iGEM 2020 — Team Chalmers-Gothenburg

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# Team:Chalmers-Gothenburg/Notebook

iGEM Chalmers Gothenburg 2020

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[Supervisors](https://2020.igem.org/Team%3AChalmers-Gothenburg/Supervisors)
[Attributions](https://2020.igem.org/Team%3AChalmers-Gothenburg/Attributions)

[Project](https://2020.igem.org/Team%3AChalmers-Gothenburg/Project)

[Description](https://2020.igem.org/Team%3AChalmers-Gothenburg/Description)
[Implementation](https://2020.igem.org/Team%3AChalmers-Gothenburg/Implementation)
[Parts](https://2020.igem.org/Team%3AChalmers-Gothenburg/Parts)
[Safety](https://2020.igem.org/Team%3AChalmers-Gothenburg/Safety)

[Human
Practices](https://2020.igem.org/Team%3AChalmers-Gothenburg/Human_Practices)

[Integration](https://2020.igem.org/Team%3AChalmers-Gothenburg/Integration)
[Science Communication](https://2020.igem.org/Team%3AChalmers-Gothenburg/Education)
[Collaborations](https://2020.igem.org/Team%3AChalmers-Gothenburg/Collaborations)
[Sustainable](https://2020.igem.org/Team%3AChalmers-Gothenburg/Sustainable)

[Wet Lab](https://2020.igem.org/Team%3AChalmers-Gothenburg/Wetlab)

[Design](https://2020.igem.org/Team%3AChalmers-Gothenburg/Design)
[Results](https://2020.igem.org/Team%3AChalmers-Gothenburg/Results)
[Measurement](https://2020.igem.org/Team%3AChalmers-Gothenburg/Measurement)
[Notebook](https://2020.igem.org/Team%3AChalmers-Gothenburg/Notebook)

[Dry Lab](https://2020.igem.org/Team%3AChalmers-Gothenburg/Model)

[Metagenomics](https://2020.igem.org/Team%3AChalmers-Gothenburg/Metagenomics)
[GEM](https://2020.igem.org/Team%3AChalmers-Gothenburg/GEM)
[Kinetic Model](https://2020.igem.org/Team%3AChalmers-Gothenburg/ODE)
[Reproducibility](https://2020.igem.org/Team%3AChalmers-Gothenburg/Reproducibility)

[For Judges](https://2020.igem.org/Team%3AChalmers-Gothenburg/ForJudges)

[Engineering Success](https://2020.igem.org/Team%3AChalmers-Gothenburg/Engineering)
[Awards And Medals](https://2020.igem.org/Team%3AChalmers-Gothenburg/Awards)
[Contribution](https://2020.igem.org/Team%3AChalmers-Gothenburg/Contribution)

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# Notebook, Protocols and Primers

For details about the wet lab procedure, check out the lab notes below! For each week in the lab,
methods and results are presented for all our experiments. The method description commonly refers to
a standard protocol, all of which are included below as well.

The primers used in the experiments are referred to as numbers in both lab notes and protocols.
Under “Primer Excel Sheet”, a summarizing description of the primers and their numbers can be found.

**Protocols**

[Cell lysis and DC assay »](https://static.igem.org/mediawiki/2020/6/6f/T--Chalmers-Gothenburg--DCAssay.pdf)
[FastDigest »](https://static.igem.org/mediawiki/2020/9/9f/T--Chalmers-Gothenburg--fastDigest.pdf)
[Fluorescence microscopy »](https://static.igem.org/mediawiki/2020/7/75/T--Chalmers-Gothenburg--fluoroMicro.pdf)
[His-Tag purification »](https://static.igem.org/mediawiki/2020/0/08/T--Chalmers-Gothenburg--hisTagPur.pdf)
[Gel electrophoresis and gel extraction »](https://static.igem.org/mediawiki/2020/1/1b/T--Chalmers-Gothenburg--gelElec.pdf)
[Gibson assembly »](https://static.igem.org/mediawiki/2020/d/d2/T--Chalmers-Gothenburg--gibsonAss.pdf)
[PCR amplification of block segment »](https://static.igem.org/mediawiki/2020/2/2e/T--Chalmers-Gothenburg--PCRAmpli.pdf)
[PCR product purification »](https://static.igem.org/mediawiki/2020/a/ae/T--Chalmers-Gothenburg--PCRproduct.pdf)
[Plasmid miniprep »](https://static.igem.org/mediawiki/2020/c/c2/T--Chalmers-Gothenburg--miniprep.pdf)
[Plate reading »](https://static.igem.org/mediawiki/2020/e/e9/T--Chalmers-Gothenburg--plateRead.pdf)
[Creating competent cells »](https://static.igem.org/mediawiki/2020/7/7a/T--Chalmers-Gothenburg--CompetentCell.pdf)
[Preparation of media and plates »](https://static.igem.org/mediawiki/2020/7/76/T--Chalmers-Gothenburg--plateMediaPrep.pdf)
[Promoter characterization »](https://static.igem.org/mediawiki/2020/3/33/T--Chalmers-Gothenburg--promoterChar.pdf)
[Transformation »](https://static.igem.org/mediawiki/2020/6/6a/T--Chalmers-Gothenburg--Transformation.pdf)
[Western Blot & SDS-Page »](https://static.igem.org/mediawiki/2020/8/8d/T--Chalmers-Gothenburg--sdsWb.pdf)

