Contribution

Introduction

Following our extensive design and troubleshooting process, we wanted to contribute insight into our experiences and knowledge to the wider science community facilitating a stronger foundation in Synthetic Biology, scaffold design and educational outreach. We have documented a comprehensive range of tools to help the future iGEM community to strive.

Here at Renervate Therapeutics we believe in excellence and collective growth.

Bioprinting Contribution

Understanding the Fabrication Process

Prior to printing our scaffold, we had to consider all aspects of the printing and design process. This involved reviewing the material choice, common printing techniques and subsequent methods of testing the printed scaffold. We have developed this guide to walk you through the key design considerations we had to complete in engineering our 3D-printed scaffold.

Developing a Log-Pile Scaffold CAD Design

This guide was developed to educate members of the iGEM community in the creation of a CAD design of a scaffold using the Autodesk Fusion 360 software. In designing our scaffold we created a log-pile 3D-printed scaffold, which was an essential progression in our engineering cycle as our initial design involved the use of complex gyroid unit cell geometry which was too complicated for our pre-printing STL file processing. As a result, we troubleshot our design and incorporated the following steps in our design process.

MFP Contribution

After acknowledging the lack of adhesive ability of our scaffold, we decided upon the use of a mussel foot protein to aid the adhesion of our scaffold to the site of injury in the spinal environment, as well as to promote neuronal regrowth across the scaffold. More information about mussel foot proteins can be found here. Below we have documented a number of guides, parts and research we have developed throughout our project which we hope will be of use to other iGEM teams and the wider scientific community.

Parts

A detailed description of our parts can be found on our Parts page, but a table summarising our parts can be found below.

Our Phase II Parts

For our Phase II project this year, improvements were made to our Phase I parts to optimise certain aspects of our expression. Unfortunately, due to limited laboratory access this year we were unable to provide experimental evidence for these developments.

In Phase I, we created a DNA sequence for our mussel foot protein, PVFP-5, inspired by the paper, ‘Recombinant mussel protein Pvfp-5β: A potential tissue bioadhesive’, (Santonocito et al., 2019). This year, we derived a new DNA sequence for PVFP-5 from UniProtKB (U5Y3S6) (Guerette et al., 2013). This sequence is 40 amino acids longer than our initial sequence and contains an additional 7 tyrosine residues to improve the adhesive potential of our protein. This sequence was also codon optimised for expression in E. coli K12, and a 6xHis tag and TEV site were added to assist with purification via Ni-NTA chromatography.

Furthermore, the tyrosinase enzyme we decided to use was secreted by Bacillus megaterium. The sequence for this enzyme was designed by the 2019 UM Macau iGEM team and was obtained from the iGEM registry under the code BBa_K3033013, however for our project, a stop codon was also added and the sequence was codon optimised for expression in E. coli K12.

Introducing New Parts

This year, we have designed a third basic part for our expression system: thermostabilised Chondroitinase ABC (ChABC). ChABC plays an important role in breaking down CSPGs present in the central nervous system following an SCI, which inhibit axonal regrowth. However, ChABC is limited by its thermosensitive nature, exhibiting reduced efficacy at 37°C. For this reason, we decided to mutate the original ChABC DNA sequence (accessed via UniProtKB (P5N807)) after speaking with Dr Marian Hetteriatchi, who previously performed random mutagenesis on the ChABC sequence to find mutations to increase its thermal stability. We introduced eight point mutations at Dr Hetteriatchi’s recommendation.

Table 1. A list of our basic and composite parts.

Part Name	Part Number	Description	Type	Length (bp)
PVFP-5 CDS	BBa_K3794000	PVFP-5 insert with stop codon and N-terminal 6xHis tag+TEV site.	Basic	417
Tyrosinase CDS	BBa_K3794002	Tyrosinase insert with stop codon.	Basic	897
ChABC CDS	BBa_K3794004	ChABC insert with stop codon and N-terminal 6xHis tag+TEV site.	Basic	3042
Lac Regulator	BBa_R0010	Lac Promoter encoding CAP Binding site, Lac promoter and Lac operator. Sensitive to CAP and LacI	Regulatory	200
Terminator	BBa_B0010	rrnB T1 Terminator	Terminator	80
PVFP-5 Composite Part	BBa_K3794001	Translational unit for PVFP-5. Encodes Lac regulatory system, as well as PVFP-5 with upstream 6xHis Tag and TEV cleavage site	Composite	617
Tyrosinase Composite Part	BBa_K3794003	Translational unit for Tyrosinase encoding Lac regulatory system, Tyrosinase coding sequence and rrnB1 T1 Terminator.	Composite	1177
ChABC Composite Part	BBa_K3794005	Translational unit for ChABC encoding Lac regulatory system, ChABC coding sequence with upstream 6xHis tag and TEV cleavage site. rrnB T1 Terminator found on 3’ end.	Composite	3322

Contribution to Greater Science

Mussel foot proteins are of current interest throughout science for use as bioadhesives, particularly in the fields of biotechnology and therapeutics. Our research surrounding the synthesis, purification and supply of mussel-inspired bioadhesives will provide a significant contribution to these sectors. Additionally, our structural modelling guides and protocols will prove to be invaluable to the wider scientific community in various aspects of research. We hope that our project, research and laboratory work will provide a significant contribution to other iGEM teams and scientists worldwide.

