1.1 Problem and Background
Enzymes are required for almost all RNA/DNA detection methods including the RT-qPCR assay and the RT-LAMP assay. These enzymes include reverse transcriptases for converting viral RNA to DNA as well as various DNA polymerases for amplifying DNA to detectable levels. These assays are used for detection of the genome of the SARS-CoV-2 coronavirus. Both resource-limited and developed nations are facing shortages under the high global demand for commercial enzymes and reagents. This project aims to help by developing enzyme production protocols that can lead to the creation of kits for distribution.
The Open Bio Economy Lab has curated a panel of 84 off-patent enzyme genes. The BioBricks Foundation through their FreeGenes program have synthesized the first collection of 42 enzyme genes and are in the process of synthesizing the remainder. These genes are released under the OpenMTA which permits unencumbered distribution and use of the genes.
A subset of this collection includes various enzymes used in the different diagnostic assays. The specific enzymes required for the various assays include reverse transcriptases (for converting RNA to DNA), thermostable DNA polymerases (for Polymerase Chain Reaction (PCR) amplification of DNA), or for Loop-Mediated Isothermal Amplification (LAMP).
1.2 Solution summary in simple terms
Since the discovery of the structure of DNA, enzymes have proved to be powerful and useful tools for health, biotechnology and scientific inquiry. Open Enzyme Production and Purification is a collaborative consortium with each lab focusing on different aspects of the challenge.
- Team OSN (Canada): Purifying diagnostic enzymes traditionally and as cellular reagents
- Team BioBlaze (USA): Exploring protein tags and inducible promoters for low cost purification
- Team epiLAB (Germany): Exploring enzyme variants for optimizing activities
- Team Bionet (USA): Building expression plasmids for low-cost protein purification
To enhance affordability and distribution of non-commercial enzymes worldwide, this collective proposes to develop strategies and open protocols for purifying and formulating various IP-free Open Enzymes that are essential for use in covid-19 diagnostic assays. Beyond the focus on covid-19, these open tools and strategies are applicable for other DNA/RNA-based diagnostic assays to help detect and treat a great many diseases as well as to lead innovation in open scientific enquiry and biotechnology.
1.3 Solution summary in technical terms
Team OSN will use IPTG-induced expression of His-tagged enzymes as the first enzyme production and purification method, against which we will benchmark subsequent autoinduction, low-cost purification, and cellular reagent production protocols. Many diagnostic-relevant Open Enzyme Collection genes, like reverse transcriptases, thermostable hi-fidelity DNA polymerases and isothermal DNA polymerases, have already been introduced into expression vectors. Enzymes produced with these plasmids will be purified using nickel resin and as cellular reagents. Enzyme quantity and purity will be measured with PAGE gels and Lowry Protein Assay. Enzyme function will be assessed with the appropriate assay.
Nickel resins and IPTG are expensive, so a second generation of lower-cost plasmids and protocols will be built, tested and validated by Teams Bionet and BioBlaze. We and our collaborators on the FreeGenes and OpenEnzyme projects have already designed and ordered an off-patent collection of tags that bind to lower-cost chromatography substrates (including silica, chitin/chitosan, starch/amylose, and cellulose), along with self-cleaving intein domains. These tags will be assembled with diagnostic enzyme genes and fuGFP tags, transformed into E. coli expression strains, grown in lactose-rich autoinducing media, and screened by fluorescence for the best-expressing enzyme/tag combinations. These plasmids will be cultured at larger scale, and purification with each of the above low-cost chromatography media will be tested, with purification yield rapidly measured by GFP fluorescence, and purity assessed with PAGE.
Team epiLAB will test the activity of the homebrewed and purified enzymes in detection assays such as RT-qPCR and LAMP. Additionally, Team epiLAB will run site-directed mutagenesis on the functioning expression plasmids for different diagnostic enzymes, in order to introduce off-patent point mutations known from the scientific literature to increase stability and activity of some of the diagnostic enzymes (e.g. MMLV Transcriptase).
