iGEM 2024 — Team uppsalauniversity

{% extends "layout.html" %} {% block title %}Notebook{% endblock %} {% block
lead %}On this page we've documented everything that was done during each week
of the project.{% endblock %} {% block page\_content %}

# Team Notebook

## Week 17 (April 22-26)

The start of iGEM 2024! We had the first meeting with the team and
started brainstorming project ideas for the summer. Professor Anthony
Forster and Margareta Krabbe joined the meeting to introduce themselves
and the course.

## Week 19 (May 6-10)

During this week's meeting, we all presented what we had learned after
researching the different project ideas suggested last week.

## Week 20 (May 13-17)

For this week, we were all tasked to submit a project proposal for one
of the different project ideas. After presenting the proposals, we voted
on which projects we thought were the most interesting and viable. We
also had an insightful lecture on biosensors with Professor Helena
Danielson.

## Week 21 (May 20-24)

We sent our project proposals that we worked on last week to Margareta
for feedback. Other than that, not a lot was done this week since many
of us were studying for exams and wrapping up the school year before
summer, before the start of the experimental part of the iGEM course.

## Week 23 (June 3-5)

The start of the experimental part of the iGEM course! During the first
few days, we were in the process of getting integrated into the lab. We
got a safety introduction from our PI and other faculty working in our
corridor. We synthesized LB to create LA plates with and without
kanamycin and SOB medium, to be used for creating competent cells and in
transformation experiments. We also continued researching the two
surviving project ideas; bioremediation of PET and PFAS.

## Week 24 (June 10-14)

We started the week by continuing the work in the lab: we made LA
plates, competent E.coli cells, and transformed mRFP1 plasmids into the
competent cells. This week, we were also divided into six different
subgroups; bioinformatics, human practices, wiki, social, graphical
design, and fund-raising. We continued researching the PET and PFAS
project ideas, and added a third option; filtering out gold/heavy metals
from electronic waste. They were all presented to iGEM-related
professors, teachers, and advisors (Margareta Krabbe, Anthony Forster,
Dianna Zeleskov, and Professor Gunnar Johansson). After valuable
feedback, we decided on terminating the PFAS project as it was not
deemed to be viable.

Later in the week we were kindly invited to listen to two
researchers from the Department of Cell and Molecular Biology talk about
their ongoing research on colorful psychrophiles. From this meeting, a
new project idea was born: determining what causes these organisms to
express color, in an effort to find new biodyes.

## Week 25 (June 17-20)

This week we finally selected our project for the summer; finding novel
biological colors. We had a waste management tour and a lecture by
Professor Gunnar Johansson about separation techniques for our project.
Later in the week we chose our village; Fashion & Cosmetics, and
submitted a safety and check-in form to iGEM. We looked into the
methodologies and found protocols for both the environmental strains and
the green chromoprotein maturation sub-projects. Our first version of
the project description was put on the wiki. The funding subgroup worked
on drafts for sponsorship emails, while the human practices group
discussed possible avenues.

## Week 26 (June 24-30)

The week started off with a project briefing meeting together with
Margareta Krabbe, Anthony Forster, chromoprotein expert Letian Bao, and
iGEM board member Adam Hallberg. We presented our project plan for the
two parts; extracting novel colors from environmental samples and
modifying an existing green chromoprotein. We got some valuable feedback
and answers to current questions.

We re-did the transformation experiments from the beginning of June, to
confirm the competence of our E.coli cell strains. We also transformed
the green chromoprotein plasmid into competent cells. We went through
our inventory to check for the chemicals we need for upcoming
experiments, designed primers for the site-directed mutagenesis and
error prone PCR experiments we would do, and got more acquainted with
the chosen protocols and assays. The bioinformatics subgroup continued
working on the pipeline for the environmental strains and developing a
strategy to find possible sites in the green chromoprotein in which we
can introduce mutations to improve maturation.

During the week we all submitted project name suggestions and decided on our groupname.
The graphical design subgroup
started working on the homepage design of our wiki. We took some really
nice team pictures to publish on the wiki and on social media.

At the end of the week, half of the team (Samuel F, Samuel J, Thanos,
Andreas, Alex and William) traveled to Lund to attend the Swedish iGEM
Conference to meet our fellow Swedish iGEM teams, present our project
and exchange ideas.