**Primer Excel Sheet**

[Primers.xlsx »](https://static.igem.org/mediawiki/2020/7/70/T--Chalmers-Gothenburg--primers.xlsx)

# Week 28

Our first week in the lab! Some preparatory work such as preparing selective media and agar
plates, producing competent MG1C655
cells and noting the concentrations of ordered DNA was done. We also proceeded with the
promoter
characterization from previously and, once the primers arrived, amplified each gene using
PCR.

[Read PDF
»](https://static.igem.org/mediawiki/2020/3/32/T--Chalmers-Gothenburg--Week28.pdf)

# Week 32

Send positive colonies for sequencing and more screening for this week. Beginning of the
week, we tried PCR but we shifted
the method to digestion. Since almost all colonies were checked and got result as negative,
we
tried again from Gibson Assembly as well. They will be screening for next week.

[Read PDF
»](https://static.igem.org/mediawiki/2020/d/de/T--Chalmers-Gothenburg--Week32.pdf)

# Week 36

A week consisting of intense digest screening in a last attempt to find positive colonies.
We also ran another protein extraction
and Western blot, this time with quite good results. Found protein expression for A3 but
that
they aggregate to a quite large extent.

[Read PDF
»](https://static.igem.org/mediawiki/2020/3/35/T--Chalmers-Gothenburg--Week36.pdf)

# Promoter characterization

In this document we describe the laboratory procedure for our contribution, in which we characterized three promoters from the parts registry: BBa\_J23104, BBa\_J23106 and BBa\_J23115. In the notebook, these are referred to as A, B and C, respectively. The process includes introducing each promoter in pSB1C3 upstream of an RFP gene, assembling the plasmid, transforming and cultivating bacteria, and studying the fluorescent levels in fluorescence microscopy and in a plate reader. Details about data analysis are found in the contribution page.

[Read PDF
»](https://static.igem.org/mediawiki/2020/2/23/T--Chalmers-Gothenburg--Note-PromoterChr.pdf)

# Week 29

We proceeded by purifying the amplified block segments and started trying to assemble the
gene block plasmids through Gibson
assembly. Different alternative methods to get better results from the amplification of some
of the genes were attempted. As problems arose with the Gibson assembly of the gene blocks,
fusion
PCR was attempted as an alternative assemble method. The genes were amplified with primers
introducing
his-tags as a result of the re-design of the project.

[Read PDF
»](https://static.igem.org/mediawiki/2020/e/e3/T--Chalmers-Gothenburg--Week29.pdf)

# Week 33

More screening, sending positive colonies for sequencing, and tried to measure the
concentration of protein. This protein
concentration measurement methods will be used for western blot which we will do later of
this
project.

[Read PDF
»](https://static.igem.org/mediawiki/2020/9/97/T--Chalmers-Gothenburg--Week33.pdf)

# Week 37

This week we tried to get protein expression of BphB and decrease the aggregation of BphC by
cultivating bacteria to different
growth phases before extracting the proteins. Both bacteria were cultivated to exponential
and
stationary phase respectively and run in western blot.

