Team:Uppsala/Parts


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Parts

Below are two tables of iGEM Uppsala 21’s basic and composite parts. Our favourite parts, which we nominate to the best parts awards, are marked with a ❤. Our parts were created by first designing the FGF2 fusion protein using SnapGene. Then they were synthesized by Integrated DNA Technologies (IDT). This was followed by performing BioBrick assembly using PCR amplification, restriction enzyme digestion and ligation to insert our FGF2 gene on a pET backbone [1],[2]. Some of the parts were finalized using PCR mutagenesis.

Basic Parts

Favourite Name Type Description Designer Length
❤ BBa_K3934005 Coding Enhanced bovine growth factor FGF2 designed to increase affinity to the FGFR2 receptor, making it induce cell proliferation. with higher efficiency. Emelie Oscarsson 468 bp
BBa_K3934000 Coding Bovine growth factor FGF2wt which induces cell proliferation. Diogo Filipe 468 bp
BBa_K3934001 Coding Thermostably enhanced bovine growth factor which induces cell proliferation. Diogo Filipe 468 bp
BBa_K3934002 Coding Chimera of bovine growth factors FGF2 and FGF1 with increased stability, decreased heparin dependency and decreased trypsin degradation compared to FGF2 wt. Daniel Lake 467 bp
BBa_K3934003 Coding Enhanced bovine growth factor FGF2 designed to increase affinity to the FGFR2 receptor, making it induce cell proliferation, with higher efficiency. Emelie Oscarsson 468 bp
BBa_K3934004 Coding Enhanced bovine growth factor FGF2 designed to increase affinity to the FGFR2 receptor, making it induce cell proliferation, with higher efficiency. Emelie Oscarsson 468 bp
BBa_K3934006 Coding Enhanced Bovine growth factor FGF2 designed to increase affinity to the FGFR2 receptor, making it induce cell proliferation, with higher efficiency. Emelie Oscarsson 468 bp
BBa_K3934008 Coding Thioredoxin used as a fusion protein to increase solubility. iGEM Uppsala 21 327 bp
BBa_K3934015 Tag 6xHis-tag for protein purification. iGEM Uppsala 21
BBa_K3934016 Kinase site Enterokinase site used to cleave fusion proteins. iGEM Uppsala 21

Favourite Basic Part

For the best basic part we nominate a mutant version of the bovine FGF2 protein (BBa_K3934005). FGF2 is a mammalian growth factor which amongst other things signals cell proliferation [3]. It is one of the most important, and most expensive, components of serum free growth medium used in cellular agriculture to cultivate meat [4]. To decrease the costs of cultivated meat production in order to make it available on the food market, the growth medium needs to be optimized. This would enable a more environmentally friendly alternative to meat consumption. This part has 2 point mutations in the site where the protein binds to its receptor FGFR2 to induce cell growth. These point mutations were designed in PyMOL [5] using rational design. Free energy perturbation (FEP) was performed using the molecular dynamics software package Q [6] to evaluate the effects of these single point mutations in silico. The mutations were shown to increase the binding affinity to its receptor and we hypothesize that this will lead to increased mitotic signalling so that less FGF2 will be needed in the serum free growth medium for cultivating meat, decreasing the cost of the process. The part has been successfully expressed.

Composite Parts

Favourite Name Type Description Designer Length
❤ BBa_K3934007 Composite Bovine growth factor FGF2 wt which induces cell proliferation. Diogo Filipe 943 bp
BBa_K3934013 Composite Enhanced bovine growth factor FGF2 designed to increase affinity to the FGFR2 receptor, making it induce cell proliferation with higher efficiency. Emelie Oscarsson 943 bp
BBa_K3934009 Composite Chimera of bovine growth factors FGF2 and FGF1 with increased stability, decreased heparin dependency and decreased trypsin degradation compared to FGF2 wt. Daniel Lake 942 bp
BBa_K3934010 Composite Enhanced bovine growth factor FGF2 designed to increase affinity to the FGFR2 receptor, making it induce cell proliferation with higher efficiency. Emelie Oscarsson 943 bp
BBa_K3934011 Composite Enhanced bovine growth factor FGF2 designed to increase affinity to the FGFR2 receptor, making it induce cell proliferation with higher efficiency. Emelie Oscarsson 943 bp
BBa_K3934012 Composite Thermostably enhanced bovine growth factor which induces cell proliferation. Diogo Filipe 943 bp
BBa_K3934014 Composite Enhanced bovine growth factor FGF2 designed to increase affinity to the FGFR2 receptor, making it induce cell proliferation with higher efficiency. Emelie Oscarsson 943 bp

Favourite Composite Part

For the best composite part we nominate the bovine FGF2wt gene (BBa_K3934007). This part encodes for a biobrick part of the bovine FGF2 wt protein used in growth medium for cultivated meat in the field and industry of cellular agriculture. Our FGF2 biobrick enables researchers and companies to more economically access FGF2 for research and protein engineering, which can be used towards an optimized FGF2. The part has been successfully expressed and purified on both a small scale and a large scale. By making FGF2 more accessible, further research can be more easily conducted to one day reach the goal of a global lifestyle where what we eat does not harm our planet the way it does today.

