iGEM 2021 — Team Uppsala

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# Team:Uppsala/Notebook

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[Notebook](#pagecontent)

---

# Lab Introduction

This year's iGEM journey began in February for the Uppsala team. During that time a virus, tiny in size but huge in impact, made it impossible for us to meet each other in real life. Instead we had to make due with seeing each other over zoom. As we were getting to know each other online we were also spawning and researching project ideas. We voted for a group of ideas and a selection of 6 ideas from 20 in total were chosen to be further researched in groups. Together in these groups we wrote reports and made presentations of our ideas that were given feedback to from professors and faculty at Uppsala University. With that as the basis we made a final vote and decided that reducing the cost of cultivated meat through protein engineering was our project. From then on we all started to work towards the same goal and we were impatiently waiting to finally enter the lab.

During the summer, iGEM Uppsala worked in three different lab groups with three different approaches. Lab group 1 wanted to express the wild type FGF2 and mutate it to increase binding affinity. Lab group 2 had a mission to create a chimeric protein of FGF1 and FGF2 which is called FGFC and lab group 3 wanted to express a hyperstable variant of FGF2. During the first two weeks and also the last weeks we worked together at BMC since we all had similar laboratory tasks to do. Also during one and a half weeks of August we had the opportunity to work at TESTA Center in order to upscale our project.

This is a summary of every lab week for iGEM Uppsala 2021. For more detailed information about the laboratory practices see [Experiments](https://2021.igem.org/Team%3AUppsala/Experiments).

# Week 1-2 (7 June - 20 June)

## Team BMC

The team finally entered the lab at Uppsala Biomedical Centre (BMC), but first and foremost iGEM Uppsala had a tour of the lab and a safety introduction in order to prevent accidents. The first week at BMC mainly focused on planning for the summer and creating appropriate protocols. In terms of actual lab work the team prepared LB liquid media and LB agar plates with chloramphenicol to practice for the weeks ahead.

Second week also consisted of practicing important laboratory techniques. We transformed plasmids into competent DH5alpha cells. We made more LB agar plates with kanamycin and ampicillin and followed protocols on making DH5alpha competent cells. The iGEM Uppsala team practiced streaking for single colonies on the transformed cells. There was also designing and ordering of primers and planning regarding lab cleaning.

# Week 3-4 (21 June - 4 July)

## Labgroup 1

Lab group 1 performed transformation of wild type FGF into DH5alpha cells. Afterwards, the plasmid was amplified using PCR. Lastly the PCR product was purified and sent for sequencing

During the fourth week the sequencing results were analyzed and they were satisfactory, which enabled us to start the purification of the plasmid. We performed PCR to add the terminator to the FGF2 plasmid. Labgroup 1 also tried to digest the pET vector and ligate it with FGF2. Due to low concentrations we redid it a few times. Finally we sent them for sequencing.

## Labgroup 2

Lab group 2 performed transformation of the BioBrick pET plasmid into TOP10 competent cells. Then, transformation of the plasmid with FGFC into DH5alpha cells was done. The success of the transformations into the DH5 alpha cells were confirmed through a colony PCR and subsequent gel electrophoresis.

The following week we did plasmid prep with several purifications. We tried to confirm transformation with PCR and gel electrophoresis but was unsuccessful twice. We did transformations but unfortunately with no cell growth. We were hoping to send sequencing but after observing the agar plates without any colonies, it was not an option. Instead new overnight cultures were made and the procedure was started once more.

## Labgroup 3

Lab group 3 made a transformation of the BioBrick pET plasmid into TOP10 competent cells and also transformed the IDT plasmid containing the hyper stable FGF2 into DH5alpha competent cells. The results of the two transformations were verified using colony PCR and running the amplified products on a gel electrophoresis. The PCR products also got sent to sequencing and glycerol cell stocks were made of the DH5alpha competent cells with the plasmid inserted.

The fourth week lab group 3 purified the pET plasmid and the IDT plasmid using plasmid miniprep kits. We also had multiple attempts of adding the terminator to the hyper stable FGF2 by using PCR. After each PCR the samples were tested on a gel electrophoresis but with no success. Lastly alternative approaches on how to add the terminator that might work better were planned.

# Week 5-6 (5 July - 18 July)

## Labgroup 1

This week labgroup 1 redid digestion and ligation and ran the product on a gel. Then overnight cultures were grown and pET plasmid from a band on the gel electrophoresis was cut out and purified. Next step was transformation of ligated pET and wild type FGF2 was also transformed into DH5alpha. A colony PCR was performed, plasmids were purified using kits and more attempts of digestion using NdeI and SpeI were made.

We continued to try to digest and ligate the biobrick parts, but with limited success. Therefore we scheduled a meeting with Anthony Foster and came to the conclusion that we need to add base pairs outside the restriction sites on the FGF2 primers in order for the restriction enzymes to be able to cut the DNA. Therefore lab coordinators and co-coordinators designed new primers with 6bp overhangs which arrived the following monday.

## Labgroup 2

This week started with trying to confirm that we had amplified the FGFC-terminator by using an agarose gel electrophoresis. It seemed to have failed with no clear bands, therefore the PCR to add terminator to FGFC was repeated. It wasn’t successful at this point as well which made us repeat the previous steps once again. After that one sample on the gel showed promising results and we went on with PCR purification. However, a new PCR was also performed with a gradient in order to get an idea of what the most optimal annealing temperature could be. From this point, we continued with DpnI digestion of the FGFC-term construct to get our gene without methyl groups.

The next week the DpnI digested FGFC-term went on to be digested with the restriction enzymes NdeI and SpeI separately. This was followed by ligation into the PET-vector and transformation in DH5alpha. Colony PCR was run the day after and observed in gel electrophoresis, with no visible bands therefore PCR was repeated once more. FGFC-term was double digested with the same restriction enzymes, followed by ligation. Transformation was repeated and in the following day, the colonies were purified which showed very low DNA concentration. We went back to the first step and purified the PUC-vector.

## Labgroup 3

Week 5 mainly consisted of continuing to try to add the terminator to the hyper stable FGF2 gene and testing different PCR programs and fresh reagents in order for the PCR reaction to work. Finally we tested with a new DNA polymerase with fresh buffer and nucleotides and managed to successfully add the terminator with the correct restriction sites.

