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’.

Beckman Echo

The automated Golden Gate assemblies are performed using Beckman Coulter Echo™ (Echo 525 and 550) which are capable of transferring liquid droplets with superior precision and speed using acoustic droplet ejection technology. 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.

Goals of the partnership

- 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

Setting up a partnership

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 (https://2021.igem.org/Team:Marburg/Partnership/Bettencourt, https://2021.igem.org/Team:Paris_Bettencourt/Partnership) 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.

Acquiring the assembly parts

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 in ‘Shipping’ section). 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 [1, 2]. Therefore, all the parts used in the assemblies were sourced from the Subtitoolkit developed by Joaquin Caro Astorga [3].


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 (3), 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 [4, 5]. 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.

Improving existing protocols

To further familiarise ourselves with the Echo 525 and 550 liquid handlers and to improve on the existing Golden Gate protocols we reached out to Beckmann Coulter. We received tremendous support from them in the form of a two-part training. The first online training explained how the Echo works and how to build Cherry pick files for Golden Gate assemblies. The second training was held on site at Imperial College London with the help of a field applications scientist and was streamed to the Paris-Bettencourt team. We completed a demo assembly using the MoClo CIDAR kit [1].

During the on-site training we optimised the golden-gate protocol via selecting the appropriate Echo source plate (384PP, PP-0200), and via performing a calibration experiment. The calibration experiment allowed us to check the appropriate calibration setting for each liquid reagent used in the Golden Gate reaction, depending on their viscosity and surfactant concentrations. We have updated the golden-gate protocol initially created by the 2021 Paris-Bettencourt/Marburg Partnership (https://2021.igem.org/Team:Paris_Bettencourt/Partnership) and we also created protocols for these calibration experiments. We suggest that every team participating in this platform performs these calibration experiments, as reagent formulation may differ depending on different brands.
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We also created a new protocol for a water-shooting test, which serves to calibrate the destination plate and check that the fluid transfers land correctly in the wells.

Protocol for Golden Gate Assembly using the Echo Liquid Handler [golden gate protocol]

Protocol for Fluid Calibration [fluid calibration protocol]

Protocol for Destination Plate Calibration [destination plate calibration protocol]
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Completing the assemblies

The final assembly were designed via Imperial College London providing the Subtitoolkit part selection to Costa Rica and Costa Rica chose the parts appropriate for their use. A Benchling folder was created to simulate the assemblies as well as a Cherry Pick file for the Echo by Imperial College London. The assemblies were performed by both Paris-Bettencourt and Imperial College London, to ensure reproducibility. The workflow started with preparing mini-preps for the parts used in the assembly. To increase automation the Imperial College London team used the Beckmann Coulter Biomek i7 Automated Workstation, while the same process were done manually by Paris-Bettencourt. The parts and other reagents were added to the Echo source plate and the Echo performed the individual reactions in the destination plate. The destination plate was added to the thermocycler for the Golden Gate reaction. The resulting constructs were transformed into E.coli. After transformation, the colonies were screened via colony PCR at Imperial College London and the selected colonies were mini-prepped and sent for sequencing. At Paris-Bettencourt, the colonies were directly screened via Mycrosynth E.coli NightSeq and the correct colonies were mini-prepped. Both teams shipped their parts to Costa Rica following the shipping guidelines via locker routes.

Shipping to Costa Rica

In Costa Rica and other Latin American teams, the request for genetic sequences becomes a real problem in order to start laboratory work, which has led to teams not being able to access these sequences to obtain results. As an example, the Costa Rica iGEM Team from 2021 hasn’t been able to evict their genetic sequences from customs offices, which translates into a great economic and scientific loss and iGEM projects highly affected by discontinuation. This is due to the existence of strict regulatory policies in customs offices with long bureaucratic processes. These processes not only require the investment of a lot of time, but a great deal of money to dislodge these packages. For this year, iGEM has had the incredible sponsor from companies such as IDT and Twist Bioscience and for iGEM Costa Rica team to be able to use these base pairs was fundamental for the development of the project. To order genes, it is possible to do the procedure by the locker route through companies such as FedEx, JetBox, others, who take care of the whole logistical process and bring the package to the university. In this specific case, we have access to the company's lockers in the United States, which allows a fast delivery to these branches and from there the courier company is in charge of all the procedures. This allowed us to obtain the genetic sequences in less than 3 weeks from order to arrival in Costa Rica, while the requested IDT sequences have been kept in customs offices for more than 1 month, not counting the shipping process from IDT and the handling through DHL. Legal procedures for shipping

Network

To help connect teams that would be interested in collaborating and building partnerships surrounding remote lab automation we built a Lab Automation Network on the Just One Giant Lab (JOGL) platform. JOGL functions as a type of social media, which is particularly suited for sharing and collaborating in science research with participants from around the world. 