[Read PDF
»](https://static.igem.org/mediawiki/2020/0/0d/T--Chalmers-Gothenburg--Week37.pdf)

# Week 30

Problems were identified regarding assembling the block segments and were solved by
amplification of the plasmid backbone
used in the promoter characterization, as this sequence corresponded to the sequence used
when
designing the overlaps between the his-tagged block segment and the plasmid backbone for the
Gibson assembly. This assembly into a new backbone was attempted for all the his-tagged
genes.
Further attempts to use fusion PCR to assemble the gene blocks were done.

[Read PDF
»](https://static.igem.org/mediawiki/2020/1/17/T--Chalmers-Gothenburg--Week30.pdf)

# Week 34

This week, more screening for positive his-tagged block segment transformants was done, as
well as sending the positive colonies
for sequencing. We also did our first protein extraction and western blot of three confirmed
positive colonies.

[Read PDF
»](https://static.igem.org/mediawiki/2020/9/9f/T--Chalmers-Gothenburg--Week34.pdf)

# Week 38

No lab done this week, and as a result we have no notes to present.

# Week 31

This week, we tried to establish the gene block A plasmid, tried to fix the problem about
backbone (pSB1C3), constructed
each his tagged strain and screening for them. First, we tried to do normal colony PCR but
it
had so much noise to check them, we change the methods to extract plasmid before start PCR.
We
could get some positive colonies so we will send them for sequence for next week and need
more
screening for other strains.

[Read PDF
»](https://static.igem.org/mediawiki/2020/5/5f/T--Chalmers-Gothenburg--Week31.pdf)

# Week 35

Further screening of transformants with different his-tagged block segments was the focus of
this week. For some of the block
segments, new Gibson assembly and transformation were done as all colonies seemed negative.
A
new protein extraction and Western blot was attempted for all the confirmed positive
colonies.

[Read PDF
»](https://static.igem.org/mediawiki/2020/4/44/T--Chalmers-Gothenburg--Week35.pdf)

# Week 39

We ran another protein extraction and made another attempt to measure the activity of BphC
(A3).

[Read PDF
»](https://static.igem.org/mediawiki/2020/a/ae/T--Chalmers-Gothenburg--Week39.pdf)

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# Team:Chalmers-Gothenburg/Engineering

iGEM Chalmers Gothenburg 2020

[Home](https://2020.igem.org/Team%3AChalmers-Gothenburg)

[People](https://2020.igem.org/Team%3AChalmers-Gothenburg/People)

[Team](https://2020.igem.org/Team%3AChalmers-Gothenburg/Team)
[Supervisors](https://2020.igem.org/Team%3AChalmers-Gothenburg/Supervisors)
[Attributions](https://2020.igem.org/Team%3AChalmers-Gothenburg/Attributions)

[Project](https://2020.igem.org/Team%3AChalmers-Gothenburg/Project)

[Description](https://2020.igem.org/Team%3AChalmers-Gothenburg/Description)
[Implementation](https://2020.igem.org/Team%3AChalmers-Gothenburg/Implementation)
[Parts](https://2020.igem.org/Team%3AChalmers-Gothenburg/Parts)
[Safety](https://2020.igem.org/Team%3AChalmers-Gothenburg/Safety)

[Human
Practices](https://2020.igem.org/Team%3AChalmers-Gothenburg/Human_Practices)

[Integration](https://2020.igem.org/Team%3AChalmers-Gothenburg/Integration)
[Science Communication](https://2020.igem.org/Team%3AChalmers-Gothenburg/Education)
[Collaborations](https://2020.igem.org/Team%3AChalmers-Gothenburg/Collaborations)
[Sustainable](https://2020.igem.org/Team%3AChalmers-Gothenburg/Sustainable)

[Wet Lab](https://2020.igem.org/Team%3AChalmers-Gothenburg/Wetlab)

[Design](https://2020.igem.org/Team%3AChalmers-Gothenburg/Design)
[Results](https://2020.igem.org/Team%3AChalmers-Gothenburg/Results)
[Measurement](https://2020.igem.org/Team%3AChalmers-Gothenburg/Measurement)
[Notebook](https://2020.igem.org/Team%3AChalmers-Gothenburg/Notebook)

[Dry Lab](https://2020.igem.org/Team%3AChalmers-Gothenburg/Model)

[Metagenomics](https://2020.igem.org/Team%3AChalmers-Gothenburg/Metagenomics)
[GEM](https://2020.igem.org/Team%3AChalmers-Gothenburg/GEM)
[Kinetic Model](https://2020.igem.org/Team%3AChalmers-Gothenburg/ODE)
[Reproducibility](https://2020.igem.org/Team%3AChalmers-Gothenburg/Reproducibility)