1.4 State of advancement of the project
The first set of Open Bioeconomy Lab’s curated open source Open Enzyme Collection genes is now available through Free Genes. Synthesis by Free Genes of the second collection of an additional 42 curated IP-free genes has commenced. In addition, Free Genes has placed an order to Twist for His-tagged, lactose/IPTG-inducible expression plasmids for all diagnostic enzymes in the collection, and will soon place an order for the low-cost purification tags. Open Bioeconomy Lab is also conducting a crowdsourced survey of production and purification methods for the full list of enzymes in the collection.
1.5 Project Timeline
● Week 1 - Acquisition of supplies and reagents. All teams order items on our budgets once the funds have been acquired. Teams Bionet and BioBlaze begin design of low-cost expression plasmids, and identify any JOGL-, FreeGenes-, and Open Bioeconomy-affiliated labs interested in collaborating in testing/replicating low-cost purification protocols. Team epiLAB designs mutational expression plasmids.
● Week 2 - Begin experimentation. Team OSN and Team epiLAB transform His-tagged enzyme plasmids into E. coli BL21 stain and confirm clones through standard mini-prep and restriction mapping. BL21 will act as a bio factory to synthesize the enzymes we are interested in. Prepare buffer solutions and nickel columns. Team BioBlaze tests construction and flow-through of chromatography columns with low-cost purification chromatography media, and distributes low-cost purification reagents to any collaborating labs. Upon receipt of low-cost purification tag gene collection from Twist, Team Bionet assembles parts with enzyme genes into experimental expression plasmids and transforms constructs into E. coli BL21.
● Week 3 - Staggering production of the different enzymes. Team OSN & epiLAB sets up small bacterial cultures of the first candidate and induce with either IPTG or autoinduction media at the appropriate time point. Lyse the E. coli and attempt to purify the enzymes using different protocols. Collect samples at the various stages of production for SDS-PAGE analysis. Team BioBlaze designs and shares planned purification protocols for low-cost protein purification using the different tags, chromatography media, and tag cleavage domains. Team Bionet completes assembly, transformation, and sequence verification of experimental low-cost expression plasmid library, and distributes strains/plasmids to the consortium members and other collaborating labs.
● Week 4 - Test enzymes produced in previous week in appropriate assays. Team OSN starts experimenting with cellular reagent approach to enzyme production using lyophilization and other low cost approaches. Teams Bionet, BioBlaze and collaborators begin high-throughput testing of experimental expression plasmids in deep 96-well culture plates with auto-induction media, measuring enzyme production with GFP fusion tags. Different incubation temperatures (37ºC, 30ºC, 25ºC) and incubation times (overnight, 24 hours, 36 hours) are tested. High-expressing enzyme-tag combinations (at least one for the different low-cost chromatography media) are selected for larger-scale (200 mL) expression and purification. Team epiLAB starts implementing benchmarking systems for the optimized enzymes against commercial alternatives on synthetic samples.
● Week 5 - Consortium members and collaborators continue screening experimental low-cost expression plasmids and auto-induction expression conditions, replicating and validating any conditions that yield particularly high GFP tag fluorescence for each enzyme. Lysis with B-PER solution is tested, and enzyme yield in the soluble fraction is measured by GFP fluorescence.
● Week 6 - Consortium members and collaborators select best performing plasmid/auto-induction protocol pairs for larger-scale (200 mL) culturing. Lysates are run through different chromatography media, and enzyme retention on the chromatography columns are measured by fluorescence.
● Week 7 - Consortium members and collaborators continue testing low-cost culturing and purification protocols. Protocols to elute enzymes from the different low-cost chromatography columns using glutathione, high salt concentrations, and epilamhomebrew SUMO protease are tested. Eluted enzyme yields are quantified with fluorescence.
● Week 8 - Consortium members and collaborators will finalize the first set of low-cost expression constructs and protocols. Non-GFP-tagged enzyme variants are expressed and purified using the optimized protocols developed in weeks 4-7, with yield and purity quantified by PAGE. Low-cost purified enzymes are shipped to Team epiLAB and Team OSN, to begin testing activity in different diagnostic assays. Validated plasmids, strains, expression protocols and lists of reagents are shared with anyone who wants to use and improve them.
