On this page we've documented everything that was done during each week of the project.
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.
During this week's meeting, we all presented what we had learned after researching the different project ideas suggested last week.
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.
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.
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.
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.
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.
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.
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-š¼. 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.
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.
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.
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
didnāt 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 amilCPās chromophore to water and oxygen.
This week we also had another meeting discussing the game with the other
Swedish iGEM teams.
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.
This week we rounded up the wetlab activities. We sent our mutants for sequencing, and repeated the PCR of Ile195.
Since we didnāt 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 -80ā 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.
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.
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.
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.