[For Judges](https://2020.igem.org/Team%3AChalmers-Gothenburg/ForJudges)

[Engineering Success](https://2020.igem.org/Team%3AChalmers-Gothenburg/Engineering)
[Awards And Medals](https://2020.igem.org/Team%3AChalmers-Gothenburg/Awards)
[Contribution](https://2020.igem.org/Team%3AChalmers-Gothenburg/Contribution)

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# Engineering Success

**Introduction**

Our project was born out of the ambition of having a positive impact in society. We conducted extensive
research on different types of waste and their geographical, societal and environmental impact and found
that, for several types of waste, solutions within synthetic biology were already being developed.
Further research pointed towards the source of a huge amount of waste that typically accumulates in
landfills: the textile industry (read more in the [Description](https://2020.igem.org/Team%3AChalmers-Gothenburg/Description) section).
Figure 1 illustrates the Design-Build-Learn cycle, which we have implemented when developing our project.
![DPL cycle](https://static.igem.org/mediawiki/2020/8/8b/T--Chalmers-Gothenburg--Engineeringsuccess--Figure1.png)
**Figure 1.** Graphic summary of our Design-Build-Learn system. We started by
designing our constructs and
transform them in E. coli. We built our strain and planned our implementation. However, when testing
this in silico and receiving feedback from our stakeholders, we realized our approach was not optimal.
Therefore, we re-designed our project to overcome the problems that we were presented with.

**Research and imagine**

We started gathering information from companies, municipalities and fashion actors to comprehend the
full picture and figure out why textiles are not recycled to a greater extent (read more in the [Integration](https://2020.igem.org/Team%3AChalmers-Gothenburg/Integration) section). We
found that the main problem is the blending of different materials. For example, in jeans, cotton cannot
be recycled due to the presence of the synthetic fibre Elastane. Digging deeper into this issue, an idea
started to form. We imagined a biological degradation of the Elastane fibre, by engineering a strain
that would consume the elastane fibre out of the textile.

**Design**

To realize our idea, we designed two gene blocks containing four and five genes respectively. This
design was based on being able to make three bacterial strains; one that degrades the soft segment of
the fibre, one that degrades the hard segment and finally one strain that could be transformed with both
gene blocks. The reasoning behind these two gene blocks was that it might be of industrial interest to
degrade the soft and hard segments separately, as the final products are different and might be usable
for other purposes. Our original implementation design was to grow the bacteria in a bioreactor using
the plastic fibres as a carbon source. One of the reasons behind this was also our ambition to add value
to the recycling process: theoretically it should be possible to engineer our strain further to produce
a chemical, such as succinate, from the plastic degradation.

**Build, test and learn**

We built our constructs and transformed them into E. coli, but no transformants survived in the
selective media. This was likely due to some assembly problem, maybe the plasmids turned out to be too
large or our Gibson mix simply could not assemble that many fragments at once. A discussion on how to
move forward after the transformation also emerged. When planning activity measurements for each
individual enzyme, it came to our attention that there was no easy way to purify them.

While we were conducting the first experiments, the modelling team started simulating the growth of the
recombinant bacteria in different carbon sources, to test whether they should be able to grow on the
plastic substrates and potentially produce useful chemicals (read more in the [Dry Lab](https://2020.igem.org/Team%3AChalmers-Gothenburg/Modeling) section). From the genome scale
modelling (GEM), we found out that the yield for E.coli growing on polyurethane, PEG, and PET, which are
the plastic fibres that conform elastane, was quite low. Furthermore, according to our simulations it
would also be hard to engineer the strain to produce a chemical.

Simultaneously, as we progressed with Human Practices and gathered more information from stakeholders,
we learned that having a bacterial strain that degrades the Elastane fibres might not be the best
option. First, such an implementation would be hard to fit in the current recycling infrastructure that
many companies already have. Second, cultivating bacteria on a large scale outside of laboratory
environment comes with several issues that might render the process ineffective. We also considered
ethical aspects of our project in collaboration with the iGEM team Lund ([the SynthEthics initiative](https://2020.igem.org/Team%3AChalmers-Gothenburg/Collaborations))
and found that using the bacteria immediately in the textile brings ethical
concerns regarding the spread of GMO.

