Estimating the Carbon Cost of E. coli Transformations
Over the past year, Célia Algros took part in a project to discover microbial enzymes that could metabolize nucleosides in an internship at New England Biolabs. In an offshoot of this project, she also surveyed energy consumption, consumables, and waste generated to estimate the carbon cost of her E. coli transformations. The effort gave her a new appreciation for both the complexity of calculating carbon footprints and the sizable environmental impact of experiments.
Carbon Accounting in a Microbial Genetics Research Lab
The Berkmen Lab at New England Biolabs conducts basic microbial genetics research to identify novel enzymes and solutions to express challenging recombinant proteins. Célia Algros recently completed her master’s degree in biotechnology engineering, with a focus on bioprocessing, at École Superieure de Biotechnologie de Strasbourg (ESBS) in France. Her studies incorporated a six-month internship in the Berkmen Lab. Senior Scientist Mehmet Berkmen, Ph.D., and researcher Emily McNutt, Ph.D., mentored Célia to discover enzymes that could metabolize modified nucleosides using a genetic selection approach.
The Berkmen Lab at New England Biolabs conducts microbial genetics research to identify novel enzymes and solutions to express challenging recombinant proteins. B.Sc. student intern Jessica Khani, scientist Emily McNutt, Ph.D., Célia Algros, and P.I. Mehmet Berkmen, Ph.D. are shown left to right.
The project included screening genomic plasmid DNA libraries from various organisms, including bacteria, archaea, and metagenomic libraries. The libraries were transformed into E. coli purine/pyrimidine auxotroph host strains and then plated on minimal media supplemented with a modified nucleoside. When colonies grew, the plasmid could be isolated and sequenced to identify the gene for the enzyme responsible for recycling the modified nucleoside and allowing E. coli to grow in these conditions. “I tested around thirty modified nucleosides with seven different libraries. I did a lot of transformations, which made me think that the transformation protocol had a high carbon footprint within my project,” explained Célia. She added an objective to calculate the energy cost of a laboratory experiment based on a suggested workshop from her ESBS degree program.
“I tested around thirty modified nucleosides with seven different libraries. I did a lot of transformations, which made me think that the transformation protocol had a high carbon footprint within my project.”
Célia understood upfront that the scope must be limited to make it achievable. As you can imagine, calculating the precise carbon footprint of an entire cloning project, or indeed E. coli transformations only, would be a massive undertaking. A typical transformation protocol via electroporation or heat shock involves many types of lab equipment and materials originating from around the world. The list of energy-consuming equipment includes everything needed to culture, prep for competency, and transform E. coli cells. Then you could add in freezing the strains and nucleic acids for cloning. In some cases, freshly made competent cells are needed for constructs. This workflow requires running the cold centrifuge for two hours to prepare for the transformation, instead of pulling frozen stocks of competent cells. Technically at a minimum, transformations include the use of an autoclave, ULT freezer, electroporation instrument, shaker incubator, and gel electrophoresis power supply. You’re using either pre-cast or lab-made agarose gels, electroporation cuvettes, microcentrifuge tubes, pipette tips, toothpicks, and Petri plates. In the end, the waste generated must be handled to meet biosafety regulations which is a big footprint in itself. Altogether, it would be an extensive parallel investigation to include all these carbon footprint contributing factors. Célia simplified the objective by narrowing the scope to exclusively electroporation and plating.
