About

Resource inequalities among iGEM teams are undeniable. While certain high-profile teams have access to state-of-the-art lab equipment and large amounts of funding, other teams have to make do with much less. These disparities disadvantage many iGEM teams regardless of their capabilities. To tackle this problem, and alleviate such discrepancies, the  ‘Remote Lab’ project idea was born and builds on the partnership of the 2021 Paris-Bettencourt and Marburg iGEM teams. This year the Paris-Bettencourt, Imperial College London and the Costa Rica iGEM teams have teamed up to participate in the development and expansion of the ‘Remote Lab’ platform to make it more accessible to share biotechnological resources to teams around the globe.

WHAT IS THE REMOTE LAB ?

The ‘Remote Lab’ platform allows teams to remotely design and execute high-throughput combinatorial Golden Gate assemblies with the help of the ‘assembly teams’. Whenever a team has limited cloning capacity and/or funding, but would like to fine-tune the expression of a specific construct, the team can submit a high-throughput assembly order to the ‘assembly teams’ to execute. The ‘assembly team’ can modify and implement automated protocols for the designed assemblies, and perform them using state-of-art liquid handlers on site. The final validated constructs can then be shipped back to the ‘receiving team’ to be characterized. This automated cloning service could provide a fast and reliable solution to expand the project scope for teams with limited funding and equipment, while it also minimizes the workload on the ‘assembly teams’.

High-throughput Golden Gate assembly using the Beckmann Echo

The automated Golden Gate assemblies are performed using Beckman Coulter Echo™ (Echo 525 and 550) which are capable of transferring liquid droplets using acoustic droplet ejection with superior precision and speed. The 2021 Marburg and Paris-Bettencourt teams have started to develop standard protocols to facilitate this process. This year, the iGEM teams of Imperial College London, Costa Rica and Paris-Bettencourt decided to put the existing framework to the test and set up the first overseas remote project. During the partnership we have managed to complete the assembly pipeline, improved on the existing protocols, increased the scope of the platform as well identified and solved key bottlenecks for future collaborations.

• Include overseas teams and share state-of-the-art equipment
• Create a pipeline to allow a remote team to use the Echo liquid handler for golden gate assembly
• Show reproducibility of the assembly pipeline
• Divide tasks of assembly
• Facilitate inclusion of countries with strict shipping restrictions

In our partnership with Imperial and Costa-Rica, we aimed to address the inequalities surrounding the type of laboratory equipment that iGEM teams around the world have access to. In March 2022, the Paris and Imperial team met and discussed their interest in working together on making our lab equipment available from a distance by teams outside of Europe and the USA. A common machine that both teams have access to is the Beckman Coulter Echo™ liquid handler that has been characterized in a previous partnership (link) for high-throughput golden-gate assembly. Our teams wanted to prove that we could make our machine available to other teams, however far away. The Costa-Rican team showed interest in using our machine, due to their difficulties in accessing the materials needed for genetic engineering, and our three teams worked together for several months to demonstrate how it is possible to share resources cross-continentally. The Costa-Rican team thus became the “receiving” team and Imperial and Paris-Bettencourt the “assembly” teams.

The original plan was that Costa Rica would create and synthesize their parts of interest and these would be sent to the other assembly teams. However, when it came to implementing this idea, some inconveniences arose. Firstly, the importation of DNA to Costa Rica is often hindered at the customs offices, requiring a large amount of paperwork and economic investment (see in more detail below). This occurs mainly with the parts that are requested through IDT's donated credit, which means that the Costa Rican team cannot have direct access to these donated bases. It is worth noting that Costa Rica tried to import parts synthesized by IDT for the development of its project, but they are still held in customs, requiring about $600 and the contract of a legal customs agent to be able to take these parts to the laboratory. In fact, past Costa-Rican teams have historically had difficulty in receiving orders from IDT, where their orders remain blocked at the borders for up to one year or more, reflecting the difficulty that some teams may have to access the resources provided by sponsors and iGEM.

To circumvent this issue, we contacted representatives from IDT to change Costa-Rican’s delivery address to Paris. This would allow synthesized parts ordered from the Costa-Rican to reach the Paris and Imperial teams directly for assembly. A representative responded and together we created a separate iGEM member account specially for the partnership and credit transfer. Although the processing time for the credit transfer took too long to be included in this year's pipeline, this exchange with IDT demonstrated how IDT credit transfers can be possible, which paves the way for future iGEM teams which may have similar issues to Costa-Rica in receiving synthesized parts.

To be able to proceed without the synthesised parts, Imperial has managed to arrange access to a currently unpublished Golden Gate assembly kit specific to Gram-positive bacteria. This was a crucial achievement as Costa Rica uses the gram+ Lactobacillus casei as their engineering chassis. Most assembly kits available on the market are designed specifically for E. coli and other gram- bacteria, and would have been unsuitable for Costa Rica. Therefore, all the parts used in the assemblies were sourced from the Subtitoolkit developed by Joaquin Caro Astorga.

To increase the chances of successful assemblies and to make them more reproducible, the Imperial team shared the toolkit with the Paris-Bettencourt team. The entire kit was shipped as agar stabs from London to Paris on a 96-well plate, along with sequencing primers and plasmid DNA of the most important parts. After reception of the plates, Paris successfully replicated the plate and produced glycerol stocks for long-term storage. This demonstrated how agar stabs are an efficient way to transport samples intercontinentally. The introduction of the Subtitoolkit has also increased the scope of future collaborations as from now on the current ‘assembly teams’ can cater to ‘receiving teams’ using a variety of engineering chassis.

Based on the Gram+ toolkit, provided by Joaquin Caro-Astorga from Imperial College London (data not published yet), Costa Rica chose different parts that could be used in their project. Since Costa Rica's project is based on the use and characterization  of Lactobacillus casei as a chassis in synthetic biology for industrial application, the promoters and RBSs chosen from the kit were based on the potential they would have if used in this bacteria.

Among the chosen parts, a strong promoter was selected in order to compare its effect in expression levels with the force of the constitutive promoters designed by Costa Rica. Likewise, due to the potential of Lactobacillus in the food industry, it was also decided to use sugar-inducible promoters such as xylose and IPTG inducible promoters, for the exploration of recombinant protein expression during the production of foods such as yogurt using L. casei. Finally, multiple RBSs with different ranges of strength (medium, weak and very strong) were chosen, with the objective of comparing differences in expression when using these RBSs with respect to those designed by Costa Rica as part of their toolbox for Lactobacillus. Also, the vector used to assemble these parts and characterize them in L. casei was pSTK. This vector has a ColE1 origin site and a repB gene for its replication in E. coli and Bacillus, respectively, functioning as a shuttle vector between Gram-negative and Gram-positive bacteria. In addition, reporter genes that could be expressed by Gram-positive bacteria such as L. casei were selected from the kit and used in the assembly.