The idea to have a centralized platform came from the difficulty of contacting teams for partnerships and knowing what materials and machines each team had access to. Imperial and Costa-Rica both responded to requests for partnerships, but there are probably many other teams that would be interested in sharing resources. Using the Lab Automation Network would make it easier to collaborate on remote lab projects with teams from around the world. This network also makes it easy to advertise what materials and machines your team has access to and to respond to requests.

To construct this Lab Automation Network Paris reached out to the CEO of JOGL, Thomas Landrain, and he was enthusiastic about our idea of creating a page dedicated to collaborations around liquid handling machines. He allowed us access to a Beta-version of JOGL where we were able to build a page from which collaboration groups can form.

Here is the link : https://app.jogl.io/space/iGEMRLN and we invite teams to contribute to the group.

We hope this would help increase inclusivity in iGEM by alleviating the unequal access to genetic engineering technology. We hope this Lab Automation Network can be used by future iGEM teams to find and connect teams and alleviate unequal access to resources.

Conclusions

In this three team partnership we demonstrated how we could perform Golden Gate assembly cross-continentally and we hope that future teams will feel encouraged in the future to improve inclusivity by sharing materials and machines to teams in remote places. Key points that we have retained from this partnership are to take a lot of advance because of the time it takes to arrange administrative procedures. We recommend contacting the providers of the liquid-handling machines and the gene synthesis companies because their help can be crucial in overcoming difficulties and enhancing protocols.

Contributions

Costa Rica contributed selecting the parts for the assembly and contacting IDT to transfer base-pair credit to Paris-Bettencourt. They found ways to import DNA in easier and faster ways and worked on understanding how custom offices work at Costa Rica for the importation of genetic sequences. Imperial contributed in the acquisition of the Subtitoolkit to cater to the engineering chassis of Costa Rica and sharing the parts with Paris-Bettencourt. They organised the hands-on Echo training. They assembled the constructs, validated them by sequencing and sent them to Costa Rica. Paris-Bettencourt contributed in designing the demo assembly, creating the JOGL plateform, arranging the IDT credit transfer and writing the calibration protocols. They also assembled the constructs, validated them by sequencing and shipped them to Costa Rica.

Acknowledgement

During our partnership we received help from external members which greatly enhanced the overall pipeline. Thank you to Zoe Pincemaille for establishing the first contact and sharing her learnings from the 2021 Paris-Bettencourt / Marburg partnership. Thank you to Alexis Casas for mentoring the teams and permitting Imperial to access the Echo. Thank you to Maria Savino, Kristie Najir and Dominik Zahr from Beckmann Coulter for the training sessions. Thank you to Mitchell Ryckmans from IDT customer care for the credit transfers. Thank you to Thomas Landrain for allowing us access to JOGL to create the network. And thank you to Joaquin Caro Astorga for providing the Subtitoolkit for this project.

References

[1] Moore SJ, Lai H, Kelwick RJR, Ghee SM, Bell DJ, Polizzi KM, et al. EcoFlex: A Multifunctional MoClo Kit for E-coli Synthetic Biology. ACS Synth Biol 2016 OCT;5(10):1059-1069.
[2] Iverson SV, Haddock TL, Beal J, Densmore DM. CIDAR MoClo: Improved MoClo Assembly Standard and New E-coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology. ACS Synth Biol 2016 JAN;5(1):99-103.
[3] Caro Astorga J Subtitoolkit. Unpublished 2022
[4] Perez-Arellano I, Zuniga M, Perez-Martinez G. Construction of compatible wide-host-range shuttle vectors for lactic acid bacteria and Escherichia coli. Plasmid 2001 SEP;46(2):106-116.
[5] Roeland Boer D, Angel Ruiz-Maso J, Rueda M, Petoukhov MV, Machon C, Svergun DI, et al. Conformational plasticity of RepB, the replication initiator protein of promiscuous streptococcal plasmid pMV158. Sci Rep 2016 FEB 15;6:20915.