Protocols

For our Phase II project this year, we validated our research from Phase I by designing and implementing various protocols to express, purify and analyse our protein, PVFP-5, in two cell lines of E. coli, and ChABC in one cell line. Our protocols are available for use and adaptation by other iGEM teams, and can be found here:

Modelling

A Guide to Molecular Dynamics using GROMACS

We have developed this tutorial to guide you through the process of a general GROMACS molecular dynamics simulation. We utilised GROMACS to assist in the formation of our predicted disulphide bonds, as well as to improve the overall quality of our PVFP-5 structural model.

MFP-PCL Binding Calculator

Our calculations about MFP-PCL binding work by extrapolating from the values of the areas of the scaffold and a unit of protein. The JavaScript version of this calculator is available directly here:

Codon Optimizer

Optimizer Information:

What is this?

A codon optimizer is a tool that translates a protein sequence in reverse. This translation is done so that the resulting DNA sequence will contain the codons most used by an organism for a particular amino-acid. This in turn can significantly increase protein production.

How to use it:

Input
The input is the protein sequence that is to be reverse-translated. The format it should be input in is standard one-letter amino-acid code.
Choose an organism
This codon optimizer can use the codon preferences for four organisms: E. coli bacteria, D. melanogaster flies, mice, and humans. The choice can be made from the drop-down menu below the input field.
Optimizer
The output is the 5' to 3' DNA sequence that has been optimized based on the selected organism's codon frequencies.
Warnings
There are two warnings that may appear. If you see 'Invalid Value!', that means that the input contains a non-standard one-letter amino-acid code. If you see 'No stop codon!', that means the input protein sequence does not have a '*' to signify termination at the end of it.

Optimized DNA sequence:

Input Protein Sequence

Our Excellence in Another Area

This year we have worked extensively on developing our educational outreach programme, Biologix and encourage other iGEM teams to take inspiration from our work!

Biologix Competition

Our competition, Biologix, aims to promote widening participation in STEM and synthetic biology to secondary school students from 16 to 18 years of age.

We developed a website to act as a platform for all of the information required by participants, such as important dates, recorded lectures and submission points. Our KCL iGEM team members that were directly involved in the competition all underwent DBS checks and safeguarding training.

In collaboration with the Manchester and the St Andrews’ iGEM teams, we created lectures to expand the participants’ knowledge in the applications of synthetic biology to solve a range of problems, as we do during the iGEM competition. We liaised with our sponsors to create prizes to reward participants for the completion of the competition. To ensure the continued running of Biologix, we have launched it under the King’s College London Biotechnology and Synthetic Biology Society and encourage the future KCL iGEM teams to carry on our work.

Our participants’ feedback clearly conveys a more developed understanding of the field of synthetic biology following our competition and we are looking forward to continuing running Biologix annually to have the same or even greater impact. We encourage other iGEM teams to help promote and collaborate with us in the future to create content for Biologix so we can further widen participation in STEM in the coming years. We also would highly recommend other teams to take inspiration from how we developed and launched Biologix to start their own competition to further encourage interest in STEM and synthetic biology.

More information about our competition, its development and outcomes, can be found on our Education page.

Other Contributions

Guide to Creating a Team Virtually

With the context of forming our KCL iGEM 2021 team during the pandemic, we had to find new methods of putting together our team. In comparison to previous years where we held interviews in person, this year, we conducted our application process completely virtually. Following our redesign of this process to fit the remote nature of working while still being effective, we have created this guide to help future iGEM teams form their teams virtually as we did.

References:

Guerette, P. A., Hoon, S., Seow, Y., Raida, M., Masic, A., Wong, F. T., ... & Miserez, A. (2013). Accelerating the design of biomimetic materials by integrating RNA-seq with proteomics and materials science. Nature biotechnology, 31(10), 908-915.
Santonocito, R., Venturella, F., Dal Piaz, F., Morando, M. A., Provenzano, A., Rao, E. et al. (2019). Recombinant mussel protein Pvfp-5β: A potential tissue bioadhesive. Journal of Biological Chemistry, 294(34), 12826-12835. doi:10.1074/jbc.ra119.009531

Team:KCL UK/Contribution