## Week 27 (July 1-5)

We started off the week with a project briefing meeting to discuss where
we are at with the different parts of the project, and get some feedback
from teachers. The people who went to Lund also shared their experiences
from the Swedish iGEM Conference.

In the wet lab we evaluated transformation results and transformed
superfold-mutant plasmids into competent DH5-𝛼. We re-streaked and
inoculated the environmental strains that got approved from iGEM and
made growth curves for the liquid cultures. We designed more primers for
site directed mutagenesis of the green chromoprotein, and ordered them.
Later on, we made glycerol stocks of strains for the chromoprotein
maturation sub-project, as well as the environmental strains that showed
good growth in liquid medium, to be stored in -80 degrees Celsius. A
lysis buffer for next week was made. Lastly we made plasmid
preparations, and transformed plasmids with promoters we want into
competent cells.

The bioinformatics team finished the environmental strains pipeline and
ran the strains that have been sequenced. We analyzed results for
chromoprotein and pigment enzymatic synthesis candidates. Later in the
week we had a meeting with Professor Hugo Gutierrez de Teran to discuss
the script and possible avenues moving forward, as well as a meeting
with Professor Jan Komorowski about using machine learning. We also got
access to Uppmax to be used for computational power.

This week we reached out to many local companies to ask for funding. We
also started our individual presentations on our Instagram and
(re)decided on a team name; ColorFold. Additionally, we researched
previous teams human practices and the whole group visited Testa Center
to find out more about their operations.

## Week 28 (July 8-12)

We have made a lysis of environmental strains, and found that after
centrifugation only 6 had color in the supernatant and the rest only had
color in the pellet, indicating potential hydrophobic color molecules.
We have also made and run agarose gels on one lysate with no good
results. We have made even more plasmid preps and also digested the
plasmids that will be used in BioBrick assembly for the amilCP project.
Agarose gels were also run on the intact and digested plasmids, with
varying results. We also started working on the maturation assay for
amilCP.

We had a meeting with Nils-Krister Persson for the human practices work.
We also had a meeting with the other Swedish iGEM team and planned a
collaboration within the education aspect of the competition.

## Week 29 (July 15-19)

This week we have conducted three different PCR experiments on
AmilCP\_green. We started with PCR for one point and two point mutation
at position 37-39 that will resemble previous known superfolding
chromoproteins (SF\_AmilCP\_PM1(TQT), SF\_AmilCP\_PM2(TQN)). This experiment
was successful and we have transformed the products. Some cultures are
now growing on plates. Next, we tried PCR for all point mutations at
position Ile157Xxx (Gln, His, Asp, Trp, Pro, Gly), but these experiments
failed as no bands were visible on the gel after PCR. We will repeat the
PCR in week 30. We also had success with PCR for all point mutations at
position Leu195Xxx (Gly, Val, Ala), specifically for the mutations
Leu->Gly and Leu->Val. We also performed an error prone PCR which was
successful and will be transformed during week 30.

In addition, we have performed SEC runs with three of our environmental
samples. We started by running SEC with mRFP1 and bromophenol blue to
demonstrate the separation of pigments and proteins. Next, we performed
an SEC run with E.S. against mRFP1 and bromophenol blue as a control.
Samples 91, 153 and 350 showed potential protein/protein bound pigments.
To concentrate the flow from previous SEC runs, we used a spin column on
samples 91 and 350.Sample 153 was found to be slimy like to keep
purifying . We have set up new overnight cultures for week 30 for
further SEC and spin column runs.

Human Practicing had a meeting with all the Swedish iGEM teams about
collaborating on a board game as an outreach.

## Week 30 (July 22-26)

This week in the lab we redid PCRs of our color-change mutations and
ligated and transformed these as well as our error prone PCR product
from last week. We also started two new error prone PCRs, one where we
didn’t include the promoter in the amplified sequence and one where we
used lower concentrations of manganese.

For the environmental samples, we worked on protein concentration
through size exclusion chromatography and concentration columns. We also
conducted some experiments of mRFP1 since the bands we got on SDS-PAGEs
were at odd places. This included measuring growth curves for
IPTG-inducible mRFP1 as well as running SDS-PAGEs of inducible mRFP1
that had been exposed to IPTG for different lengths of time. The
inducible mRFP1 was also run on a SDS-PAGE together with concentrated ES
91 and 350, non-inducible mRFP1 and amilCP, after which we could
conclude that the mRFP1s were the same.

