking conditions, other favorable binding pockets were
identified. Overall, the results support the potential of both binding domains to mediate chitin
adhesion, while also emphasizing the need for experimental validation to confirm the modeled
interactions. A more detailed description and interpretation of the modeling and docking analysis can be
found on our [model page](./model#results).

## Project outlook

While this project did not manage to test the adhesion of *B. subtilis* to modified *S.
cerevisiae*,
it has laid the groundwork for future research to pick up where we left off. If future efforts are made,
it will be interesting to see if a modified transformation protocol for *B. subtilis* is necessary,
and if it is possible to make it bind to chitin. It would also be interesting to see this combined with
an increased Iturin A production, as was done by Yue H. et al. [1](#ref1),
to study if that and/or the adhesion affects the survival of the target
cells. Our specific area aside, we are looking forward to following
the development of using bacteria to deliver drugs and if adhesion to target cells will be a large part
of those efforts. Maybe the adhesion could be adapted to including a receptor which could cause the
release
of apoptosis-triggering molecules on to the yeast, much like how the immune system battles cancers
[2](#cancer).

Further into the future, it would be interesting to see this applied to actual *C. albicans* cells,
both in single cell and hyphal form, as well as in larger models in clinical trials.

Future work in the dry lab could expand on our results by investigating molecular docking with a more
advanced fungal cell wall model, in which chitin structures are accurately represented within the
overall architecture. Such a model would enable a more realistic assessment of the adhesion properties
of CHI92 and SP1 to the fungal cell wall, both in the yeast and hyphal morphologies.
Additionally, further simulations could explore alternative ligands or docking partners to better
understand binding specificity and optimize interaction strength [3,](#ref2)[4,](#ref3)[5](#ref4).

When modeling skin infections, it will be important to consider alternative infection models, since
structural differences in hair follicles and epidermal layers between species can lead to varying
infection behaviors among fungal pathogens such as *C. albicans* and *Candida auris*
[6](#ref5).

Exploring these antifungal therapies for animals is also highly relevant from a global health
perspective. Such treatments could contribute to food security and help mitigate the spread of
antimicrobial resistance, a growing issue linked to the extensive use of antibiotics in livestock.
Furthermore, advanced interventions in animals could reduce the risk of major zoonotic
diseases—such as salmonellosis, campylobacteriosis, and pathogenic *E. coli* infections that can
be transmitted from animals to humans [7](#ref6).

## References

1. Yue H, Zhong J, Li Z, Zhou J, Yang J, Wei H, et al. Optimization of iturin A production
   from *Bacillus subtilis* ZK-H2 in submerge fermentation by response surface methodology. 3
   Biotech. 2021 Feb;11(2):36.
2. Kristenson, L. NK cell recognition of malignant cells - a CRISPR approach to define novel mediators.
   Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska Academy,
   University of Gothenburg. 2025.
3. Arkowitz RA, Bassilana M.
   [Recent advances in understanding Candida albicans hyphal growth.](https://f1000research.com/articles/8-700/v1)
   F1000Res. 2019 May 21;8:F1000 Faculty Rev-700. doi: 10.12688/f1000research.18546.1. PMID: 31131089;
   PMCID: PMC6530606.
4. Chaffin WL, López-Ribot JL, Casanova M, Gozalbo D, Martínez JP.
   [Cell wall and secreted proteins of Candida albicans: identification, function, and expression.](https://journals.asm.org/doi/10.1128/mmbr.62.1.130-180.1998)
   Microbiol Mol Biol Rev. 1998 Mar;62(1):130-80. doi: 10.1128/MMBR.62.1.130-180.1998. PMID: 9529890;
   PMCID: PMC98909.
5. Chaffin WL.
   [Candida albicans cell wall proteins.](https://journals.asm.org/doi/10.1128/mmbr.00032-07)
   Microbiol Mol Biol Rev. 2008 Sep;72(3):495-544.
   doi: 10.1128/MMBR.00032-07. PMID: 18772287; PMCID: PMC2546859.
6. Saskia Seiser, Hossein Arzani, Tanya Ayub, Trinh Phan-Canh, Clement Staud, Christof
   Worda, Karl Kuchler, Adelheid Elbe-Bürger,
   [Native human and mouse skin infection models to study Candida auris-host interactions,
   Microbes and Infection](https://doi.org/10.1016/j.micinf.2023.105234),
   Volume 26, Issues 1–2, 2024, 105234, ISSN 1286-4579
7. Anee, I.J., Alam, S., Begum, R.A. et al.
   [The role of probiotics on animal health and nutrition.
   JoBAZ 82, 52 (2021).](https://doi.org/10.1186/s41936-021-00250-x)

{% endblock %}