Synthesized by Ginkgo Bioworks, Shared by Addgene: SARS-CoV-2 Plasmids for Many Expression Systems

Posted by Various Addgenies on May 19, 2020 9:15:00 AM


By Will Arnold and Shreya Vedantam

Accelerating research by improving the availability of materials is a core part of our mission. In no situation is this more true than the current global pandemic in which scientists race to find vaccines, therapeutics, and other treatments to stem the spread of COVID-19. That’s why we’re proud to be working with Ginkgo Bioworks to synthesize and share SARS-CoV-2 plasmids to help academic and industrial labs accelerate research and to help find a faster solution to the COVID-19 pandemic. By the time the project is complete, Ginkgo will have synthesized approximately 3,000 constructs to support SARS-CoV-2 research.

Though we’ve confirmed the expected features and sequences in each plasmid, none of these constructs have been functionally tested by Ginkgo Bioworks or Addgene. If you used plasmids from this collection in your experiments and published your data, please fill out this form and we’ll add your citation to the plasmid page on our website. Contributing your results helps other researchers using these plasmids and will give your research more visibility. 

Sars-CoV-2-particle-with-globe
Figure 1: Illustration of SARS-CoV-2 from Philippa Steinberg for the IGI.

What SARS-CoV-2 plasmids from Ginkgo Bioworks are currently available for request?

Currently, plasmids containing all SARS-CoV-2 protein coding genes in E. coli and S. cerevisiae expression vectors are available at Addgene. A set of mammalian expression vectors containing various spike protein variants are also available. Plasmids are continually added as they complete QC and you can find all available plasmids listed on Ginkgo’s collection page

Bacterial expression plasmids

For those constructs to be used in bacterial expression systems, Ginkgo synthesized SARS-CoV-2 expression plasmids in a high copy backbone with a T7 promoter driving multiple combinations of WT, codon optimized, untagged, cleavage sites (thrombin and TEV), and C-terminal tags (StrepII, 3xFLAG, 6xHIS, MBP, Myc). These constructs include proteins like nonstructural proteins 1 to 16, spike proteins, envelope small proteins, and membrane proteins. Although all viral components are available in bacterial expression plasmids, glycoproteins and other proteins that require post-translational modification may not be easily expressed in non-mammalian cells.

Yeast expression plasmids

The yeast expression constructs contain protein coding genes, tags, truncations, and codon optimizations similar to those found in the bacterial expression plasmids. Each combination is present in two sets of backbones which drive expression using either TEF1 or GAL1 promoters. These vectors are intended for the high level expression and purification of SARS-CoV-2 proteins for downstream experiments and offer flexibility in tag, cleavage, codon usage, and expression system.

Mammalian expression plasmids

Finally a set of wildtype and codon optimized, full length and truncated versions of SARS-CoV-2 S (spike protein) cloned into a CMV driven expression vector are also available. These contain no tags and are intended to be used in the production of pseudotyped VSV particles (Fukushi et al., 2008). 

What plasmids are coming soon?

Ginkgo is depositing new constructs weekly as they are synthesized (300-400/week) over the next several weeks. They have provided a well organized, tabular, summary of all the constructs they plan to synthesize including:

  • additional mammalian expression constructs 
  • truncated spike protein constructs for expression and for making VSV pseudovirus SARS-CoV-2 
  • plasmids containing larger segments of viral DNA fragments under T7 promoter
  • additional bacterial and yeast expression vectors containing truncations of SARS-CoV-2 protein coding genes

Browse the Ginkgo Bioworks COVID-19 Plasmid Collection

Quality control at Addgene in the midst of COVID-19

We received Ginkgo’s first deposited set of plasmids containing SARS-CoV-2 protein coding genes in E. coli and S. cerevisiae expression vectors in late March. The first set of 383 constructs went online just three weeks following initial receipt on site. 

To get these plasmids available for scientists speedily meant that we had to revamp some of our quality control processes. For the many other COVID-19 related plasmids in our collection, we’ve relied on Sanger sequencing as a first pass QC. The lab team manually transforms, innoculates, and performs DNA minipreps for small batches of samples. As soon as that process is complete, QC scientists prepare Sanger reactions by mixing this isolated plasmid DNA with appropriate sequencing primers, and hand delivers them to our Sanger provider. These results come back within 8-12 hours and are analyzed first thing the following day. Nothing like waking up to fresh data! These same plasmids are then added into our normal full plasmid NGS queue in order to bring them inline with our slower, more traditional, QC process. While this has worked well for the early sets of SARS-CoV-2 materials, it will not scale well to the several thousand plasmids from Ginkgo.

To balance the need for speed while ensuring our rigorous QC standards are met, we are taking a unique hybrid approach compared to our standard pipeline. For the first ~50 plasmids we perform our standard full plasmid sequencing by NGS. Addgene’s QC scientists immediately screen these plasmids to confirm they contain the expected features and are free of mutation. 

Once the samples arrive, each group of plates will have select samples prepared and screened by NGS to spot check the samples, the rest will be put into a status we call “Archived.” This status allows us to store plasmids and make them available to scientists without performing the full suite of time consuming, costly, and laborious QC for all several thousand plasmids at once. Any samples that are requested by scientists, will immediately be screened by our QC scientists to confirm the plasmids contain all expected features and meet our QC standards. If any concerns are identified, we can quickly put a hold on all orders and alert any scientists who requested the plasmid. By leveraging our core strengths of intensive screening and the use of high throughput platforms, Addgene and Ginkgo are striking a balance by modifying certain processes in both core operations.

As it has been in much of the world, we have found ourselves adapting to a new way of doing things. These new processes have been instrumental in achieving our mission to help science move faster in the middle of a global pandemic. And the reach of these plasmids is indeed global. Helping scientists speed up research is what we’re here for and we’re glad to be able to do so during this critical time.

Visit our COVID-19 Plasmids and Resources Page!

Graphic that says sharing science just got easier... Subscribe to the blog


References

Fukushi S, Watanabe R, Taguchi F (2008) Pseudotyped Vesicular Stomatitis Virus for Analysis of Virus Entry Mediated by SARS Coronavirus Spike Proteins. In: Methods in Molecular Biology. Humana Press, pp 331–338 https://doi.org/10.1007/978-1-59745-181-9_23

Additional resources on the Addgene blog

Resources on Addgene.org

Topics: Plasmids, COVID-19

Click here to subscribe to the Addgene Blog
 
Subscribe

 

All Topics

see all

Recent Posts