University of California Riverside | Chemical and Environmental Engineering
Justin Hoover, Undergraduate Student
The Jinkerson lab is interested in a 250 µL and a 2.5 ml syringe to improve the accuracy of our lipid extraction protocol, making the protocol easier to learn and master for myself and the other undergraduate students in the lab. We will be able to perform the protocol with both precision and accuracy, providing greater confidence in our data and more potential to create publishable data. We will also be able to perform the protocol quicker which will allow us to spend more time learning how to analyze our data and developing other lab skills.
In pursuit of optimizing the current and future production of algal biofuels, the Jinkerson lab is screening a knockout mutant library of the model algae, Chlamydomonas reinhardtii, to identify genes that affect lipid production. Utilizing flow cytometry, gas chromatography, and metabolomics, we will identify genes that influence TAG accumulation. This knowledge will be critical for engineering strategies to make algae a better biofuel feedstock, bringing us closer to economically viable algal biofuels. This knowledge can also be applied to other oil-producing organisms such as oil seed crops, potentially having an impact on food production.
In order to test the lipid concentration (TAG, triacylglyceride) of mutants identified in a preliminary screen, we perform a base saponification followed by an acid catalyzed transesterification to convert TAG into fatty acid methyl esters (FAMEs) which are then extracted from the solution with hexane for analytical separation and profiling. This conversion allows the lipid of interest to transition from liquid to gas without suffering thermal decomposition and prevents column adsorption issues during separation by reducing the polarity of the underivatized FAME (TAG). The most critical of these lipid extraction steps is the addition of 1.25 mL of extraction reagent (hexane) to the sample, as this is directly correlated to the concentrations of lipids present in the sample quantified by the GC-FID. Thus, transfer precision and reproducibility is pivotal as the experimental goal requires accurate quantification of mutant TAG production. Additionally, imprecision reduces the conclusivity of the learning experience as meaningful progress and publishing potential suffer. The final step for analysis is to siphon 150-microliters from the top phase (hexane) of the liquid-liquid extraction as a sample for input into the GC-FID. The choice of analytical instrument is based upon the gas chromatographs quick, precise, and replicable separation performance, and upon the flame ionization detectors ability to quantify FAMEs.
However, the volumes and needles currently available in the laboratory induce additional error as multiple transfers of small volumes must be made and use generally involves each individual to have a different volume syringe. For example one undergraduate utilizing the fume hood may be using a 500-microliter 700 series syringe to transfer 1.25 mL of extraction reagent while the other uses 1 mL gastight syringe, this introduces additional variation into the experiment and hinders reproducibility as uniformity is no longer guaranteed.
The syringes received from the Hamilton Syringe Grant would allow all lab members to have the same experience in precision liquid transfer, and increase both the precision and accuracy of every experiment in which small volumes are utilized. As an undergraduate student, this grant would greatly increase my experimental confidence by providing the instruments necessary to properly continue development of my analytical lab skills. This allows for constructive contribution to the experimental goals of the lab, where progress often promotes new questions that require critical application of traditional undergraduate book learning, extending my educational experience beyond routine classroom assignments. For all undergraduates, having quality laboratory experience and the ability to perform protocols with confidence is an asset that promotes career viability after graduation as well as being a stepping stone in obtaining a graduate degree. For the graduate students in the Jinkerson lab, the opportunity to achieve enhanced experimental accuracy and precision augments the viability of procured data and thus increases the probability of obtaining a publication. Additionally, we would have enough syringes so that when working together, we would not have to wait for a turn with the syringe and could each have our own.
The Jinkerson lab, headed by Robert Jinkerson Ph.D., is a multidisciplinary lab with over ten members specializing in a diverse range of fields, from biology to chemical engineering, and comprised of both undergraduates and graduate students. This allows incorporation of ideas developed from collaborations not frequently found. It is not uncommon for the conglomerated knowledge of all lab members in a group meeting to be sufficient to carry a discussion over an hour, removing the need for references and google searches to make progress on certain topics. For example, as a chemical engineer by concentration, I can offer process and design options to a colleague concentrated in biology with a need to assess a production question. This not only allows me to apply my scholastic knowledge but also provides me with the opportunity to develop new understandings of the fundamentals upon which engineering is based, as course depth was sacrificed in many areas to allow for progression to fluid studies. The intermingling also provides traditional biology and chemistry concentrated individuals with a resource that can provide fundamental engineering knowledge typically requiring additional years of coursework to combine. Current projects involve the described forward genetic approach pursuing identification of the genes regulating TAG production, metabolic algae engineering for optimization of fatty acid production, and inquisitions into the symbiosis between coral and algae. I will be graduating with a B.S. in chemical engineering in Spring 2019 and I have the desire to make a positive impact in this lab as the research being pursued is globally beneficial and participation in such an opportunity is a merit that not every student is able to obtain, but qualified UCR students are eligible to. Lastly, I feel confident when using Hamilton products, and words are not necessary to know that my colleagues do as well. Overall, this grant would enhance the educational experience of all lab members for years to come.