**Improve**

Based on both the results of our simulations, the ethical concerns and the advice of stakeholders, we
improved our design by building his-tagged block segment plasmids instead. The new design meant having
each gene in its own plasmid, and each plasmid being transformed into its own strain. Furthermore, six
histidine residues were added downstream of the gene, before the terminator sequence so that the enzymes
could be purified. The advantages proved to be numerous; we could now work with the enzymes at different
stages in the laboratory process (as soon as one transformation was successful we could proceed with
screening and protein extraction for that enzyme alone), they could be purified and measured
individually (read more in the [Measurement](https://2020.igem.org/Team%3AChalmers-Gothenburg/Measurement) section) and the assembly included fewer fragments, which greatly facilitated
the assembly.

The [Proposed Implementation](https://2020.igem.org/Team%3AChalmers-Gothenburg/Implementation) was also edited according to our findings during this second round of
research. We went from aiming to produce Elastane-eating bacteria to producing an enzymatic cocktail,
which could be produced entirely within laboratory environment. Only the GM enzymes will be used in
recycling facilities, an option that was preferred in both ethical discussions and by industrial actors.
Furthermore, it will be easier to fine-tune the enzyme activity to achieve an efficient degradation. The
integration with the existing recycling network is also favoured, since it is easier to sell an
enzymatic cocktail than a bioreactor. The design is also more convenient for future projects, since
changing the composition of the enzymatic cocktail by adding enzymes that act on other plastic fibres
would expand the range of plastics that could be degraded.
We believe that our methodology: using in silico simulations to test the efficiency of the process,
researching the available recycling infrastructure and asking the different industrial actors for
feedback, has been an efficient tool to comply with the Design-Build-Test-Learn principle and has helped
us to re-imagine our project in order to be more realistic in the lab and to fit better into society’s
needs.

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# Team:Chalmers-Gothenburg/Experiments

iGEM Chalmers Gothenburg 2020

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[Team](https://2020.igem.org/Team%3AChalmers-Gothenburg/Team)

[Attributions](https://2020.igem.org/Team%3AChalmers-Gothenburg/Attributions)
[Supervisors](https://2020.igem.org/Team%3AChalmers-Gothenburg/Supervisors)
[Advisors](https://2020.igem.org/Team%3AChalmers-Gothenburg/Advisors)

[Project](https://2020.igem.org/Team%3AChalmers-Gothenburg/Description)

[Design](https://2020.igem.org/Team%3AChalmers-Gothenburg/Design)
[Entrepreneurship](https://2020.igem.org/Team%3AChalmers-Gothenburg/Entrepreneurship)
[Sustainable](https://2020.igem.org/Team%3AChalmers-Gothenburg/Sustainable)
[Parts](https://2020.igem.org/Team%3AChalmers-Gothenburg/Parts)
[Engineering Success](https://2020.igem.org/Team%3AChalmers-Gothenburg/Engineering)
[Awards And Medals](https://2020.igem.org/Team%3AChalmers-Gothenburg/Awards)

[Human Practices](https://2020.igem.org/Team%3AChalmers-Gothenburg/Human_Practices)

[Educational Outreach](https://2020.igem.org/Team%3AChalmers-Gothenburg/Education)
[Collaborations](https://2020.igem.org/Team%3AChalmers-Gothenburg/Collaborations)
[Partnership](https://2020.igem.org/Team%3AChalmers-Gothenburg/Partnership)

[Wet Lab](https://2020.igem.org/Team%3AChalmers-Gothenburg/Experiments)

[Measurement](https://2020.igem.org/Team%3AChalmers-Gothenburg/Measurement)
[Results](https://2020.igem.org/Team%3AChalmers-Gothenburg/Results)
[Proof Of Concept](https://2020.igem.org/Team%3AChalmers-Gothenburg/Proof_Of_Concept)
[Primers](https://2020.igem.org/Team%3AChalmers-Gothenburg/Primers)
[Existing Part Improvement](https://2020.igem.org/Team%3AChalmers-Gothenburg/Improve)
[Contribution](https://2020.igem.org/Team%3AChalmers-Gothenburg/Contribution)
[Notebook](https://2020.igem.org/Team%3AChalmers-Gothenburg/Notebook)

Dry Lab

[Design/idea](https://2020.igem.org/Team%3AChalmers-Gothenburg/Modeling)
[ODE](https://2020.igem.org/Team%3AChalmers-Gothenburg/ODE)
[Metagenomics](https://2020.igem.org/Team%3AChalmers-Gothenburg/Metagenomics)
[GEM](https://2020.igem.org/Team%3AChalmers-Gothenburg/GEM)