2.0 Project Implementation
Our collaborative consortium envisions a collection of functional and optimized open enzyme expression vectors and protocols to permit anyone to produce useful diagnostic enzymes (and hopefully, almost any enzyme). Potential beneficiaries include any researchers, medical institutions, and communities facing shortages of critical diagnostic enzymes during the COVID-19 pandemic. This consortium consists of the following team leads. Collaborators are most welcome to join this initiative.
- Team OSN (BC, Canada): Scott Pownall (OSN) with support of Nico Crudele
- Purifying diagnostic enzymes traditionally and as cellular reagents
- Team BioBlaze (Chicago, USA): Isaac Larkin with support of Sarah Ware
- Exploring protein tags and inducible promoters for low cost purification
- Growing collaborative network of biolabs to test and replicate low-cost manufacturing of enzymes
- Team epiLAB (Germany): Kathrin Hadasch (Technical university Darmstadt)
- Exploring enzyme variants in silico & in vitro for optimizing activities
- Team Bionet (Stanford, USA): Keoni Gandall (Free Genes, BioBrick Foundation)
- Building expression plasmids for low-cost protein purification on low-cost automated liquid handling and DNA sequencing hardware.
We hypothesize that low cost and open solutions to enzyme production and distribution can mitigate the impact of SARS-CoV2 in low resource settings around the world. Two primary barriers to the low-cost production of enzymes are the protein purification protocols and the enzyme optimization steps.
We hypothesize that the cost of enzyme production and purification could be dramatically reduced by combining (1) automatic induction of recombinant protein expression using cheap lactose media, (2) purification domains that bind to cheap and widely available materials such as starch, cellulose, chitin or silica, and (3) highly specific recovery of tag-free purified enzyme using either self-cleaving intein domains or SUMO protease cleavage domains. All of these components are now off-patent.
If we were to test only a small number of enzyme/purification tag/cleavage domain combinations, we suspect we would be unlikely to generate working protocols for high-yield protein expression and purification. This is because newly designed genetic constructs often don’t work as intended, and different enzymes can require expression at different temperatures and for different lengths of time in order to generate high yields of soluble and functional protein. Happily, members of our teams and collaborators at the FreeGenes project and Open Bioeconomy Lab have already designed a MoClo-compatible GoldenGate assembly standard (called FG ProClo) for appending inducible promoters, terminators, and composite purification/cleavage/reporter tags to protein-coding gene sequences in customized expression vectors; and are in the process of ordering from Twist a collection of free-to-distribute inducible promoters, tags and diagnostic enzyme sequences compatible with this assembly standard--the Research in Diagnostics Collection (assembly standard outlined in the 3rd tab of that Google Sheet). We hypothesize that the FG ProClo assembly standard will enable us to use Team Bionet’s home biofoundry to quickly and easily assemble hundreds of custom vectors expressing diagnostic enzymes with different tag combinations. We further hypothesize that the ability to append fuGFP reporter tags to either end of any enzyme gene in these assemblies will make screening for the best-expressing vector/enzyme/tag combinations, the best induction and incubation protocols, the most effective and high-retention low-cost purification chromatography media, and the best tag-cleavage and enzyme elution methods, a simple matter of measuring the fluorescence (or just eyeballing the yellow-green color) of the solutions, pellets, supernatants, columns, and filtrates at each step.
- Reproduction and testing of off-patent mutations already published by various groups around the globe
- Further screening for mutational sites by directed evolution strategies
- Optimizing reaction buffer configuration for SARS-CoV-2-detection performance
The short term goal is to establish an open source and cost-efficient production pipeline of fully functional enzymes for use in diagnostic assays, like RT-qPCR and RT-LAMP, which perform similar to the commercial alternatives.