The bioinformatics team performed molecular dynamics simulations to test
the accessibility of amilCP’s chromophore to water and oxygen.

This week we also had another meeting discussing the game with the other
Swedish iGEM teams.

## Week 31 (July 29-August 2)

Our final full lab week! We started off the week in the wet lab with
some digestions, ligations and transformations of the new error prone
PCR as well as the Leu157 mutation PCR done last week. We started our
first maturation assay of our superfolder-mutations, turbo-mutations and
error prone mutations, which continued for 48 hours. Alongside this
assay, colony PCR was done on interesting candidates, in preparation for
sequencing. To evaluate if the ligation and transformation in our error
prone experiment worked, a plasmid prep was carried out from an
overnight culture. After running the purified sample on an agarose gel,
no clear band was visible, indicating that the assembly of the amplified
amilCP insert into the pSB1K3 backbone likely was unsuccessful. Because
of this, we redid ligation and transformation of 0,1 mM and 0,2 mM error
prone PCR. To validate mutations created in PCR, we also prepared oligos
containing the whole amilCP sequence, with the mutations we wish to
introduce.

For the environmental samples 91 and 350, we attempted new semi-native
SDS-PAGE runs since previous ones gave a smeared band. After a couple of
tries, we achieved a pretty clear band at roughly 25 kDa.

## Week 32 (August 5-9)

This week we rounded up the wetlab activities. We sent our mutants for
sequencing, and repeated the PCR of Ile195.

## Week 33 (August 12-16)

Since we didn’t get any good results from last week's sequencing, we did
some testing to find the problem. We froze in colonies that are of
interest, the same ones that we will sequence, in the -80℃ freezer for
long time storage. We also conducted colony PCR of the turbo mutations
valine and glycine, and did PCR, digestion and ligation of isoleucine.

## Week 34 (August 19-23)

Since the results from last week's sequencing came back and were
successful, we commenced with sequencing all the rest of our mutants of
interest. We also transformed the isoleucine mutation that was prepared
last week.

We assisted High School students that visited the university to conduct
a laboratory in biotechnology. We also held a presentation about iGEM
and our project to them.

In the subgroups, we kept working on analyzing results, summarizing
everything and updating wiki pages.

## Week 35-40 (August 26-October 2)

These weeks we worked on summarizing everything we have done this summer
on the wiki, in preparation for the wiki. We have worked on the
promotion and presentation video, and finished up the human practices
parts of our project.

## Week 39 (September 23-27)

This week we performed some final experiments to characterize our parts,
and get nice pictures for the wiki and registry. This included
restreaking the mutants, lysis of liquid cultures and absorbance
spectrum measurements.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}{% endblock %}
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#### The engineering cycle is made out of four distinct steps; design, build, test and learn. Throughout the cycle, an idea can be turned into a prototype which can be tested. Experience from this test can then be used to inform the next iteration, until a version working as intended is achieved. On this page, we have illustrated the engineering cycle we went through with one part of our project, the creation of new colors of amilCP.

---

# Color mutations - the engineering cycle

## Design

---

In this part of the project, we wanted to find mutations that caused amilCP to take on different colors. We decided to use the E.coli strain DH5-α to express the protein, since they are safe, grow quickly and are well equipped for transformation and thus a good choice for this purpose. To introduce the mutations, we decided to focus on the amino acid position N157 in the protein (not GFP-numbering). We opted to design primers to introduce various specific amino acid substitutions via PCR, as well as design primers with completely random bases at amino acid position N157. Inverse PCR was selected for this purpose since it avoided having to assemble the various parts, thus reducing the complexity of the design and improving its probability of success.
For our backbone, we chose pSB1K3. This was partly because it was convenient as we had access to the gene in this backbone. However, the fact that the plasmid was high-copy suited our experiments, as it made it easier to clearly see if new colors developed. Kanamycin as the selection marker was chosen over chloramphenicol since our control mRFP1 had the same resistance, enabling restreaking them on the same plates.
To test whether the design worked, we chose to firstly visually evaluate the color of the colonies. If they obtained a different color than E.coli that doesn’t express any chromoproteins or other pigments and a different shade than the already discovered blue, green and pink variants of amilCP, we would know that the implementation was successful. In that case, we would move forward with further characterization, with an assay of maturation time, to evaluate the viability of the modified parts, as well as measure the absorbance maximum of the chromoprotein.
The primers were designed in such a way that the forward and reverse primers were positioned right next to each other (Figure 1), which enabled amplification of the whole plasmid through inverse PCR. We designed the primers in SnapGene, and calculated annealing temperatures using Thermo Fisher’s Tm calculator. The difference in melting points in primer pairs, the GC content, probability of the primers forming hairpin loops or self-dimers were all calculated using IDT’s OligoAnalyzer and taken into account when designing the primers.