The other week we started by purifying the plasmid that contained the FGF2hs and the terminator. Then we continued with digestion in order to remove a piece of the insert to try to complete our construct. Afterwards we tested the results by running the product on a gel electrophoresis. Then we purified the pcr product and sent it for sequencing.

# Week 7-8 (19 July - 1 August)

## Labgroup 1

Week 7 started off by adding 6 base pair overhangs to the FGF2 with the newly designed primers. This was done by using the old FGF2 as a template when using the new primers in the PCR.
It was sent for sequencing and the result was promising. We therefore started to digest and ligate it with the pET vector. This time it worked excellent on the first try, compared to the two weeks before, where the experiment continually failed. They were sent to sequencing that friday.

The following week the sequencing result came back correctly! Therefore we started the mutagenesis project. We created 4 different mutations, FGF2 Q54K, FGF2 V88I, FGF2 L98M and FGF2 Q54K + L98M. These were all created using a mutagenesis protocol designed by Anthony Foster. Primers for mutagenesis were used in a PCR to create these mutations. For the double mutation, FGF2 Q54K was exposed to the FGF2 L98M mutation protocol. All mutations were sent for sequencing and everything was correct! Afterwards we created glycerol cell stocks.

## Labgroup 2

We decided to divide into two groups. The first group did a PCR with new overhangs that was designed the week before. The old FGFC sequence was used as the basis for this experiment. PCR purification was performed, however the final DNA concentration was insufficient.
The other group did a PCR on FGFC and the terminator separately. These were then digested with PstI. This made them compatible with ligating to each other, which gave a sufficient DNA concentration of the wanted product. Lastly the ends of the construct were cut with NdeI and SpeI.

The next week started off a colony PCR. Then we continued with our plan to add the terminator as an overhang on the primers and use PCR. After the PCR, the samples were put on a gel electrophoresis and the samples were purified by using a kit. DpnI digestion was performed and from the gel electrophoresis a band was extracted in order to purify the plasmid. Finally we performed transformations into BL21 cells for our FGFC.

## Labgroup 3

This week we used new primers that had 6 added base pairs on each end of the hyper stable FGF2-Terminator sequence. We ran the results on a gel to verify the results. We also tried to do a single digestion, which means that we first cut with NdeI and then purify and then cut with SpeI. Afterwards we ligated the product into the pET vector. However due to low yield we instead did a double digestion and then purified and ligated it into the pET vector. Afterward we transformed the cells and made overnight cultures and a colony PCR to see if the bacteria had our gene of interest. This was verified with a gel electrophoresis. Lastly we purified our samples with a purification kit and sent them for sequencing.

Last week we sent the final construct for sequencing and the sequencing results were great! Therefore overnight cultures were made and we purified the plasmids using a plasmid purification kit. The plasmids were transformed into a BL21 E. coli strain for both the wild type FGF2 and the Hyperstable FGF2. Afterwards we let the transformed cells grow on agar plates and performed Colony PCR. We then purified the PCR samples for both the FGF2 wt and the FGF2hs and sent them to sequencing. Towards the end of the week we made glycerol cell stocks in order to have the proper strain with the correct construct that could be used to produce FGF2.

# Week 9-10 (2 August - 15 August)

## Team TESTA

For two days this week and the upcoming week six members of our team got the opportunity to work at TESTA Center in order to scale up our project! The work started with transformation of all our FGF2 plasmid variants into E. coli BL21 strains. Then it was time to grow bacteria in small E-flasks and induce the cells with IPTG to test optimal conditions to express our different FGF2 variants. Afterwards we lysed the cells and ran an SDS PAGE.

Week 10 at Testa Center started with preparation of the two bioreactors each with a total volume of 2.5L media that we are using to express our protein on a larger scale. We picked the hyper stable FGF2 and the wild type FGF2 to grow in the bioreactors since the team at BMC had the most success with these two variants. After the cells had grown in the bioreactor we harvested the cells then we lysed them by using a french press and finally used IMAC to purify our protein. The purified protein was tested on an SDS PAGE to determine how successful our purification was.

## Team BMC

During this week the people over at BMC expressed all FGF2 variants and visualized the results by running samples on an SDS PAGE. We also selected colonies which still contained the pLysS plasmid after transformation, which is needed for proper overexpression of FGF2.
Afterwards we picked FGF2hs and wt FGF2 and continued on with lysing the cells in a french press. Then we purified these samples by using a small IMAC column. The purified samples were then put on an SDS PAGE and we saw clear bands however the bands were slightly shorter than expected. Lastly the BL21 E. coli cells were transformed with several FGF2 variants.

At BMC the following week the team prepared all the buffers necessary for IMAC purification. Then overnight cultures were grown of FGFC, FGFhs, FGF wt and also FGF2 Q54K. The cells were then induced with IPTG at 0.6 OD and grew for 6 hours. Afterwards purification with IMAC was performed and an SDS PAGE was used to verify the overexpression. Lastly we tested to transform our different FGF2 variants into new BL21 competent E. coli cells and expression was performed.

# Week 11-12 (16 August - 29 August)

## Team BMC

Week 11 began with inoculating overnight cultures and inducing them at OD 0.6 to start the expression of FGF2 for all variants. The samples were then lysed in a french press and the lysed cells were purified using IMAC. Afterwards the samples got placed on an SDS PAGE but no bands were visible. After the purification the samples got spin concentrated in order to remove imidazole and to concentrate the protein. FGF2 was also put on a heparin column in order to look at the activity of the protein and to purify it further. Finally the protein concentrations were determined with Bradford Assay.

Now we have reached the final week of laboratory work and we are ready to finally tie it all together. We started overnight cultures of FGF2 wt, FGFhs, FGFC and FGF2 L98M and induced them at OD 0.6. Then the cells grew for 6 hours and a small sample was taken to run an SDS PAGE to look at the overexpression. The rest of all the samples were lysed in a french press. After the cells were lysed, the protein was purified by using an IMAC nickel column. Then another SDS PAGE was performed to look at the lysate, the flow through from the column, the washed IMAC product and finally the elution. Afterwards all the samples were snap freezed using liquid nitrogen and then placed in a -80 degrees celsius freezer.