In the middle term, distribution of the optimized protocols and physical plasmids coding for the enzymes will be organized. Since RT-LAMP is a lower cost method suitable for detecting all RNA- or DNA-based pathogens, the global-scale implementation of SARS-CoV-2 test stations based on RT-LAMP is a tremendous gain for global-health monitoring in the long term.
In addition, having these resources available in the future will increase the speed of response if, and when, a new epidemic or pandemic were to arise. In this case only a few adjustments would be needed once the pathogen’s genome sequence became available (e.g. testing and optimizing new primer-sets). In addition, other contamination-monitoring problems can be addressed by this method. Water and food safety, vermin investigation in agriculture and waste water testing are prominent examples.
Concluding, this project offers a strong and permanent foundation for many health monitoring challenges. With a methodology that generalizes to a lot of applications beyond the scope of COVID-19 and enables all covered nations, especially in middle to low-resource contexts, to conduct efficient and exhaustive investigation in a huge variety of unsolved problems.
We collectively have basic open source expression plasmids, many from the Open Bioeconomy Lab’s Open Enzyme Collection but not exclusively. Most of the genes code for the wild-type enzymes or basic truncations used in the various diagnostic assays including RT-qPCR and the LAMP assay. These enzymes will be expressed in the BL21(DE3) E. coli strain or variants. We have expression plasmids that contain IP-free genes for various reverse transcriptases used in diagnostic assays to convert RNA such as the SARS-CoV2 RNA genome to DNA as well as various DNA-dependent DNA polymerases for quantitative PCR or isothermal LAMP amplification of the viral DNA to detectable levels. The RNAse Inhibitor enzyme will also be purified.
Team OSN will express the basic poly-histidine-tagged enzymes and use, as a benchmark, nickel-resin-based chromatography protocols optimized for each enzyme for small scale purification. This involves culturing the DNA containing bacteria and inducing gene expression using either IPTG or the less expensive autoinduction media. Cells overexpressing the enzymes will be collected and sonicated to break them open. The enzymes will be purified from lysates using nickel-immobilized metal affinity chromatography (IMAC). Samples collected from various stages of purification (lysate, pellet, flow through, washes, eluted protein, concentrated protein) will be analyzed using SDS-PAGE gel electrophoresis to check protein size and purity. Protein will be quantitated using the Lowry Protein Assay. The different enzymes produced will be functionally tested using either PCR and RT-qPCR. The same enzymes will also be produced using the Cellular Reagent approach which involves lyophilizing or desiccating whole E. coli cells. This approach aims to reduce the cost of enzyme production, storage and distribution.
Off-patent mutations will be inserted consecutively by Team epiLAB using site-directed mutagenesis to optimize enzyme function. For the basic expression plasmid design see the benchling links in the documents section. These mutants will be Histidine-tagged so they can be purified using Nickel-based & vivaspin concentration for testing. The benchmarking of these mutants in comparison to the wildtype enzyme will be done on non-infectious synthetic SARS-CoV-2 test models (e.g. transfected HEK-cells, pure RNA-DNA samples or SARS-CoV-2 Virus like particles). The optimized kit will be tested on patient samples at last, in collaborating certified testing laboratories. Ethical clearance and other permissions would be dealt with by the collaborating lab and is effectively an independent project using the materials produced during this one (if successful).
