- Slack channel in JOGL OpenCovid19: #proj-mammoth-multiplier
- Main Google Drive Doc: https://drive.google.com/drive/u/0/folders/1LlqxWKWCEr9vzwA95jdYGtbv6TOrzJ9l
Problem and Background
The COVID-19 pandemic continues to be a global threat, with the majority of countries worldwide reporting cases and a high case-fatality ratio of 3%. Critical to mitigating the spread of the virus is widespread and effective detection, which allows for effective quarantine and the development of herd immunity. However, the detection of the virus through traditional methods is challenging for many countries that lack the expensive laboratory equipment necessary for the recommended RT-PCR method. This renders the testing kits currently made available by the CDC potentially ineffective for many critical regions. Low cost and timely testing protocols based on isothermal amplification methods and lateral flow assays emerge as a potential solution to this issue. However, the reagents necessary for the tests themselves are time and temperature sensitive and reliant on cold chain delivery networks. Consequently, we seek to utilize synthetic biology methods in combination with Mammoth Biosciences’s CRISPR-based DETECTR protocol to devise a truly accessible and open source diagnostic test.
Solution summary in simple terms
Bacterial plasmids allow for the rapid and cost-effective production of proteins that would otherwise be inaccessible to many. Mammoth Bioscience’s testing strategy has great potential for widespread dissemination to increase the availability of testing, an essential part to controlling the spread of the pandemic. However, the proteins necessary to run the test can be difficult to acquire for those who need it. So, our group hopes (a) to validate the Mammoth Biosciences test and (b) to utilize engineered E. coli to easily produce and transport these proteins so that the Mammoth Bioscience’s test can be made available to a greater audience and play a role in widespread detection.
Solution summary in technical terms
We will clone an array of oligonucleotides into a high-yield/low copy number pET-29 expression vector. These will include reverse transcriptase, LbCas12a, a large-fragment Bst polymerase, and RNA sequences from a synthetic SARS-CoV-II genome.
Transformants with these vectors will be produced in the form of multiple biological replicates of E. coli. They will then be verified by kanamycin/ampicillin resistance (included in our vector) and PCR. Cellular reagents isolated from these transformants can then be employed without a time-consuming purification step.
We will finally use them to validate the Mammoth protocol, which consists of an RT-LAMP isothermal amplification reaction and uses a free reverse transcriptase vector known as MASHUP-RT. LbCas12a RNPs will be used to compensate for RT-LAMP’s lack of specificity at low reactant concentrations. Finally, a lateral flow strip assay for the presence of viral envelope and nucleoprotein genes will be performed.
State of advancement of the project
We are in the process of completing a comprehensive and reproducible protocol for our experiment. Currently, there are a few design points that require clarification, but these are relatively minor in nature. We have also completed an initial design of all experimental components. Our next step is to order the primers and inserts necessary to begin our experiments.
- 05/01 - Begin preparing cells and plasmids, pending material availability.
- 05/08 - Preparatory work complete. Begin cloning experiments.
- 05/15 - Results analysis, begin protocol testing.
- 05/22 - Results analysis. If the experimental protocol was successful, we will start additional preparation for a second round and/or implement any changes necessary. IWe will also be contacting a BSL-2 lab at this phase to conduct further testing.
- 05/29 - Results analysis from above. Kit design (what all is included and how/how can we ship it) Obtain already made test kits, ideally ship one to BSL-2 lab.
- 06/05 - We anticipate a week for them to give us feedback.
- 06/12 - Write-up (if good feedback).
- 06/20 - Publish protocol.
Research: Describe hypothesis and research objectives (1000 words max)
We will be testing two hypotheses in this research effort. First, we hypothesize that the Mammoth DETECTR test can accurately and specifically detect SARS-CoV-2. Second, we hypothesize that we can produce working solutions of Bst polymerase, Cas12a + crRNA, and MASHUP reverse transcriptase (critical reagents for the Mammoth DETECTR test) at a low enough cost to be useful.
Objective 1: Develop a protocol for low-cost reagent production.
- We will produce Cas12a complexes, Bst polymerase, and MASHUP RT in E.coli and develop a reliable method to prepare these proteins for use in the Mammoth DETECTR protocol.
Objective 2: Validate test on artificial controls.
- We will confirm the Mammoth DETECTR protocol, modified as needed to accept our reagents, works on artificial DNA and RNA samples with partial SARS-CoV-2 sequences.
Objective 3: Cooperate with other labs to reproduce and refine our protocol.
- We will reach out to labs with the capacity to carry out our protocol and work with them to ensure its reproducibility. Furthermore, as we only have access to a BSL-1 lab at present, this will be necessary to validate the test on patient samples.
Methodology (500 words max)
- Running the original Mammoth protocol. We will first validate the Mammoth protocol with commercially available reagents and SARS-Cov-2 RNA controls. The Mammoth protocol, to our knowledge, remains unsubstantiated independently. Additionally, while there is precedent for RT-LAMP and Cas12a in concert, it remains somewhat untested with regard to SARS-CoV-2.