This marks the end of the lab work which iGEM Uppsala did during the summer of 2021.

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# Team:Uppsala/Engineering

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[Engineering Success](#pagecontent)

---

# Cycle 1: Design of the strains

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## Design

FGF2 is the limiting factor for cell growth in cellular agriculture companies, as it is unstable and therefore expensive. In order to make cellular agriculture cheaper and more accessible, we decided we would try to express more stable and therefore cheaper variants of FGF2, we also tried to scale up production of FGF2 which would in turn also lower the cost. The team examined literature that had been published by research groups who had expressed FGF2 in different organisms and decided that E. coli would be the simplest to culture. We then looked at research papers which had improved protein stability and found two promising variants we wanted to test, namely hyperstable FGF2 (FGFhs) [1] and FGF chimera (FGFC) [2]. In addition, we also expressed FGF2 wild type as well as other mutant variants, which we had designed ourselves [3]. We conducted extensive modelling in order to design FGF2 mutants which would have a stronger binding affinity to fibroblast growth factor receptors (FGFRs), you can read more about our modelling efforts [here](https://2021.igem.org/Team%3AUppsala/Model).

Three FGF2 constructs were initially ordered from Integrated DNA Technologies (IDT) and were initially designed to be compatible with a non-inducible vector found in the iGEM distribution kit.
Previous studies on expression of FGF2 had all used inducible vectors, as it seemed that the FGF2 gene could potentially be toxic to the cell if its expression was unchecked, which is the case with non-inducible vectors. The vector was therefore changed to the inducible pET 28a+ vector, as pET vectors had previously been used for expression of FGF2 in E. coli [3]. Our FGF2 inserts which we ordered from IDT were not compatible with the new pET vector. In order to make them compatible, primers were used to add the correct restriction sequences to the 5’ and 3’ end of the FGF2 inserts through PCR. A terminator sequence was also added to one of the primers, this was done for all of the constructs making a roughly 90 bp reverse primer.
One of the problems highlighted in literature was that FGF2 has low solubility and stability [4]. A complementary protein was needed to increase the solubility of FGF2 and perhaps increase its stability. We would also need to design a construct which would allow this to be purified and removed so that we only end up with the FGF2 variants in the end. The protein we decided to use was called thioredoxin, and once FGF2 had been cleaved from thioredoxin, it could be extracted through a heparin column.

## Build

The desired plasmid construct would be inducible and have the T7 promoter, lac repressor, ribosome binding site (RBS), our inserted FGF2 sequence and a terminator in that order. One advantage with the pET promoter is the ability to control the expression of FGF2 to avoid harming the E. coli strain. The FGF2 construct contained thioredoxin which was added to increase solubility of the protein, followed by a his tag, an enterokinase site and then the FGF2 protein variant. The histidine tag was used so that the protein could be separated with affinity chromatography and the enterokinase site’s purpose was so thioredoxin could be separated from FGF2.

## Test

Agarose gel electrophoresis of the PCR products proved that the PCR was successful for most of the FGF2 constructs, meaning that the primers had successfully attached to our inserts and added biobrick sites and terminators. However when trying to ligate the insert into the pET plasmid all groups remained unsuccessful after multiple attempts. In addition, some of the lab groups could not successfully add the terminator, shown by an absence of right sized bands on the agarose gel after multiple PCRs.

## Learn

The FGF2 constructs we ordered from IDT had restriction sites at the beginning and at the end of the construct, but there was no flanking DNA on one side of each restriction site. It was discovered that biobrick restriction sites need some unspecific flanking DNA sequence in order to be cut properly by endonucleases, therefore, new primers were ordered which met this criteria. Instead of ordering even larger primers than the 90 bp we had previously ordered, we ordered smaller primers to add flanking biobrick sites to the ends of the FGF2 sequences where we had added the terminator. All FGF2 inserts were successfully cloned into the plasmid, as proven by the sequencing results. Furthermore, it was discovered that some lab groups were more successful using another brand of DNA polymerase when performing the PCR, where they finally got bright and thicker bands after many failed attempts.

# Cycle 2: Induction of FGF2 expression

## Design

Once the FGF2 sequence was ligated into the pET vector, the constructs were transformed into DH5 alpha cells for storage, this is as DH5 alpha is much less likely to mutate the plasmid DNA than other E. coli strains. Correct FGF2 sequences were inserted into DH5 alpha E. coli cells through transformation, as these are optimised for transformation efficiency. For the protein expression of the FGF2 variants, another E. coli cell line optimised for protein expression was used called BL21.

## Build

The DH5 alpha cells transformed our construct successfully which was proven by sequencing results. They were stored in a -80°C freezer so we could have access to the correct plasmid if we needed to transform into BL21 again. The plasmid constructs were then transformed into BL21 successfully, and confirmed with sequencing.

## Test

BL21 were induced with IPTG at varying concentrations (0.5 M, 0.1 M, 0.01 M) and the results were displayed on a gel. The gels did display bands of the correct size, though some were faint. Higher levels of IPTG did not necessarily produce thicker bands for each of the constructs, some of the constructs even had more expression at lower concentrations of IPTG. Furthermore, though the constructs were induced with the same level of IPTG, the visibility of the bands was not the same and varied considerably.

## Learn

Previous attempts at PCR had shown that certain products which were used for PCR had produced negative results. It was therefore theorised that there may be some success if we tried another brand of BL21 cells or expressed them at different temperatures or concentrations.

## Improve

We therefore tried another brand of BL21 and the bands for FGF2 improved even when the experimental conditions for expression remained the same. Although we did not have time to test all variants with the new brand of BL21 cells, we did get expression on FGFC, FGFhs, FGF2 wt and the mutant variants, although the bands were not extremely clear. The wild type FGF2 had the most clear expression.