Team BioBlaze, in collaboration with Keoni Gandall of Team Bionet, will design new expression vectors for the diagnostic enzymes that employ a strong lactose-inducible promoter, low-cost purification tags (R5 silica binding domain, MBP amylose/starch binding domain, chitin binding domain, or CipA cellulose binding domain), cleavage domains (SUMO domain, thiol-cleavable intein domain, or salt-cleavable intein domain), and reporter domains (fuGFP fused to the N-terminus, C-terminus, or absent). Team Bionet’s OpenTrons foundry will be used to construct an expression vector library containing every combination of purification/cleavage/reporter tags assembled and appended to the genes for each of the following proteins: MMLV-RT and HIV-RT (used in RT-LAMP and RT-qPCR), Bst-LF and OpenVent DNA polymerases (used in RT-LAMP and RT-qPCR respectively), T7 RNA polymerase (used to make RNA control sequences from DNA templates), fuGFP and mRFP (reporter positive controls for production and purification protocols), and SUMO protease (required to recover SUMO domain-tagged enzymes during purification chromatography). For each of the ~288 fuGFP-tagged constructs in this library, enzyme expression after autoinduction under different incubation temperatures and durations in 2 mL cultures in deep 96-well plates will be rapidly measured quantitatively and qualitatively (by plate reader and UV lamp) using the fluorescence of the fuGFP tags. To test recovery of soluble enzyme in crude extract in high throughput, induced cultures will be lysed by mixing with B-PER solution in the deep 96-well plates, spun down in a plate centrifuge, and supernatant fluorescence will be measured with a plate reader and imaged with a UV lamp. Purification chromatography columns containing different tag-binding substrates (silica, chitin, starch/amylose, and cellulose) will be prepared with both research-grade reagents and cheap, food-grade or commodity reagents--for instance, cellulose chromatography columns will be built with pure microcrystalline cellulose, cheap methyl cellulose powder, and ultra-cheap cellulose media homemade by finely shredding toilet paper in water in a miniature food blender. Enzymes immobilized on the columns will be recovered either by incubation with glutathione (for the thiol-cleavable intein domain), 1.5-2 M sodium chloride solution (for the salt-cleavable intein domain), or SUMO protease (for the SUMO domain).
2.4 Results/Expected results
We expect to be able to produce highly functional and stable open source enzymes, and protocols to produce them, for use in open source rapid diagnostic tests to detect COVID-19 viral genomes in clinical samples with the same sensitivity as the commercial solution. The protocols and optimized expression plasmids in appropriate bacteria will be made freely available for anyone needing them. Distribution of these resources is beyond the scope of this project as global shipping of functional diagnostic kits would require additional support but we will work closely with the JOGL, FreeGenes and Open Bioeconomy teams to make that happen.
3.0 Safety, quality assurance and regulation
3.1 What steps have you taken to ensure your solution’s safety? How advanced are you in this process?
This project is in full compliance with the OpenCovid-19 Initiative's Biosafety and Biosecurity Guidelines. Human or clinical samples should only be run in settings with access to appropriate biosafety facilities, of course. Testing of enzyme function does not require clinical samples.
3.2 Have you planned the conduct of your manufacturing process that ensures quality, what are the steps you have taken? How advanced are you in this?
These protocols will provide research grade enzymes suitable for testing in the various diagnostic assays. The designs for the his-tagged, lac-inducible enzyme expression vectors were based heavily on previously validated expression vectors for these enzymes, and will be used to benchmark the performance of the alternative, lower-cost tags and inducible/auto-inducible promoters. We have budgeted for both research-grade and food/commodity-grade enzyme induction and purification reagents, and will test the yield and purity of the low-cost expression and purification systems using both.
3.3 Will you need assistance with the regulation system?
3.4 Have you talked to medical staff about the feasibility of your project? What did they say?
Kathrin is in close contact with Doctors Without Borders Germany. They are excited that a project with our scope exists and confirmed the applicability of our objectives in most resource lacking regions of the world.
Florian Schulte, a MD from Germany who is participating in the JOGL community, was very open to the idea of our open source approach to purification of our non-commercial enzymes. He has suggested that this research could prove worthwhile towards exhaustive testing capabilities for frontline healthcare workers.
3.5 Have you planned the testing, verification and validation of your solution? How advanced are you?
The enzymes produced will be tested directly in JOGL sister projects that are working with LAMP assays using commercial enzymes. There is also the possibility of testing on patient samples in collaborating certified BSL2 laboratories in Erlangen (Germany). Moreover, Isaac Larkin, Keoni Gandall and Scott Pownall’s connections to the Open Bioeconomy Lab, BioBricks Foundation and Covid Testing Scaleup Slack open the possibility that some of the expression vectors and protocols developed in this project could be tested, replicated, validated and improved by numerous other community and academic labs.
