Our Contribution to Future iGEM teams
Rhodococcus Fact Sheet
The 2022 East Coast BioCrew was inspired to create a Rhodococcus fact sheet to explain the required conditions for Rhodococcus to be used by other iGEM teams. Rhodococcus is not well characterized, so we also wanted to assist future teams who select it.
The Rhodococcus Fact Sheet includes information on:
- Sourcing
- Culture conditions
- Safety precautions
- How Rhodococcus can be transformed
- Where Rhodococcus is derived from
- BioBricks compatible with Rhodococcus
We expect this fact sheet to be useful, as at least ten iGEM teams have used Rhodococcus in the past. Rhodococcus has significant potential as a chassis organism due to the wide variety of environments it lives in, including soil and water. This fact sheet contains all the information we have learned from working with Rhodococcus. With this information, future iGEM teams can take full advantage of its capabilities.
Rhodococcus Fact Sheet
Overview
We were inspired to create a Rhodococcus fact sheet to further explain the required conditions for Rhodococcus to grow. Rhodococcus is not well-characterized or commonly used as a chassis by iGEM teams, so we also wanted to assist future teams who select it.
Where is Rhodococcus Naturally Found?
Rhodococcus, a soil-borne organism, is often found in natural environments and can thrive in both soil and bodies of water. Rhodococcus is found in soil, water, and eukaryotic organisms. Rhodococcus equi was found in the soil of horse farms and horse feces.
Sourcing
- Several species of Rhodococcus are available from ATCC (https://www.atcc.org/), including Rhodococcus erythropolis (ATCC #4277 and #15903) and Rhodococcus sp. (ATCC #55899) which are BSL-1 and Rhodococcus fascians (ATCC #21057)which is BSL-2.
- We chose to use Rhodococcus jostii RHA1, which is a soil bacterium that can naturally degrade PCBs. We couldn't find this from a commerical supplier, so, like us, you could find published papers that have used Rhodococcus and request samples from the investigators
Safety Precautions
Biosafety level 1 safety procedures are recommended. Some strains are BSL-2, so check the appropriate BSL level before starting work.
Culture Conditions of Rhodococcus jostii
- Grows well on LB (Luria-Bertrani) media and many other media both in liquid and on a plate
- Usually takes 2 days from when the liquid or plate cultures are incubated at 30 degrees Celsius, which is the optimal temperature
- Growth of Rhodococcus jostii RHA1 takes 5-7 days or more on minimal media
- Resistant to Nalidixic acid (which can be used to prevent contamination)
- No special precautions are needed when freezing, and you can use glycerol or DMSO
Growth of Cells
We determined the growth of Rhodococcus at the optimal growth temp of 30C compared to E. coliby growing cells in LB media and minimal M9 media and measuring the optical denisty at 600nm every 30 min.
Using the Doubling Time Calculator [1], we calculated the doubling time of Rhodococcus jostii to be 189.8 min in minimal media at 30C and 190.6 min in LB media at 30C.
Transforming Rhodococcus
It is possible for Rhodococcus to be transformed by electroporation [2].
Rhodococcus Plasmids
A “copy-controlled integrative plasmid”, pRIME, is available. See Round et al. [3]. This paper also describes a series of promoters that works in Rhodococcus, hybrid promoters, tetracycline-inducible promoters, and integrating vectors.Shuttle plasmids that work in E. coli and Rhodococcus are available including Biobricks [4]. You can get the plasmids from Addgene [5]. Cecropin A, an insect antimicrobial peptide, works as a selectable marker.
BioBrick Parts Designed For or That Can Be Expressed In Rhodococcus
Several teams have used Biobricks in Rhodococcus previously.| Team | BioBrick | Description |
|---|---|---|
| iGEM09_Washington | BBa_K215091 | Generates OpdA enzyme; degrades nerve agents |
| iGEM16_Edinburgh_OG | BBa_K1968023 | Green fluorescent protein for expression in multiple hosts (incl. Rhodococcus) |
Other Rhodococcus Information
- Gram positive bacteria
- Some strains naturally degrade polychlorinated biphenyls [6].
- A complete genome sequence is available [7].
- Some Rhodococcus strains can perform phytoremediation [8]
Why We Selected it As Our Chassis
We are trying to degrade and detect PCBs in lakes, rivers, and estuaries. We chose to use a chassis that:
- Is aerobic because we want to work with it in our lab
- Is biosafety level 1
- Lives in shallow water or soil, where we are targeting degradation
- Has many genetic tools are available to introduce new genes
- Can thrive in environments with high levels of PCBs
For these reasons, we have chosen to assemble the genes and pathways for PCB degradation in Rhodococcus.
References
[1] Roth V. 2006 Doubling Time Computing, Available from: http://www.doubling-time.com/compute.php[2] Round, J. W., Robeck, L. D., & Eltis, L. D. (2021). An integrative toolbox for synthetic biology in Rhodococcus. ACS Synthetic Biology, 10(9), 2383–2395. https://doi.org/10.1021/acssynbio.1c00292
[3] Shao, Z., Dick, W. A., & Behki, R. M. (1995). An improved eschehchia coli-rhodococcus shuttle vector and plasmid transformation in Rhodococcus spp. using electroporation. Letters in Applied Microbiology, 21(4), 261–266. https://doi.org/10.1111/j.1472-765x.1995.tb01056.x
[4] Ellinger, J., & Schmidt-Dannert, C. (2017). Construction of a BioBrick™ compatible vector system for Rhodococcus. Plasmid, 90, 1–4. https://doi.org/10.1016/j.plasmid.2017.01.004
[5] BioBrick™ compatible plasmids for Rhodococcus. Addgene. (n.d.). Retrieved October 13, 2022, from https://www.addgene.org/browse/article/8897
[6] Seto, M., Kimbara, K., Shimura, M., Hatta, T., Fukuda, M., & Yano, K. (1995). A novel transformation of polychlorinated biphenyls by Rhodococcus sp.. strain RHA1. Applied and Environmental Microbiology, 61(9), 3353–3358. https://doi.org/10.1128/aem.61.9.3353-3358.1995
[7] McLeod, M. P., Warren, R. L., Hsiao, W. W., Araki, N., Myhre, M., Fernandes, C., Miyazawa, D., Wong, W., Lillquist, A. L., Wang, D., Dosanjh, M., Hara, H., Petrescu, A., Morin, R. D., Yang, G., Stott, J. M., Schein, J. E., Shin, H., Smailus, D., … Eltis, L. D. (2006). The complete genome of Rhodococcus SP.. RHA1 provides insights into a catabolic powerhouse. Proceedings of the National Academy of Sciences, 103(42), 15582–15587. https://doi.org/10.1073/pnas.0607048103
[8] Vergani, L., Mapelli, F., Suman, J., Cajthaml, T., Uhlik, O., & Borin, S. (2019). Novel PCB-degrading Rhodococcus strains able to promote plant growth for assisted rhizoremediation of historically polluted soils. PLOS ONE, 14(8). https://doi.org/10.1371/journal.pone.0221253