# Cycle 3: Purification of FGF2

## Design

After we got bands on most of the variants of FGF2, we decided to use histidine tag chromatography in order to purify them. This was initially performed at the university on a small scale and then upscaled at a large lab facility using 5 L bioreactors. To stop the protein from degrading once purified, we were advised to snap freeze it. We decided to lyse the cells in order to extract the protein and then use a spin concentrator to concentrate our protein in a protein lysis buffer (which is a simple buffer used in a french press machine which lyses cells). The next step would be to separate the thioredoxin from the FGF2 through using enterokinase. However, it was theorised that the FGF2 bound to thioredoxin would still be functional and cause mitogenesis, as a scientific paper had proven [3]. To prove we had successfully purified FGF2, we decided to run the purified FGF2 wild type on a heparin column, with thioredoxin still attached.

## Build

Two variants, FGF2 and FGF2hs were upscaled at a large up-scaling facility as they had the clearest bands. The other variants were tested on a small scale.

## Test

FGF2 wild type was the only variant which seemed to have successful expression on a large scale, FGF2hs did not produce bands of the right size. When FGF2 wild type was run on the his tag column it showed an elution peak at a high concentration, meaning that a protein had bound strongly to the column. The other variants were purified on a small scale and SDS-PAGE gels showed that 4 of the variants had bands of the right size after purification, meaning that a protein was of the same size as our FGF2 thioredoxin construct.

## Reflection

Our team went through a total of 3 engineering cycles over the course of our lab period, and went on to successfully express all FGF2 variants by the end of the summer. The problems we encountered in the lab were completely new to us, but were overcome through applying what we had learnt from the previous design cycle or through troubleshooting. In some cases when we were unsure about the root cause of the problem , control experiments were conducted to test hypotheses and redirect our thinking to the right track. Through using the scientific method and eliminating possibilities, we were able to move on to the next steps of the design cycle, and improve our designs until we were eventually successful in producing all FGF constructs.

Through our engineering successes we managed to create our FGF2 basic parts, which you can read about [here](https://2021.igem.org/Team%3AUppsala/Part_Collection)!

# References

[1] Dvorak P, Bednar D, Vanacek P, Balek L, Eiselleova L, Stepankova V, Sebestova E, Kunova Bosakova M, Konecna Z, Mazurenko S, Kunka A, Vanova T, Zoufalova K, Chaloupkova R, Brezovsky J, Krejci P, Prokop Z, Dvorak P, Damborsky J. Computer-assisted engineering of hyperstable fibroblast growth factor 2. Biotechnol Bioeng. 2018 Apr;115(4):850-862. doi: 10.1002/bit.26531. Epub 2018 Jan 24. PMID: 29278409.

[2] Motomura K, Hagiwara A, Komi-Kuramochi A, Hanyu Y, Honda E, Suzuki M, Kimura M, Oki J, Asada M, Sakaguchi N, Nakayama F, Akashi M, Imamura T. An FGF1:FGF2 chimeric growth factor exhibits universal FGF receptor specificity, enhanced stability and augmented activity useful for epithelial proliferation and radioprotection. Biochim Biophys Acta. 2008 Dec;1780(12):1432-40. doi: 10.1016/j.bbagen.2008.08.001. Epub 2008 Aug 12. PMID: 18760333.

[3] Gasparian ME, Elistratov PA, Drize NI, Nifontova IN, Dolgikh DA, Kirpichnikov MP. Overexpression in Escherichia coli and purification of human fibroblast growth factor (FGF-2). Biochemistry (Mosc). 2009 Feb;74(2):221-5. doi: 10.1134/s000629790902014x. PMID: 19267679.

[4] Benington L, Rajan G, Locher C, Lim LY. Fibroblast Growth Factor 2-A Review of Stabilisation Approaches for Clinical Applications. Pharmaceutics. 2020 Jun 2;12(6):508. doi: 10.3390/pharmaceutics12060508. PMID: 32498439; PMCID: PMC7356611.

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# Team:Uppsala/Experiments

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[Protocols](#pagecontent)

---

# Lab preparation protocols

During the two first weeks of summer the team prepared all upcoming lab work. All necessary solutions were prepared, such as LB, SOB, and agar plates with different antibiotics. The team also got the opportunity to practice basic lab techniques under aseptic conditions, which included creating competent cells, streaking, and re-streaking agar plates. More details on how we worked each week can be found on the [Notebook page](https://2021.igem.org/Team%3AUppsala/Notebook). Down below, protocols of all the lab preparation work can be found.

[Lab preparation protocols.pdf](https://static.igem.org/mediawiki/2021/3/39/T--Uppsala--lab-prepV2.pdf)

# Biobrick assembly protocols

The primers arrived and the team started with the biobrick assembly process. The goal was to create seven different versions of bovine FGF2. Together with our PI, Professor Anthony Forster, and our TA, Konrad Gras, different experiments were designed to create these versions. We cannot thank Forster and Gras enough for their help with guiding us through these experiments. More details of the parts we created can be found on the [Parts page](https://2021.igem.org/Team%3AUppsala/Part_Collection). Down below, protocols of all the biobrick assembly work can be found.

[Biobrick assembly protocols.pdf](https://static.igem.org/mediawiki/2021/c/ce/T--Uppsala--biobrickV2.pdf)

# Small scale purification protocols

When these seven different versions of bovine FGF2 had been created, we had to purify them. First we decided to do it at a small scale in the lab. Here we could troubleshoot and adjust the process for when we were doing an upscaled purification. For more information of the purification results, see the [Results page](https://2021.igem.org/Team%3AUppsala/Results). Down below, protocols of all the small scale purification work can be found.

[Small scale purification protocols.pdf](https://static.igem.org/mediawiki/2021/1/10/T--Uppsala--smallscaleV2.pdf)

# Large scale purification protocols

The team also did a large scale purification at Cytiva Testa Center AB. The team successfully upscaled the purification process of the bovine FGF2 wild type protein. We want to thank Cytiva Testa Center AB for giving us the chance to upscale our project. Thanks to all of the employees and experts working at Cytiva for helping us design the experiments and purify the protein. A big thank you to Chandra Sekhar Mandava for helping us with the Testa Center part of our project. All protocols followed at Cytiva Testa Center AB can be found down below.