4.0 Impact, issues and risks
4.1 What impact do you feel your project could have?
Exhaustive covid-19 testing in industrialized countries is a huge challenge. However, the Global South is struggling even more with the lack of material, knowledge and infrastructure. Recent reports have indicated that Chile, for example, has all but exhausted their reagent supply for diagnostic testing of SARS-CoV2. This is occurring throughout the world in countries that don’t have the resources to compete against the wealthier nations. With open source enzymes, protocols and test-kits we can enable doctors and biologists globally to cope with this pressing problem.
4.2 What do you think would make your project a success?
We are already in a good place with all the collaborations between each other, the other JOGL-teams and their local networks. What is needed right now is funding to purchase the reagents and labware that will enable us and other labs to actually test protein expression and purification from these open-source vectors and genetic parts.
4.3 Please list the known issues, potential risks, grey-areas, etc in your project
There needs to be a lot of systematic testing of enzyme activity and function as well as ensuring sensitivity of these enzymes in COVID-19 detection assays before we launch the test. Some enzyme-fusion proteins may not function as expected. We are considering solutions and teaching materials to assist users in producing enzymes locally with a mind to contamination containment of the test-tubes and the environment as well.
5.1 What other projects on JOGL are like yours? Search for them and Link them!
We are already in close collaboration with following JOGL projects which use commercial enzymes for their assays. Our work complements theirs and our enzymes will be tested with them.
Optimizing the NEB LAMP test (Created March 26)
https://app.jogl.io/project/181 Do-It-Together SARS CoV-2 Detective (April 03)
This project focuses on developing cellular reagent enzymes specifically for the Mammoth Biosciences’s CRISPR-based DETECTR protocol. We are happy to provide them with our already existing enzymes for testing in their system.
Encapsulating the Mammoth Biosciences Test in E. coli (April 18)
This project focuses on creating toehold and ribozyme sensors for detecting SARS-CoV2.
Cell-Free systems for seq-specific sensing of SARS-CoV-2 (April 05)
This project focuses on creating DIYbio hardware that uses commercial enzymes. Our enzymes would complement their device.
Using DIYBio tools to improve RT-LAMP Protocols (April 15)
This project focuses on creating a wave bioreactor and using light activated expression of genes. Our enzyme could be used in their system.
prgm-bioreactor (March 22)
These two new projects are by the funded bioreactor group (Project 156).
Homebrew your Test Kit reagents (May 11)
Affordable protein biosynthesis in 20L reactions (May 11)
5.2 Is this an innovative project? What makes this project different if it’s unique on JOGL?
If all the elements of this project are successful, the outcome will be not just the sharing of plasmids and protocols for manufacturing diagnostic enzymes across the entire global DIYbio community; but also the sharing of vectors and DNA parts that make the inexpensive production and purification of almost any enzyme available to anyone around the world. Because JOGL is a platform connecting the global democratized biotech community, obtaining JOGL’s support and running it on JOGL will greatly enhance the diffusion of these plasmids and protocols throughout the world. Having experienced the global transformations in innovation which resulted from the creation and development of the open source software we feel these early days of open source biotechnology will be innovative and impactful to many aspects of our collective human endeavour and our environmental stewardship.
5.3 Is there already an open source version of this project?
This is the open source version of the project! Almost all DNA expression plasmids have been released under the OpenMTA and are off-patent or IP-free. The Open Enzyme Collection is expanding with more open source genes, curated by the team and collaborators of Open Bioeconomy Lab, having been sent by FreeGenes to be synthesized. In addition, the Open Bioeconomy Team, its collaborators and FreeGenes are currently developing an open assembly standard for creating backbone plasmids suitable for use in a wide variety of biological systems.
6.0 Team experience
6.1 Please cite your team members and their roles in the project. (if applicable) If the project involves several locations or labs, list them too.