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# Team:Chalmers-Gothenburg/Results

iGEM Chalmers Gothenburg 2020

[Home](https://2020.igem.org/Team%3AChalmers-Gothenburg)

[People](https://2020.igem.org/Team%3AChalmers-Gothenburg/People)

[Team](https://2020.igem.org/Team%3AChalmers-Gothenburg/Team)
[Supervisors](https://2020.igem.org/Team%3AChalmers-Gothenburg/Supervisors)
[Attributions](https://2020.igem.org/Team%3AChalmers-Gothenburg/Attributions)

[Project](https://2020.igem.org/Team%3AChalmers-Gothenburg/Project)

[Description](https://2020.igem.org/Team%3AChalmers-Gothenburg/Description)
[Implementation](https://2020.igem.org/Team%3AChalmers-Gothenburg/Implementation)
[Parts](https://2020.igem.org/Team%3AChalmers-Gothenburg/Parts)
[Safety](https://2020.igem.org/Team%3AChalmers-Gothenburg/Safety)

[Human
Practices](https://2020.igem.org/Team%3AChalmers-Gothenburg/Human_Practices)

[Integration](https://2020.igem.org/Team%3AChalmers-Gothenburg/Integration)
[Science Communication](https://2020.igem.org/Team%3AChalmers-Gothenburg/Education)
[Collaborations](https://2020.igem.org/Team%3AChalmers-Gothenburg/Collaborations)
[Sustainable](https://2020.igem.org/Team%3AChalmers-Gothenburg/Sustainable)

[Wet Lab](https://2020.igem.org/Team%3AChalmers-Gothenburg/Wetlab)

[Design](https://2020.igem.org/Team%3AChalmers-Gothenburg/Design)
[Results](https://2020.igem.org/Team%3AChalmers-Gothenburg/Results)
[Measurement](https://2020.igem.org/Team%3AChalmers-Gothenburg/Measurement)
[Notebook](https://2020.igem.org/Team%3AChalmers-Gothenburg/Notebook)

[Dry Lab](https://2020.igem.org/Team%3AChalmers-Gothenburg/Model)

[Metagenomics](https://2020.igem.org/Team%3AChalmers-Gothenburg/Metagenomics)
[GEM](https://2020.igem.org/Team%3AChalmers-Gothenburg/GEM)
[Kinetic Model](https://2020.igem.org/Team%3AChalmers-Gothenburg/ODE)
[Reproducibility](https://2020.igem.org/Team%3AChalmers-Gothenburg/Reproducibility)

[For Judges](https://2020.igem.org/Team%3AChalmers-Gothenburg/ForJudges)

[Engineering Success](https://2020.igem.org/Team%3AChalmers-Gothenburg/Engineering)
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# Results

**Overview**
On this page, results from our experiments are presented in a chronological order. This includes gels
from gel electrophoresis of his-tag introducing PCRs and screening digestion, measured protein
concentrations after cell lysis and protein extraction and images of western blot membranes. The results
of the activity measurements can be found on the Measurements page.

**Establishment of His-tagged gene block fragment by PCR**

 6 histidine residues were added by PCR with overhanging primers to each ordered block segment
before the
stop codon, as shown in Figure 1.

![Placeholder](https://static.igem.org/mediawiki/2020/3/3b/T--Chalmers-Gothenburg--Figure-Wetlab-Results-1.jpg)

**Figure 1. Schematic illustration of the establishing each his-tag block segment.** Six histidine tag was inserted downstream of each nine different enzyme gene and upstream of stop codon to purify each enzyme protein later of this project.

 The amplification was confirmed successful by electrophoresis, see Figure 2. The expected size of
each His-tagged
gene block fragments is 1507 bp (BphA1), 926 bp (BphB), 977 bp (BphC), 677 bp (BphD), 1009 bp (BphA2),
934
bp (PETase), 1949 bp (PueA), 1703 bp (PEG-DH) and 1984 bp (MHETase). For more details, check out [Week 29](https://static.igem.org/mediawiki/2020/e/e3/T--Chalmers-Gothenburg--Week29.pdf) (July 17th) in the [Notebook](https://2020.igem.org/Team%3AChalmers-Gothenburg/Notebook) page!