[Large scale purification protocols.pdf](https://static.igem.org/mediawiki/2021/f/fe/T--Uppsala--LargescaleV2.pdf)

# References

[1] Liljeruhm, J., Gullberg, E., & Forster, A. C. (2014). “Synthetic biology: a lab manual”

[2] Thermo Fisher Scientific. (2012) “Thermo Scientific GeneJET Plasmid Miniprep Kit K0502, K0503” <https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2FMAN0012655_GeneJET_Plasmid_Miniprep_UG.pdf> (Accessed Sep. 22, 2021)

[3] Thermo Fisher Scientific. (2013) “Thermo Scientific DreamTaq DNA Polymerase” <https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012037_DreamTaqDNAPolymerase_5x500U_UG.pdf> (Accessed Sep. 22, 2021)

[4] Thermo Fisher Scientific. (2015) “Thermo ScientificGeneJET PCR Purification Kit #K0701, #K0702” <https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2FMAN0012662_GeneJET_PCR_Purification_UG.pdf> (Accessed Sep. 22, 2021)

[5] Eurofins Genomics. (2021) “Mix2Seq Sequncing Kit” <https://eurofinsgenomics.eu/media/1587665/tubesequencing_flyer_dinlang_8seiter_wickelfalz.pdf> (Accessed Sep. 22, 2021)

[6] New England Bioloabs (2021) “Double Digest” <https://international.neb.com/tools-and-resources/usage-guidelines/double-digests> (Accessed Sep. 22, 2021)

[7] New England Bioloabs (2021) “Ligation Protocol with T4 DNA Ligase (M0202)” <https://international.neb.com/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202> (Accessed Sep. 22, 2021)

[8] Bio-Rad Laboratories (2021) “Quick Start™ Bradford Protein Assay” <https://kirschner.med.harvard.edu/files/protocols/BioRad_proteinassay.pdf> (Accessed Sep. 22, 2021)

[9] Protocols were created together with experts at Cytiva Testa Center AB. <https://testacenter.com/>

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# Team:Uppsala/Results

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LOADING

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[Results](#pagecontent)

---

# Assembly of the constructs

The team obtained three different FGF constructs from IDT. These contained either the bovine wild-type FGF2 (FGF2 wt), a hyperstable version of the bovine wt FGF2 (FGF2hs) that was designed by the team using mutations described previously [1], or a chimera between the bovine wt FGF-1 and bovine wt FGF2 (FGFC) that was designed by the team based on an earlier publication [2]. All genes were ordered with a thioredoxin tag and a six histidine tag on their 5’ end. The genes came in pUC plasmids

## Adding the T7 terminator to the initial FGF2 constructs

Since the Biobrick pET vector [3], [4] had its terminator within the SpeI and NdeI cleavage sites, we had to add the T7 terminator and a NdeI cleavage site to the ordered sequence before digesting and ligating it to our vector of choice. This was done by using the same forward primer for all three constructs but a total of three reverse primers (Table 1), since each one was specific for one of the three constructs

Table 1 - Primer sequences used to add the T7 terminator and the NdeI site to the three constructs.

| Sequence *(sequences in red are overhangs)* | Function |
| --- | --- |
| 5’-CATATGTCTAGAATGTCTGACAAGATTATTCATCTTAC-3’ | Forward primer used for all three reactions. |
| 5’-ACTAGTAGTTCCTCCTTTCAGCAAAAAACCCCTCAAGA CCCGTTTAGAGGCCCCAAGGGGTTATGCTAGCTGCAGTT AACTCTTGGCACTC-3’ | Reverse primer for FGF2 wt. |
| 5’-ACTAGTAGTTCCTCCTTTCAGCAAAAAACCCCTCAAGA CCCGTTTAGAGGCCCCAAGGGGTTATGCTAGCTGCAGGT CCGAGGATAC-3’ | Reverse primer for the chimeric FGFC. |
| 5’-ACTAGTAGTTCCTCCTTTCAGCAAAAAACCCCTCAAGA CCCGTTTAGAGGCCCCAAGGGGTTATGCTAGCTGCAGTT ATGATTTGGCTGAC-3’ | Reverse primer for the FGF2hs. |

After weeks of attempts with different conditions and polymerases, the team was finally able to add the terminator to the construct. The successful reaction was done using Q5® High-Fidelity DNA Polymerase from New England Biolabs Inc. (NEB) according to the manufacturer’s instructions and the following PCR conditions:

Table 2. PCR conditions as described in the manufacturer’s protocol in NEB website.

| Step | Temperature | Time | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98°C | 30 seconds | 1 |
| Denaturation  Annealing  Elongation | 98°C  64°C  72°C | 10 seconds 30 seconds 30 seconds | 30 |
| Final Elongation | 72°C | 2 minutes | 1 |
| Hold | 4°C | Infinite | 1 |

By using the above conditions in the PCR reaction, the team managed to obtain bands with the expected number of base pairs, for the hsFGF (Figure 1 A) and the wildtype FGF (Figure 1 B) and later for FGFC (not shown).

![](https://static.igem.org/mediawiki/2021/0/06/T--Uppsala--res-n-f1.jpg)

Figure 1. PCR products obtained after adding the terminator. The electrophoresis was performed at 100 V for 1 hour in a 1% agarose gel. A: FGF2hs after addition of the terminator through PCR. Lanes 2-4 show products with the expected size around 900 bp. B: FGF2 wt after addition of the terminator through PCR. Lanes 1-3 show products with the expected size around 900 bp.

## Adding 6 bp to each restriction site

After adding the terminator to the three constructs, the team realized that more base pairs were needed to flank the sequences for efficient digestion. The base pairs were added using a single forward primer and three reverse primers, each one of them specific to one of the three constructs (Table 2). Our team planned four different strategies, each one requiring different primers, as a backup to save time in case something went wrong. Thus a variety of primers were designed. However, since the reaction of our first strategy for the wildtype was successful (Figure 1), only these corresponding primers were used for the remaining approaches and are showcased in Table 3.

Table 3. The primers used to add 6 base pairs to each end of the constructs to increase the efficiency of the digestion reactions.

| Sequence *(sequences in red are the extra base pairs)* | Function |
| --- | --- |
| 5’-TAGTAGCATATGTCTAGAATGTCTGACAAGATTATTCA-3’ | Forward primer used for all three reactions. |
| 5’-TAGTAGACTAGTAGTTCCTCCTTTCAGCA-3’ | Reverse primer for FGF2 wt. |
| 5’-TAGTAGACTAGTAGTTCCTCCTTTCAGCA-3’ | Reverse primer for FGFC. |
| 5’-TAGTAGACTAGTAGTTCCTCCTTTCAGC-3’ | Reverse primer for FGF2hs. |

With the new primers we were successfully able to ad 6 bp to the ends of each construct (Figure 2 A-C).