Team OSN (Vancouver, Canada):
Scott Pownall, PhD (Genetics, UBC) (JOGL user) - Co-founder & President Open Science Network Society. Scott has approximately 30 years of experience working with DNA-based technology in academia, Industry and community biology. He established Canada’s premier community biolab in Vancouver and is an active leader in the local and global community biology movement. He is an active Open Bioeconomy Lab collaborator and has worked closely with Free Genes Project since 2017 to bring open source concepts to DNA-based technologies. His Open Yeast Collection for building metabolic pathways is nearing synthesis by Free Genes. He is the project lead on the Team OSN components of this consortium.
Team Bioblaze (West Chicago, USA):
Isaac Larkin, PhD candidate, Director at BioBlaze Community Bio Lab, Developer on the Free Genes Project
Isaac is devoted to making it as cheap and easy as possible for everyone to build beautiful and useful things with biology. In his 6 years of graduate research at Northwestern University, Isaac has developed nanoparticle-based detection assays for human disease biomarkers and recombinantly expressed Cas9 for packaging into DNA-functionalized liposomes and delivery into cells. Outside of his thesis work, he has led and contributed to initiatives aimed at democratizing biotechnology, including co-founding and helping run the GeneMods student synthetic biology community, blog and podcast; co-founding ChiTownBio, the leading DIYbio community in the city of Chicago; becoming co-director of BioBlaze, the first community biotechnology lab in the Chicagoland area; and contributing wetware and recruiting volunteers as one of the core developers on the BioBricks Foundation’s Free Genes Project.
Sarah Ware, PhD, Founder & Director of BioBlaze Community Bio Lab, Co-Founder of Lizzy Blossom Ag Services
Sarah Ware has a doctorate degree from Wageningen University in The Netherlands. She has 20 years experience as a geneticist/molecular biologist in both academia and the private sector. Sarah is the founder of two independent labs: BioBlaze Community Bio Lab (501(c)3)) and Lizzy Blossom Ag Services. Within BioBlaze, Sarah leads hands-on workshops for the general public that range from a basic How to Design a Scientific Experiment to CRISPR Gene Editing. Within Lizzy Blossom, Sarah does RNA extraction and reverse-transcriptase PCR to detect RNA viruses in plants. Sarah is already an active collaborator in JOGL’s OpenCovid19 Initiative: https://app.jogl.io/project/163.
Team epiLAB (Germany):
Kathrin Hadasch, B.Sc. (TU Darmstadt) Kathrin has approximately 6 years of experience in the wet lab domain, including heavy ion microbeam experiments for chromatin remodeling at Helmholtz centre for heavy ion research in Darmstadt and patch-clamp/bilayer experiments at TU Darmstadt. She is an active member of the informatics, electrical engineering and biotech-makerspace Lab³ and founding member of IANUS Peacelab, an initiative for critical technology assessment and peace studies in citizen science. She has the full support and cooperation of the presidium of both universities in Darmstadt (TU Darmstadt & h_da), helping her to use the resources of her location to the fullest.
Team Bionet (Stanford, USA)
Keoni Gandall, Technical Lead on the Free Genes Project, BioBricks Foundation.
Keoni is a biohacking prodigy (note: he didn’t write this bio) who has been designing and engineering biology since he was in middle school. In 2013 at the age of 14 he got to meet President Obama after winning the national Broadcom Masters Science Competition by developing a method to genetically engineer a species of pink, halophilic archaea. At 17 he joined the BioBricks Foundation as the technical lead for their new 10,000 Genes (now Free Genes) project, to make a massive library of useful synthetic genetic parts freely available to the world. In his spare time, he has built a biofoundry in his home, including two OpenTrons OT2 robots and a MinION nanopore DNA sequencer, and has written software to enable automated, high-throughput setup and running of GoldenGate plasmid assembly reactions on this foundry. He is currently working on a new FreeGenes vector collection and assembly standard that will enable the genetic engineering of almost any organism.
7.0 Funding and Costs
7.1 Please provide a costing of your project be as detailed as you can.
Open Enzyme Production and Purification is a collaborative consortium with each lab focusing on different aspects of the challenge. Each group is submitting their own budgets.
Since epiLAB is the only project, that is applying for the 4th Microgrant you can only find one budget in this section.