Parts Collection

Our part collection consists of different FGF2 versions. Except for the wt FGF2, they were all designed to, in different ways, optimize this growth factor to achieve a more economically favourable cultivated meat production. This includes increased thermostability, decreased heparin dependency, decreased trypsin degradation and increased receptor binding affinity. By exploring different ways to improve FGF2 we hoped to identify a range of improvements that in the future could potentially be combined to obtain a significantly more effective FGF2.

Favourite Name Type Description Designer Length
❤ BBa_K3934005 Coding Enhanced bovine growth factor FGF2 designed to increase affinity to the FGFR2 receptor, making it induce cell proliferation with higher efficiency. Emelie Oscarsson 468 bp
BBa_K3934000 Coding Bovine growth factor FGF2 wt which induces cell proliferation. Diogo Filipe 468 bp
BBa_K3934001 Coding Thermostably enhanced bovine growth factor which induces cell proliferation. Diogo Filipe 468 bp
BBa_K3934002 Coding Chimera of bovine growth factors FGF2 and FGF1 with increased stability, decreased heparin dependency and decreased trypsin degradation compared to FGF2 wt. Daniel Lake 467 bp
BBa_K3934003 Coding Enhanced bovine growth factor FGF2 designed to increase affinity to the FGFR2 receptor, making it induce cell proliferation with higher efficiency. Emelie Oscarsson 468 bp
BBa_K3934004 Coding Enhanced bovine growth factor FGF2 designed to increase affinity to the FGFR2 receptor, making it induce cell proliferation with higher efficiency. Emelie Oscarsson 468 bp
BBa_K3934006 Coding Enhanced bovine growth factor FGF2 designed to increase affinity to the FGFR2 receptor, making it induce cell proliferation with higher efficiency. Emelie Oscarsson 468 bp
❤ BBa_K3934007 Composite Bovine growth factor FGF2 wt which induces cell proliferation. Diogo Filipe 943 bp
BBa_K3934013 Composite Enhanced bovine growth factor FGF2 designed to increase affinity to the FGFR2 receptor, making it induce cell proliferation with higher efficiency. Emelie Oscarsson 943 bp
BBa_K3934009 Composite Chimera of bovine growth factors FGF2 and FGF1 with increased stability, decreased heparin dependency and decreased trypsin degradation compared to FGF2 wt. Daniel Lake 942 bp
BBa_K3934010 Composite Enhanced bovine growth factor FGF2 designed to increase affinity to the FGFR2 receptor, making it induce cell proliferation with higher efficiency. Emelie Oscarsson 943 bp
BBa_K3934011 Composite Enhanced bovine growth factor FGF2 designed to increase affinity to the FGFR2 receptor, making it induce cell proliferation with higher efficiency. Emelie Oscarsson 943 bp
BBa_K3934012 Composite Thermostably enhanced bovine growth factor which induces cell proliferation. Diogo Filipe 943 bp
BBa_K3934014 Composite Enhanced bovine growth factor FGF2 designed to increase affinity to the FGFR2 receptor, making it induce cell proliferation with higher efficiency. Emelie Oscarsson 943 bp

References

[1] 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.

[2] L. Du, S. Villarreal, and A. C. Forster, “Multigene expression in vivo: Supremacy of large versus small terminators for T7 RNA polymerase,” Biotechnol. Bioeng., vol. 109, no. 4, pp. 1043–1050, 2012, doi: 10.1002/bit.24379.

[3] A. N. Plotnikov, S. R. Hubbard, J. Schlessinger, and M. Mohammadi, ‘Crystal Structures of Two FGF-FGFR Complexes Reveal the Determinants of Ligand-Receptor Specificity’, Cell, vol. 101, no. 4, pp. 413–424, May 2000, doi: 10.1016/S0092-8674(00)80851-X.

[4] E. Swartz. “Meeting the Needs of the Cell-Based Meat Industry,” American Institute of Chemical Engineers (AIChE), Okt. 2019. Accessed on: Okt. 04, 2021. [Online]. Available: https://gfi.org/wp-content/uploads/2021/01/Cell-Based_Meat_CEP_Oct2019.pdf

[5] The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.

[6] QresFEP: An Automated Protocol for Free Energy Calculations of Protein Mutations in Q Willem Jespers, Geir V. Isaksen, Tor A.H. Andberg, Silvana Vasile, Amber van Veen, Johan Åqvist, Bjørn Olav Brandsdal, and Hugo Gutiérrez-de-Terán Journal of Chemical Theory and Computation 2019 15 (10), 5461-5473 DOI: 10.1021/acs.jctc.9b00538