![](https://static.igem.org/mediawiki/2021/thumb/e/e6/T--Uppsala--res-n-f2.jpg/800px-T--Uppsala--res-n-f2.jpg)

Figure 2. PCR products after the addition of 6 bp to each end of the constructs to allow for a more efficient digestion by the restriction enzymes. The electrophoresis was performed at 100 V for 1 hour in a 1% agarose gel. A: FGF wt after addition of the 6 bp to each end of the construct. Lanes 1 and 2 show products with the expected size of 1300 bp. B: FGFhs after addition of the 6 bp to each end of the construct. Lanes 1-3 show products with the expected size of 1300 bp. C: FGFC after addition of the 6 bp to each end of the construct. Lanes 1-3 show products with the expected size of 1300 bp.

# Double digestion, ligation and transformation

It was decided that a double digestion would be performed since separate digestion steps resulted in low yields due to many purification steps. We followed NEB’s recommendation on a double digestion with SpeI and NdeI. After digestion, the DNA was purified and concentrated using the Monarch® PCR & DNA Cleanup Kit from NEB. Finally, each of the three constructs was individually ligated to the Biobrick pET plasmid. This was done by using T4 DNA Ligase from NEB while following the manufacturer’s instructions. In the last step, the ligation product was transformed into E. coli Dh5 alpha cells that the team had made competent earlier, see [Protocols](https://2021.igem.org/Team%3AUppsala/Experiments). The transformation protocol followed is also described on the [Protocols](https://2021.igem.org/Team%3AUppsala/Experiments) page. The success of the whole assembly was assessed by colony PCR (Figure 3 A-C) and confirmed with sequencing performed by Eurofins.

![](https://static.igem.org/mediawiki/2021/8/89/T--Uppsala--res-n-f3.jpg)

Figure 3. Gel electrophoresis of the colony PCR products. The electrophoresis was performed at 100 V for 1 hour in a 1% agarose gel. A: Products of colony PCR of FGF2hs. Lane 1 shows a lane with a product with the expected size around 1300 bp. B: Products of colony PCR of FGF2 wt. Lane 1 shows a band with the wrong size, lanes 2 and 3 show products with the expected size of 1300 bp. C: Products of colony PCR of FGFC. Lanes 1-8 show bands with the expected size of 1300 bp.

## Transformation into BL21 (DE3).

The team spent several weeks trying to get good levels of expression on several brands of E. coli BL21 (DE3) with different amounts of IPTG. In the end the cells from NEB were the ones that presented positive results so these are the ones that we are going to describe.
Since the team was able to assemble the construct on the vector, the final step was to transform it into E. coli BL21 (DE3) cells for expression. The plasmid was purified using the Monarch® Plasmid Miniprep Kit from NEB following the company’s instructions. The plasmids containing the three genes were transformed into NEB’s competent E. coli BL21 (DE3) cells using the manufacturer’s instructions. The success of the procedure was assessed by PCR and sequencing (Eurofins) as done in previous transformation steps.

# Mutagenesis

Phosphorylated primers containing the mutations were used for PCR mutagenesis to mutate the FGF2 wt gene. The mutations were L98M, Q54K and V88I. Three separate single mutants (FGF2 L98M, FGF2 Q54K, and FGF2 V88I) were made with PCR and the amplicons contained the entire mutated plasmids and were ligated using T4 DNA ligase. The mutant plasmids were then transformed into E. coli DH5ɑ cells and a colony PCR performed to confirm the PCR (Figure 4A). To confirm successful mutagenesis the amplicons were sequenced using Sanger sequencing. One double mutant was also made by repeating the process on Q54K with the L98M primers (FGF2 Q54K+L98M). All plasmids were purified and transformed into E. coli BL21 (DE3) competent cells from NEB. To confirm transformation a colony PCR and gel electrophoresis was performed (Figure 4B ).

![](https://static.igem.org/mediawiki/2021/f/fb/T--Uppsala--res-n-f4.jpg)

Figure 4. Gel electrophoresis of FGF2 mutants designed by the team. The electrophoresis was performed at 100 V for 1 hour in a 1% agarose gel. A: Bands of the right size (~1300) confirm successful amplification of FGF2 Q54K, FGF2 V88I, and FGF2 L98M in mutagenesis PCR. B: Colony PCR products of FGF2 Q54K, FGF2 V88I, and FGF2 L98M transformed into E. coli BL21 cells. Bands of the right size (~1300) confirm the successful transformation.

# Expression of the FGF2 variants

The NEB E. coli BL21 (DE3) cells that had been transformed with the pET plasmid containing either FGF2 wt, FGF2hs, FGFC, FGF2 V88I, FGF2 Q54K, or FGF2 L98M, were cultured in LB medium supplemented with kanamycin 50 ug/ml overnight at 37 ºC while shaking at 220 rpm. In the next morning, the culture was diluted by taking 2 ml of the overnight and adding it to 100 ml of LB medium supplemented with kanamycin 50 ug/ml. When the cultured achieved an OD600 = 0.6 the cells were induced using IPTG 1 mM. The cells were incubated at 37 ºC and 220 rpm for 6 hours. A culture containing the pET plasmid with the IF3 gene was used as a control. The cell cultures were then analyzed through SDS PAGE (Figure 5). The 12% polyacrylamide gel loaded the samples was submitted to 200 V and 0.04 A for 90 minutes. The gel was stained with coomassie brilliant blue

![](https://static.igem.org/mediawiki/2021/thumb/6/6c/T--Uppsala--res-n-f5.jpg/722px-T--Uppsala--res-n-f5.jpg)

Figure 5. Expression of different FGF2 variants shown on a 12% polyacrylamide gel. The samples loaded in the gel were submitted to 200 V and 0.04 A for 90 minutes and stained with coomassie brilliant blue. A: Expression of FGF2 wt, the first lane with “WT U” shows the uninduced control while “WT 10 uM” shows expression with 10 uM IPTG and “WT 1 mM” shows the expression with 1mM IPTG. B: Expression of FGF2 Q54K, FGF2hs, and FGFC. The lanes with “U” show the uninduced controls while the remaining lanes show the induced cultures. The arrow points at a band that appears to be around 30.8 kDa. C: Expression of FGF2 L98M. The “U-V88I” lane shows the uninduced control. “V88I” shows the induced culture and the arrow points at the band that seems to have a molecular weight around 30.8 kDa. D: Expression of FGF2 V88I. The “U-V88I” lane shows the uninduced control. “V88I” shows the induced culture and the arrow points at a band that appears to be around 30.8 kDa.