For the budgets from the 3rd Microgrant please go to the Documents section.
Team epiLAB's budget for Microgrant Round 4 (Google Sheet)
7.2 How is your project being funded so far?
Please scroll below to find the detailed line item budgets that are being requested by the three members of the collaborative consortium: Team OSN, Team epiLAB and Team BioBlaze. Team Bionet’s work is covered through independent funds with no contribution from JOGL.
As a collaborative consortium we already succesfully applied for 6000€ in total (2000€ each project) in the 3rd Microgrant round.
Team OSN and Team BioBlaze will be conducting research in the non-profit community laboratories of The Open Science Network and BioBlaze Community Bio Lab, respectively. Both of these labs are funded in general through memberships. The laboratory equipment, reagents, chemicals and supplies were largely acquired by the founders or through donations. Both the OSN and BioBlaze are fully-functional molecular biology labs that have an extensive collections of reagents and consumables that will support this project and have the capacity to fulfill the project requirements other than what is requested in the budget.
Team epiLAB has the full support of both universities in their sphere of action: Technical University Darmstadt and University of applied sciences Darmstadt. The laboratory equipment, reagents, chemicals and supplies used so far are all covered by various well-established academical working groups. Since some key reagents, the purification columns, are provided by the universities as items on loan, they need to be replaced somewhen soon to uphold the willingness to cooperate. All other expenses beyond that will still be covered by the cooperating working groups.
8.0 Achievement and Benefits of funding
Our progess reports can be found on the Updates&News wall of the project aswell.
Progess report on epiLAB - in silico domain: Extensive research on DNA polymerases literature was done prior to setting up the infrastructure. We managed to get a grand for the high performance computing cluster in Darmstadt, Germany and some sponsoring from AWS. On the HPC-cluster we will screen for possible mutational sides with the Rosetta Commons framework. We set up the infrastructure already, and relaxed two suiting structures of Bst-LF. Interestingly, they showed a higher stability without complexed ions, which contradicts experimental data. This finding needs further assessment, before we use this structures for mutational sampling. In parallel we set up a sequence database for mutational sampling & the AWS infrastructure for Gromacs stability assessment MD simulation runs.
Progress Report on epiLAB - in vitro domain: We ordered the e.coli codon optimized Bst-LF fragement and cloned it into a pET14b vector. Transformed in DH5alpha, 3 colonies showed no mutation on CDS and expression cassette (as far as the cassette was sequenced). Expression in BL21(DE3)pLys yielded 2.1mg/100ml of culture, with a purity of 85% (estimated by SDS gel). For Purification Alex' Brown's protocol was followed and a Ni-NTA of 1.5 ml was used. Validation of the DNA LAMP test model (SARS-CoV-2 N-protein targeting primers taken from Zhang et al 2020 with N-protein plasmid provided by a befriended laboratory) showed positive endpoint detection, but not a good result with gel electrophoresis. Awaiting the test model from Rachel Aronoff of CoronaDetective to try it again, before starting realtime evalutation and specific activity measurements. Expression with Ni-IDA columns (less expensive than Ni-NTA) showed high impurities, implicating these columns are not to be used without polishing with this protocol. However, there was a update on the purification protocol, with highly optimized buffers. Will test Ni-IDA columns again with this one and reevaluate.
For the 4th Microgrant only Team epiLAB is asking for additional 500€. We have recieved in the 3rd Microgrant Round 2000€ of the 3000€ we asked for.
For process surveillance in a realtime PCR machine of the emzyme performance an intercalating dye is neccessairy, because endpoint detection only is not suitable for assessing the enzyme performance in a whole. SYTO9 is the suitable dye, since its less inhibitory and Tm-shifting than e.g. SYBR-Green.
Additionally we would purchase 5 ml of Ni-NTA resin, for purification of the mutants.
- Short Name: #OpenEnzymeProduction
- Created on: April 2, 2020
- Last update: July 12, 2021
- Grant information: Received €6,581.00€ from the OpenCOVID19 Grant Round 3/4 on 10/18/2020