Engineering the Tools of Genetic Engineers

By: Melody Keith, Bibiana Toro, Kim Ly, Alyssa Braddom, and Eleanor Young, University of Texas at Austin undergraduate researchers.

The University of Texas at Austin’s 2018 International Genetically Engineered Machine (iGEM) team is a group of students whose aim is to use synthetic biology to solve real world problems. We are a diverse group of upper and underclassmen who come from fields such as biochemistry, biology, and neuroscience. For some of us, this was our first in-depth research experience. For others, it was a chance to apply years of experience to an exciting project (Figure 1). Not only did we utilize microbiology techniques, but we were able to collaborate with researchers from Rice University and Texas Tech University, create a visually compelling poster and oral presentation, and design a website.

Figure 1: Students at the annual iGEM conference.  Eleanor Young, Melody Keith, Alyssa Braddom, Bibiana Toro, and Kim Ly with their poster.

This summer we came together to engineer a solution to a problem many scientists face in their lab every day: how to genetically manipulate non-model organisms. Bacteria are able to accomplish feats that human technology has yet to realize. They are better at producing certain materials, they can manufacture medicines, and some survive in extreme environments. However, these organisms that have incredible potential are usually non-model, which is to say, not the ones we work with in standard research labs. Biologists generally use only a handful of organisms, such as E. coli, because they are better understood and well characterized.  Therefore, we know how to genetically engineer them. However, these organisms do not always have the internal molecular machinery necessary to produce the molecule(s) that scientists desire. Scientists must instead engineer a bacterium that is unfamiliar to them, for which good protocols may not yet be established. Opportunities for failure pervade this process.

Our solution to this common problem is the Broad Host Range Kit, a combination of plasmid parts and fully assembled plasmids, that allows a researcher to test many plasmids at once and then build their own plasmid with their own coding sequence of interest. It relies on a molecular cloning method known as Golden Gate Assembly, which allows genetic parts to be easily assembled and interchanged. These plasmid parts are classified according to their function, or type, and many hours were spent cloning desired parts from template sequences. We built assembly plasmids out of these plasmid parts, varying some parts while conserving others. These varied regions were the reporter gene, encoding a fluorescent protein or chromoprotein, the origin of replication, which allows the bacteria to make copies of the plasmid, the barcode region, a short DNA sequence identifying each plasmid, and the antibiotic resistance gene. Origins of replication were chosen that are known to be broad host range; they function in a wide variety of different bacteria.

Each assembly plasmid, which we call a “Pioneer Plasmid”, has the origin of replication coupled to a specific reporter and barcode. Therefore, when the plasmid is inserted into the bacteria of interest, the origin of replication can be determined just by looking at the color of colonies on the plate. If for some reason, the bacteria can’t express the reporter, the barcode can be sequenced, which adds a layer of redundancy to the system.

Our kit relies on the “One Tube Method”, which puts all the Pioneer Plasmids into a single tube, so the mixture must only be transformed into the non-model organism of interest once (Figure 2). The transformation is then plated onto various antibiotic plates and the origin of replication that functions can be determined by visual inspection of the plate alone (Figure 3). Out kit, therefore, speeds up the process of finding out which broad host range origin functions in non-model organisms and contains 8 fully assembled pioneer plasmids with 3 different origins and over 40 part plasmids. We’ve also test the One Tube Method in Vibrio Natriegens and Serratia Marcescens.

Figure 2: The Kit in Action.  Schematic of how the One Tube Method works.  After transformation, screening for color reveals the plasmid each colony contains. The DNA in non-colored colonies can be extracted and sequenced to identify the plasmid within.  The identity of the plasmid reveals which genetic parts are functional in your bacteria.

Figure 3: Screening Colonies.  Seven plasmids containing different reporter genes transformed into E. coli.  The plate on the left is in natural light, while the plate on the right is under blue or UV light.  The different colonies are highlighted along with the identified reporter in each.

Members of our team had the privilege to attend the iGEM “Giant Jamboree” in Boston, where over 250 teams from all around the world met to present their research, but also to network, collaborate, and share ideas. It was a stimulating and rewarding conference. For example, the team from the National University of Singapore expressed luteolin in E. coli as an eco-friendly alternative to toxic yellow textile dyes. Cornell University produced a genetic circuit that would respond to frequency variable input signals to specifically regulate expression. Both presentations inspired one of our members to investigate optogenetic regulation and engineering of photoswitches for a project currently in progress.

The scientists and undergraduate researchers we interacted with commented that engineering non-model organisms was a problem that they too encountered in their own labs daily. They provided excellent feedback and suggestions, such as showing our kit functions in a particular culture collection or using software to design a combinatorial library of Pioneer Plasmids.  We also spoke to teams interested in acquiring and using our kit. For instance, the team from the Indian Institute of Technology Madras want to use the BHR kit to engineer Acinetobacter baylyi in order to produce biofuels from the degradation of aromatic compounds.

We also had the opportunity to hear from illustrious keynote speakers Dr. Ingrid Pultz, Jason Kelly, and George Church. In particular, the way Jason Kelly talked about the field of synthetic biology, and its enormous potential to revolutionize industries from agriculture to materials to medicine, was inspirational. They made the career path many of us are on feel tangible, achievable and bursting with opportunity. To hear them speak, changing the world seemed not only possible, but just within reach.

Presenting our own team’s research was a dynamic experience that highlighted the grit and focus it takes to practice, refine, and effectively communicate a project which took months to produce (Figures 1 and 4). We left feeling empowered, knowing this experience, being a member of the iGEM team, had given us the skills to successfully achieve every step of the research process from initiation to generating results to finally synthesizing a message about the meaning and impact of that work.As we continue to improve the Broad Host Range Kit, we know that the small steps we take in the lab everyday can translate into larger benefits for both the scientific community and the world.

Figure 4: Melody Keith presenting the students’ work. Bibiana Toro (right) and Eleanor Young (not pictured) also participated in the oral presentation.

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