![Placeholder](https://static.igem.org/mediawiki/2020/9/98/T--Chalmers-Gothenburg--Figure-Wetlab-Results-2.jpg)

**Figure 2. Confirming the size of his-tagged block segments.** Each his-tagged block
segment was constructed by PCR and the size of them were confirmed in a 1% agarose gel
electrophoresis. The gel picture shows from left: 1: GeneRuler Express DNA Ladder (SM1553) 2: BphA1,
3: BphB, 4: BphC, 5: BphD, 6: BphA2, 7: GeneRuler Express DNA Ladder, 8: PETase, 9: PueA, 10:
PEG-DH, 11: MHETase, 12: GeneRuler Express DNA Ladder. The length of each fragment correspond to the
expected lengths and successful amplification was thus confirmed by this electrophoresis.

**Transformants screening**

 Each His-tagged block segment was inserted into pSB1C3 through Gibson Assembly and the resulting
plasmid
was transformed into DH5α. To screen the transformants colonies, the plasmids were extracted using
Thermo
Fisher’s miniprep kit, digested with restriction enzymes XhoI and SacI and run in a gel
electrophoresis.
The resulting gel is presented in Figure 3. Figure 3A shows the electrophoresis of BphA1, which was the
only
block segment for which the bands showing were not correct. Figure 3B shows all other digested block
segments.
Each block segment should have three fragments from this digestion as two restriction enzymes were used.
Two of the fragments should be of the same length for all block segments, these are 116 bp and 776 bp.
The
last fragment should be different for each block segment and their corresponding expected lengths are
2708
bp (BphA1), 2162 bp (BphB), 2213 bp (BphC), 2165 bp BphD), 1913 bp (BphA2), 2117 bp (PETase), 3185 bp
(PueA),
2939 bp (PEG-DH) and 3143 bp (MHETase). The plasmids with block segments for which the screening results
were positive were sent for sequencing. For BphA1, BphB, BphC and BphA2, the sequencing results confirmed
the right sequence. You can check how we got these results in our [Notebook](https://2020.igem.org/Team%3AChalmers-Gothenburg/Notebook)!
We got positive colonies expressing BphB and PEG-DH (by sequencing we noticed that PEG-DH had deletion) in [Week 31](https://static.igem.org/mediawiki/2020/5/5f/T--Chalmers-Gothenburg--Week31.pdf) (July 31st), for BphA1, BphC, and BpfA2 in [Week 33](https://static.igem.org/mediawiki/2020/9/97/T--Chalmers-Gothenburg--Week33.pdf) (August 10th), for BphD and PETase (but sequencing for them are negative) in [Week 35](https://static.igem.org/mediawiki/2020/9/97/T--Chalmers-Gothenburg--Week35.pdf) (August 26th) and for PueA (sequencing is negative) on August 27th the same week. You can see the corresponding negative bands for MHETase August 27th in the same week.

![Placeholder](https://static.igem.org/mediawiki/2020/0/09/T--Chalmers-Gothenburg--Figure-Wetlab-Results-3.jpg)

**Figure 3. Confirming the size of digested plasmid for screening.** Gels showing the lengths of the fragments resulting from digesting each his-tagged
block
segments with SacI and XhoI when run in a 1% agarose gel electrophoresis. The gel picture A shows
from
the left, 1: GeneRuler Express DNA Ladder (SM1553) 2: empty 3: BphA1 and picture B showed from left,
1: BphB, 2: BphC, 3: BphD, 4: BphA2, 5: GeneRuler Express DNA Ladder, 6: PETase, 7: PueA, 8: PEG-DH,
9: MHETase, 10: GeneRuler Express DNA Ladder. There should be three fragments each and two small
fragments
for all should be 116 bp and 776 bp. The last fragment length should be different by each strain and
they should be 2708 bp (BphA1), 2162 bp (BphB), 2213 bp (BphC), 2165 bp (BphD), 1913 bp (BphA2),
2117
bp (PETase), 3185 bp (PueA), 2939 bp (PEG-DH) and 3143 bp (MHETase) and all but MHETase were
confirmed
by this electrophoresis.