It is noticeable that the expression of FGF2 wt (Figure 5A), FGF2hs and FGFC (Figure 5B) seems to be higher than the expression of FGF2 L98M (Figure 5C) and FGF2 V88I (Figure 5D) when we compare the bands visible at around 30 kDa. However, since the constructs only differ in a single codon, we hypothesize that this might be due to differences in the starting inoculum or due to some slight differences in the handling of the different cultures by different members of the team.

# Purification of recombinant FGF2 variants.

The NEB E. coli BL21 (DE3) cells containing the pET vector with FGF2 wt, FGFhs, FGFC, and FGF2 L98M were lysed using a french press at 1 kBa after expression. The lysate was spun for 20 minutes at 17000 rpm twice in a SS-34 rotor in a Sorval RC 6 Plus centrifuge. The clear lysate was purified using a GraviTrap column following the manufacturer’s instructions. The clear lysate and elution samples were analyzed through an SDS PAGE (Figure 6).

![](https://static.igem.org/mediawiki/2021/2/21/T--Uppsala--resultpic.png)

Figure 6. The E. coli BL21 (DE3) cells containing the pET vector with FGF2 wt, FGF2hs, FGFC, and FGF2 L98M were lysed using a french press. The cleared cellular lysates (before purification with the column) and the elutions from the GraviTrap columns were analyzed by SDS-PAGE. The 12% polyacrylamide gel containing the samples was submitted to 200 V and 0.04 A for 90 minutes. The gel was stained with coomassie brilliant blue. B-C: FGFC cellular clear lysate before purification; B-HS: FGF2hs cellular clear lysate before purification; B-L98M: FGF2 L98M cellular clear lysate before purification; B-WT: FGF2 wt cellular clear lysate before purification; E-C: FGFC eluted from the GraviTrap column; E-HS: FGF hs eluted from the GraviTrap column; E-L98M: FGF2 L98M eluted from the GraviTrap column; E-WT: FGF2 wt eluted from the GraviTrap column.

From this initial step used for purification it is possible to see that we were able to purify a visible band at around 35 kDa in the lane where the sample that contained FGF2hs was loaded. We suspect that this band with a higher molecular weight might be due to aggregation of FGFhs with other proteins. However, if this were to be FGF2hs it would be a sign that we would have enough to continue purification with confidence since we had a good amount of this protein even though it was aggregated.
In the same SDS PAGE we were also able to notice faded bands of FGF2 L98M and FGF wt at around 35 kDa like we see on the FGF2hs lane. This meant that we might have lost some of the protein during the cell lysis step, the several centrifugations, or the nickel column purification that we submitted the sample to. It might also be due to the natural instability seen in FGF2 [5] which could have led to fast degradation of the expressed protein during the time that the team took to purify it.
No bands of the desired size are visible on the lane where the sample containing FGFC was loaded. We think that loss of protein and its intrinsic instability might be responsible for this.

# Upscaling at Testa Center

### Overexpression in Bioreactor

Overexpression was performed in 5 L bioreactors (Ez2 controller from Applikon). In order to maintain pH levels and keep foam from forming and clogging the bioreactor inlets, the bioreactor was coupled with an anti-foam solution and a base (25% NH3) that would regulate the pH during expression. A control tower was connected to the bioreactor to monitor and regulate pH levels and oxygen flow. A vitamin solution, trace A & B solutions and 50 µg/ml of kanamycin was added to the bioreactor before inoculation. Approximately 50 mL of overnight culture of E. coli BL21 (DE3) pLysS containing the FGF2 plasmid was inoculated in 2.5 L of cultivation media which gave a final OD600 value of 0.091. For more information on overexpression in the bioreactor, see our Lab Notebook in the [Protocols](https://2021.igem.org/Team%3AUppsala/Experiments) page.

The OD was measured every 30 minutes to monitor the growth of the bacteria. At OD600 3.12 induction with 1 mM IPTG was performed. Before induction a small sample was collected and kept in 37 °C with 150 rpm shaking as a negative control. After induction OD was measured every hour for three hours (Figure 7). The bioreactor ran for 7.5 h more to a total of 14 h.

![](https://static.igem.org/mediawiki/2021/7/7b/T--Uppsala--growthcurvewt.jpeg)

Figure 7. Optical density as a function of time for growth of E. coli cells expressing FGF2 wt.

To test if FGF2 was overexpressed, a sample from the bioreactor and the negative control was run on an SDS-PAGE gel. A 12% polyacrylamide gel containing the samples was submitted to 200 V and 0.04 A for 90 minutes. An induction band at the size of FGF2 was not clearly visible (Figure 8), in contrast to the strong induction band seen in the small-scale over-expression in Figure 5A.

![](https://static.igem.org/mediawiki/2021/f/f1/T--Uppsala--res16.png)

Figure 8. SDS-PAGE gel of FGF2 proteins expressed in E. coli (DE3) pLysS competent cells. The 12% polyacrylamide gel containing the samples was submitted to 200 V and 0.04 A for 90 minutes. The gel was stained with coomassie brilliant blue. From the left: induced FGF2, induced FGF2, induced hyperstable version of FGF2 (not relevant here), protein ladder, uninduced FGF2, uninduced hyperstable version of FGF2 (not relevant here). All induced samples are induced with 1 mM IPTG. Strong bands corresponding to the size of FGF2 (30.8 kDa) are not clearly visible for induced cells compared with uninduced cells.