- Develop cellular reagents for LbCas12a and SARS-Cov-2 specific gRNA, Bst polymerase, and MashUp reverse transcriptase. We plan to express the proteins essential for the RT-LAMP and CRISPR reactions of the Mammoth protocol in E. coli. These proteins include the CRISPR ribonucleoprotein, composed of LbCas12a and guide RNAs specific to the virus, which will be co-expressed and assembled in the cells following the strategy of Qiao et al. We will develop reagents from these engineered cells by lyophilizing the cells in a manner similar to Bhadra et al.
- Running the original Mammoth protocol with our cellular reagents. We will attempt to reproduce the test with our reagents produced in step 2. We will compare the precision and accuracy of our reagents to commercially available reagents in these reactions.
- Determining conditions that affect test reliability. Primer concentration and design, reagent concentration, and sample buffer will be assessed. Concentration, in particular, as cellular reagent production consistency has yet to be firmly established and represents a potential variable in testing efficacy.
- Patient sample verification. Our test, initially performed with synthetic RNA, will be tested with patient samples collected according to CDC guidelines. This will be done in laboratories equipped to handle such samples, according to their respective protocol.
- Replicated the Mammoth Biosciences test and verified its functionality on synthetic DNA & RNA controls
- Produced functioning Bst polymerase, Cas12a-crRNA complexes, and reverse transcriptase in E.coli and verified their functionality in Mammoth DETECTR test
- Distributed final protocol and E.coli stocks to partner labs for independent verification
Safety, quality assurance and regulation
Experimentation will occur in a BSL-1 lab with synthetic positive controls for comprehensive protection. All participating members involved in laboratory work are trained in responsible research procedures and safe handling of chemical agents. Protocol and reagents have been selected to produce effective quality and minimize safety risks.
We will not be using clinical samples, offering testing to the public, using human or animal test subjects, or environmental samples. In terms of positive controls, we will be using RNA from Twist Bioscience which is listed in the OpenCovid19 Biosafety and Biosecurity guidelines.
Lab work will mostly be taking place at the garage of Zach Mueller, Open COVID-19 co-founder. As specified in the Biosafety and Biosecurity guidelines of the project, the fragments of viral RNA that we are using in the DETECTR lateral flow portion of our protocol are short, constituting less than 66% of the SARS-CoV-2 viral genome. Furthermore, our experimental modifications are only towards the plasmid production and purification of non-viral reagents, not modifications on the detection mechanism.
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 (if applicable)?
Our manufacturing process is capable of being carried out in a BSL-1 lab by independent scientists and requires minimal technical training.
Will you need assistance with the regulation system? if not which Regulatory system do you plan on distributing the product? Please elaborate (please see: Regulatory-Strategies) (if applicable)
We have no plans to distribute any test kits at this time, so this is not relevant.
Have you talked to medical staff about the feasibility of your project? What did they say?
Because the targets of our testing protocol are laboratory staff and the protocol itself uses established micro and molecular biology techniques, we have not yet consulted medical personnel. We will consult these personnel when appropriate.
Have you planned the testing, verification and validation of your solution? How advanced are you? (if applicable)
We intend to test our protocol on synthetic viral RNA. This is substantially safer than samples isolated from patients and will mitigate risk of exposure to anyone not trained in the handling of such specimens. We hope to eventually collaborate with labs that have the ability to validate our solution with inactivated viral samples.
Impact, issues and risks
What impact do you feel your product could have?
Insufficient SARS-CoV-2 testing around the world has contributed to a global health crisis. Cheap, effective and widely available detection methods are essential for the protection of society.
If validated and developed fully, our test could be performed on-site by laboratories with minimal specialized equipment, allowing for greater accessibility and timely diagnosis. The ability to mass test frequently reduces the rate of infection and allows control of affected populations. Our product could expand access to testing at a time when accurate data collection has the potential to save lives.
What do you think would make your project a success?
Our project could be considered a success once we are able to make a protocol that generally allows for a tester to lyse cells, add other readily available reagents, and successfully detect the presence of viral RNA.
Please list the known issues, potential risks, grey-areas, etc in your project.
Before our test can be widely implemented, it would need to be verified with patient samples by a party that has the authority to do so.
Through experimentation we hope to clear up grey areas regarding enzyme purification and overexpression and interference of other cellular components.
Charlie Anderson - Lead
Claire Yang- Fundraising
David Lu - Experiment design
Eric Xia - Experiment design
Frederick Kayes - Contributor
Isaac Larkin - Contributor
JJ Wheeler - Experiment design
Liatris Reevey - Contributor
Michael Hope - Experiment design
Pauline Chane - Experiment design
Sophie Liu - Lead
Zach Mueller - Lead
- SoundBio Lab
- Zach’s Lab