**DC assay of extracted proteins**

 Once the cells had been lysed, the resulting protein concentrations were determined as presented in
Table
1. The protein concentrations differ notably between the different block segments despite having been
grown
for the same amount of time and having the same RBS and promoter. This could be due to the metabolic
burden
inflicted by the expression of the enzyme, an effect that should be further evaluated to optimize the
enzyme
expression process.

| Block Segment | Protein Concentration [mg/ml] |
| --- | --- |
| BphA1 | 5.8999 |
| BphB | 3.7254 |
| BphC | 4.7667 |
| BphA2 | 6.0224 |
| NC | 6.9106 |
| PC | 1.9039 |
**Table 1.** The concentration of the protein extracts measured by DC assay for the first
western
blot.

**Western blot gels**

 When examining the protein extracts in a western blot, in which the His-tags are utilized as
binding spots
for the antibodies, BphC (A3) was the only enzyme that showed a band at the expected length (32 kDa
[1]),
as seen in Figure 4. There seem to be some unspecific binding to some protein of approximately 36 kDa
for
all the different samples, but as this is present in the negative control as well it’s disregarded
as background. Interestingly, also another unique band for BphC is clearly visible around 145 kDa. This
band
could be the result of misfolding and aggregate formation when BphC is expressed in E. coli. Since BphC
originates
from Pseudomonas sp, it is possible that our chassis lacks chaperones necessary for the correct folding
of
this specific protein leading to the aggregation. Read more about these results in the Notebook, [Week 36](https://static.igem.org/mediawiki/2020/9/97/T--Chalmers-Gothenburg--Week36.pdf) (September 3rd).

![placeholder](https://static.igem.org/mediawiki/2020/f/f5/T--Chalmers-Gothenburg--Figure-Wetlab-Results-4.png)

**Figure 4. Western blot result.** The western blot membrane after stain with antibodies that bind to his-tags. The plasmid backbone used for all block segments with a gene coding for RFP instead of a his-tagged enzyme was used as a negative control. The positive control was a his-tagged protein we got from another researcher in our lab. Expected lengths for the block segments are 52 kDa for BphA1, 29 kDa for BphB, 32 kDa for BphC and 23 kDa for BphA2. SpectraTM Multicolor Broad Range Protein Ladder (26634) was used as a size marker.

 To further investigate the aggregation of BphC, new protein samples were extracted from cells that
were
in the exponential phase instead of the stationary phase. The same procedure was done for BphB (A2) in
case
the protein extraction at stationary phase was the reason that we couldn’t see any expression of
the
other enzymes. Table 2 shows the corresponding protein concentrations after the cell lysis.

|  |  |
| --- | --- |
| Block Segment | Protein Concentration [mg/ml] |
| BphB (OD600 = 0.5) | 2.7031 |
| BphB (OD600 = 1.2) | 5.6282 |
| BphC (OD600 = 0.3) | 1.5616 |
| BphC (OD600 = 0.9) | 2.4178 |
**Table 2.** The concentration of the protein extracts measured by DC assay for the second
western
blot.

Figure 5 shows the resulting western blot, including BphB and BphC at exponential and stationary phase,
respectively. It
also includes a positive control and a negative control (consisting of the pSB1C3 with an RFP gene as
insert).
The western blot shows clearly that BphC starts aggregating when the culture approaches the stationary
phase.
This probably happens when the abundance of enzyme gets too high and can be avoided if cells are kept at
lower OD in exponential phase. The part of the growth curve in which the cells does not seem to affect whether BphB is expressed or not. We got this result [Week 37](https://static.igem.org/mediawiki/2020/9/97/T--Chalmers-Gothenburg--Week37.pdf) (September 11th) if you want to read more about it in the Notebook.
![](https://static.igem.org/mediawiki/2020/1/15/T--Chalmers-Gothenburg--Figure-Wetlab-Results-5.png)

**Figure 5. Western blot result.** The western blot membrane after stain with antibodies that bind to his-tags of BphB and BphC at different growth phases (exponential and stationary). The plasmid backbone used for all block segments with a gene coding for RFP instead of a his-tagged enzyme was used as a negative control. The positive control is a protein solution containing a his-tagged protein we got from another researcher in our lab. SpectraTM Multicolor Broad Range Protein Ladder (26634) was used as a size marker.

1. [1] “
   *bphC - Biphenyl-2,3-diol 1,2-dioxygenase - Pseudomonas sp. (strain KKS102) - bphC gene &
   protein.*”
   <https://www.uniprot.org/uniprot/P17297>
   (Accessed on Sep. 08, 2020).

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