![colorprimers](https://static.igem.wiki/teams/5527/colorprimers-amilcp.webp)

**Figure 1:** All primers for the color mutations experiments. F = Forward, R = Reverse. One of two reverse primers were used for each specific mutant, depending on the melting point of the forward primer.

## Build

---

The primers were phosphorylated at the 5’ end to enable ligation of the amplified segment into a circular plasmid after the inverse PCR. To confirm the inverse PCR worked as expected, agarose gel electrophoresis was used. A band around 2800 bp indicated successful amplification (Figure 2). After self-ligation using T4 DNA ligase, the plasmid was then transformed into competent DH5-α E.coli. Since we chose pSB1K3 as the vector we used kanamycin as the selection marker.

![color_pcr_gel](https://static.igem.wiki/teams/5527/gelpic-colormut-better.png)

**Figure 2:** Agarose gel (1%) of PCR product after inverse PCR of the color mutations.

To confirm that the build worked as intended, in other words that the mutations were the right ones and at the right position, colony PCR was performed. Primers annealing to both ends of the amilCP gene were used, and the PCR product was analyzed by agarose gel electrophoresis to confirm the size. The PCR products were then sent to Eurofins for Sanger sequencing.

## Test

---

To test and characterize the correctly constructed builds, we measured their maturation times and absorbance maximum. The former is an important value when evaluating the protein’s usefulness, and the latter helps to more precisely and quantitatively characterize its function than the qualitative visual inspection done by eye.
The maturation assay utilized a camera to automatically take pictures of restreak LB-agar plates of the mutants at regular intervals during a 96 hour period (Figure 3). mRFP1 and green amilCP were used as controls on all plates, which helped to evaluate the accuracy of the analysis by comparing the variance between plates.

![mat-assay](https://static.igem.wiki/teams/5527/maturation-assay.png)

**Figure 3:** The plates from the maturation assay.

The final data result attained through this analysis is the chromoproteins halftime value. This is the time it takes for half of the maximum intensity to be reached.
More information about these results can be found on  [AmilCP results](https://2024.igem.wiki/uppsalauniversity/amilcp-results).

## Learn

---

Through this engineering cycle, we reaffirmed previous observations that position 157 is important for the color of the chromoprotein. Since the aim of this part of the project was to find new colors of amilCP, we can conclude that our design and the engineering cycle was successful. The new and characterized colors (Figure 4) have been submitted as new parts to the registry, and can be found [here](https://2024.igem.wiki/uppsalauniversity/contribution).

![color_mut_liquid](https://static.igem.wiki/teams/5527/results/color-mutations-liquid.webp)

**Figure 4:** Some of the colors we got from the introduced mutations.

If we were to do a similar project in the future, we would try to improve the test stage, specifically the maturation assay. The odd behaviors seen in the results from the maturation assay indicate some fine tuning of the setup is necessary for more accurate results. This would include making sure the camera settings are always the same and don’t adjust automatically, and that the lighting is good and constant.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Protocols{% endblock %}
{% block lead %}{% endblock %}
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## Protocols

In the following PDF we have gathered all the protocols we've used
throughout our project.

Your browser does not support PDFs.
[Download the PDF](https://static.igem.wiki/teams/5527/pdf/igem-protocols-2.pdf)

---

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

---

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](https://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

---

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project and what you learned from them. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort. Furthermore, you can still impress the judges by thoughtfully discussing what went wrong and how you might change things.

## Inspirations

---

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

{% endblock %}