Célia shared and qualified her findings on the carbon footprint of transformation. “According to my calculations, performing four transformations using frozen competent cells generates a carbon footprint of 6.2 kg carbon dioxide equivalent. However, this is not precise due to limitations in collecting data.” According to Célia plastic consumables were challenging in particular. “It's difficult to find out where consumables are manufactured and how they are transported in shipments.” Suppliers don’t add the geographical location of a product’s manufacturing site to labeling. In some cases, vendors did provide comparative carbon footprints between product formats. The Berkmen lab uses sterilized wooden toothpicks and platinum loops for picking colonies. However, high throughput laboratory setups and field work often incorporate single-use flexible plastic loops out of convenience or to avoid gouging agar during streaking. Célia thought it would be interesting to look at the carbon footprint associated with plastic loops. Her estimate also included pipette tips, microcentrifuge tubes, electroporation cuvettes, and lab-made agar plates. “Overall, this carbon estimate confirms what we already know is the biggest challenge in our field. Our work has a high carbon footprint, especially considering the need for single-use plastic materials. It’s very important to try to minimize plastic consumables, like pipette tips, gloves, and plastic tubes if experimental conditions allow. It helped me to realize that our impact on the environment is high, even working in a relatively small-scale project. Doing a rough estimate can prompt ideas for changes. At the same time, it’s not possible to change some things. In my case, we needed to use freshly made competent cells for genetic selections, which required running the cold centrifuge to prepare the transformants. We tend to think about the carbon footprints of organizations rather than individual processes or protocols. The goal for an estimate is to understand your personal impact, the impact of your work.”
Next Steps
Once a carbon footprint estimate is in hand, the question becomes, can it be used to identify improvements in a lab workflow?
There are a few broadly applicable steps to reduce the environmental impact of E. coli transformations. Célia Algros’ workshop demonstrated that single-use plastic is a good place to begin based on its outsized impact. Scientists can use this insight as a starting point to discuss moving to bio-based/plant-based lab plastic consumables with their procurement department. Lab plastics made with alternative, non-fossil fuel stocks reduce carbon footprints in the manufacturing stage. Bulk order formats that reduce packaging waste can also be investigated. For high-throughput cloning projects, lab groups can explore instruments that reprocess non-filtered pipette tips for reuse. Each of these steps will reduce the carbon footprint contribution of single-use lab plastics. Moving beyond consumables to laboratory energy consumption, biologists should consider stocking competent cells storage at -70⁰ C instead of -80⁰ C when a newer model ultra-low temp freezer is available, that is validated by the manufacturer for holding temperature. The caveat is that older ULT units do not hold temperature as well when doors are opened and closed to access stocks. Older units can drop to -60⁰ C long enough to reduce the transformation efficiency of competent cells. As a point of interest in the subject of equipment for E. coli transformations, sonoporation is an ultrasound-mediated alternative to electroporation for DNA delivery that is reported to have significantly higher plasmid transformation efficiencies. Bench scale sonoporation requires flat-bottom glass vials rather than single-use electroporation cuvettes. Sonoporation is widely used in other life science applications. To be fair, it’s not pleasant on your ears.
It’s intriguing to think about the potential of sustainability training in life science. Could it inspire protocol reviews in bench-scale development phases that enable resource conservation in industrial-scale processes like biomanufacturing or biopharma, down the line?
The Future of eco-responsible engineering in life science
Scientific training that prioritizes getting experiments to work, can be enhanced with engineering that factors in environmental and resource costs. Academic research, biomedical, and biotechnology sectors are seeking to attract scientific talent with this mindset. Biotechnology has emerged in commercial applications to efficiently generate bio-based fuels and prevent carbon waste in high-impact industries. We are at the forefront of a movement to tie research funding to demonstrated lab sustainability. A large part of life science is focused on biomedical solutions and medical systems are currently reviewing their own carbon footprints and that of their suppliers. Many large life science organizations have begun tracking their carbon emissions to establish software tools for good decision making. In the future, as more carbon footprint data is generated, consumable choices will be evaluated by biologists and sustainability professionals to demonstrate potential returns on investment for decarbonization. Eco-responsible engineering is a newer decarbonization skillset introduced to biotechnology and genetic engineering graduate school programs to meet all of these emerging demands.
I want to thank Célia Algros and the Berkmen lab here at NEB for sharing her learning experience with the Labconscious community! There is a shared environmental responsibility in life science, but there is also an individual responsibility. Analyzing the impact of your lab work doesn’t need to be comprehensive to derive meaningful information. Narrow the scope. Focus on conditions you can change. Begin by developing an understanding.