# Large Scale Purification

Despite the low expression, because of time limitation at Testa, purification was performed with IMAC using an ÄKTA PURE 125 system. The column was packed with Capto Chelating resin and charged with 0.1 M NiSO4. To remove any unbound nickel, the column was washed with a lysis buffer (20 mM Tris pH 7.5, 10 mM MgCl2 and 200 mM NaCl). The 2.5 L culture from the bioreactors were spun down for 30 min at 12 000 rpm twice, removing the supernatant in between. The pellet was resuspended in the lysis buffer. To break down the E. coli DNA, DNAse was added to the sample before using a french press (EmulsiFlex-C55A from Avestin) to lyse the cells. The sample was run through the french press at 800 bar 3-4 times. The now lysed cells were centrifuged again and the supernatant containing FGF2 was then run through the Ni-charged column. The flow through was collected and the column washed twice, both washes were collected. Elution was collected in fractions as the elution buffer (20 mM tris pH 7.5, 10 mM MgCl2 and 200 mM NaCl, 500 mM Imidazole pH 7.5) was added with a gradient, slowly increasing the imidazole concentration. This was done since the optimal imidazole concentration for FGF2 elution was unknown. Light absorbance, corresponding to presence of protein, was measured throughout the process to monitor when protein flowed out of the column (Figure 9).

![](https://static.igem.org/mediawiki/2021/c/c0/T--Uppsala--res17.png)

Figure 9. Elution profile from large-scale Ni column used for purification from a 5 L bioreactor. Blue line: absorbance in mAU (milli-absorbance units), y-axis values to the left. Red line: imidazole concentration, y-axis values to the right. Yellow line: column pressure.

Large amounts of protein was seen in the flow through, and two clear peaks were observed during the second wash when the imidazole concentration was 2%. During the elution there was one clear peak followed by a plateau and this was seen at imidazole concentrations ~140 mM and 220 mM respectively. To analyse in which fractions FGF2 were present, an SDS-PAGE gel was run with flow through, wash 1, wash 2 and fractions B1-C1 (Figure 10). A 12% polyacrylamide gel containing the samples was submitted to 200 V and 0.04 A for 90 minutes.

![](https://static.igem.org/mediawiki/2021/2/22/T--Uppsala--res18.png)

Figure 10. SDS-PAGE gel of purified FGF2 protein expressed on a large scale. From the left: lysate flowthrough, wash 1, wash 2, fraction B1, fraction B2, protein ladder, fraction B3, fraction B4, fraction B5, fraction C1. Bands slightly larger than the size of FGF2 (30.8 kDa) are visible in fractions B5 and C1.

No band corresponding to the size of FGF2 was clearly visible. In fractions B1-B3, corresponding to the first peak in the purification, thick bands are visible of ~39 kDa. In fractions B5-C1, corresponding to the shoulder peak, thick bands are visible corresponding to the size of FGF2. The first peak could be a protein with 3-5 His in a row, causing it to bind to the IMAC column, but eluting at a lower imidazole concentration than FGF2, which has 6 His in a row. However, the gel does not confirm that FGF2 was successfully purified on a large scale.

## Bradford Assay

The fractions potentially containing any FGF2 were spin concentrated (Amicon spin concentrators) and the imidazole washed away with a reaction buffer (20 mM Tris-HCl, 50 mM NaCl, 2 mM CaCl2 (pH 8.0)) using the same spin concentrators which were used for protein purification on the small scale. The protein concentration was measured using a Bradford Assay from Bio-Rad [6]. A standard curve was created using BSA. OD595 for the sample was measured and the standard curve used to calculate the amount of expressed protein (Figure 11). The total protein concentration was 147 mg/mL in 11 mL and the total protein yield from large scale production was 1.6 g. However, due to the impurity of the sample (see the multiple bands in Figure 10) it is impossible to say how much of the protein from the large scale purification is FGF2 and how much is protein contamination.

![](https://static.igem.org/mediawiki/2021/c/cd/T--Uppsala--res19.png)

Figure 11. Standard curve for the Bradford assay determined by measuring the absorbance of different concentrations of BSA at 595 nm.

# Future prospects

After the nickel column purification step, the team aimed to cleave the thioredoxin and histidine tags by using enterokinase. This enzyme would cut exactly on the C terminus of each FGF2 variant since we added an enterokinase cleavage site (DDDK) between the tags and the protein. Afterwards, the protein of interest could be separated from the tags in one of two possible ways. The first option would be to use a heparin column since it is known that FGF2 binds heparin [7]. This would then allow us to wash away the tags. The second option would be to use the nickel column again since the His tag, linked to the thioredoxin, would bind the nickel ions in the column and the FGF2 variants could be washed from the column.
With the growth factors purified, the team would then be ready to test them on mammalian cell lines to compare their action with the one from commercial FGF2. The team planned to grow bovine aortic endothelial cells, since endothelial cells express FGF receptors and respond well to the presence of FGF [8] and NIH/3T3 cells, since these cells had been used before to compare commercially obtained FGF2 with recombinant FGF2 produced in a research laboratory [9]. The cells would be grown in DMEM supplemented with either commercial FGF2 or one of the FGF2 variants produced by the team. Finally, through an MTT assay, we would be able to show if the cells were growing faster with our enhanced version of the growth factor [9], [10].

# References

[1] P. Dvorak et al., “Expression and potential role of fibroblast growth factor 2 and its receptors in human embryonic stem cells,” Stem Cells Dayt. Ohio, vol. 23, no. 8, pp. 1200–1211, Sep. 2005, doi: 10.1634/stemcells.2004-0303.

[2] K. Motomura et al., “An FGF1:FGF2 chimeric growth factor exhibits universal FGF receptor specificity, enhanced stability and augmented activity useful for epithelial proliferation and radioprotection,” Biochim. Biophys. Acta, vol. 1780, no. 12, pp. 1432–1440, Dec. 2008, doi: 10.1016/j.bbagen.2008.08.001.

[3] L. Du, R. Gao, and A. C. Forster, “Engineering multigene expression in vitro and in vivo with small terminators for T7 RNA polymerase,” Biotechnol. Bioeng., vol. 104, no. 6, pp. 1189–1196, 2009, doi: 10.1002/bit.22491.

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