Devin Camenares, Curriculum Vitae
Education -- Employment -- Skills -- Publications -- Awards -- Outreach

Introduction -- Teaching Philosophy -- Research Interests
Welcome to the interactive CV website for Devin Camenares, Ph.D., currently an Assistant Professor in the Department of Biological Sciences at Kingsborough Community College. In addition to downloading a PDF version of a standard CV for Prof. Camenares, you can also learn about each experience and qualification in depth, as well as learn about the research interests and teaching philosophy that motivates his academic career.

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Biographical Sketch

 They say everybody is born a scientist, but only some retain the passion and curiosity into adulthood. This passion for learning and discovery is something that has always been with me and has often extended to a thirst for knowledge that transcends the hard sciences. Beyond a drive to learn and understand, I also had a strong impulse to apply my understanding to design and create.

As a high school student, I excelled my science courses, including life science courses such as biology and anatomy. I even got a taste of independent research, completing a project that analyzed memory in a flatworm: planaria, to be precise. My project was similar to the 1955 experiments of Thompson and McConnell. It involved training planaria to learn a simple maze using an electroshock. Once the worms learnt the maze, they were bisected (cut) between the head and the tail. Each half regenerates, leading to two viable worms. As one might expect, the head-half of the planaria retained more memory after regeneration, although both halves displayed retained memory compared to a control. While I enjoyed this simple project, my interest in biology was not cemented until I took the Advanced Biotechnology course offered at Sachem High School, taught by the outstanding Fred Gilliam (now retired).

The Advanced Biotechnology course was a college level genetics and molecular biology curriculum presented to high school students. These fields, which form the core of most contemporary important life science research, left me awestruck. More than just interesting science, the implications of biotechnology were made clear in this course. At the time, I had been immersed in learning HTML and other programming languages, which was a challenging but rewarding outlet for my drive to design, create and engineer. The similarities between the world of computer programming and the digital world of DNA and protein sequences (not to mention programs of gene regulation) were not lost on me, even at a young age. Exposure to this material fostered my love of science, changing the trajectory for the rest of my career as I proceeded to study molecular biology at both Rutgers and SUNY Stony Brook. Even before the term had become popular lexicon in molecular biology, I had an interest in synthetic biology and the reprogramming of bacteria.

Within other pages of this site, you can learn more about my journey since Fred Gilliam's biotechnology course. I proceeded to conduct almost a decade of independent research across several labs, honing not only molecular cloning and purification skills, but analytical and critical thinking skills as well. Along the way, I have become exposed to a great deal of research across many fields. This has deepened my appreciation of molecular biology research, as well as informing my attitude about science in general. Despite this breadth of experience, I have continued to maintain a strong interest in synthetic biology.

Education History

 Select either of the following to find out more information regarding my undergraduate or graduate studies. Visit another section of the CV by using the menu at the top.

Ph.D. in Molecular and Cellular Biology

September 2007 - May 2013
SUNY at Stony Brook


Successfully graduated in the Spring of 2013 with a 3.74 / 4 GPA

Relevant Coursework Includes Additional Activities Corresponding Experience

B.Sc in Biotechnology

September 2003 - December 2006
Cook College, Rutgers University (SUNJ): Now know as SEBS


Successfully Completed the Degree in December 2006 with a 3.70 / 4 GPA

Relevant Coursework Includes Additional Activities Corresponding Experience

Employment History

 Select any of the following to find out more information regarding the various positions I have held during my scientific and academic career. Visit another section of the CV by using the menu at the top.

Assistant Professor, Dept. of Biological Sciences

Department of Biological Sciences at Kingsborough Community College, CUNY
March 2014 to Present Day
Summary of Experience:
In my current position as a faculty member at Kingsborough Community College, I combine teaching a range of courses (General Biology, Anatomy, Microbiology) and mentoring student research. I have collaborated with my colleagues in developing supplementary workshops for students in addition to developing active learning exercises and online content. I have taken steps towards establishing a flipped classroom by providing my lecture material with narration on my YouTube channel; I worked with other Faculty on a pedagogical study / program ('Crossroads') and developed case studies and content for the 'Community of Biological Leaners' workshop from 2015 - 2016. I also received training in the use of the Paxton Patterson laboratory modules and applied this training to the 2015 STEM Bootcamp, introducing rising freshmen to careers in Life Sciences.

I am the receipent of an intramural research award which has supported independent work on bacterial translation and co-evolution. I received an innovation award for developing Kingsborough’s iGEM team, an undergraduate research program in synthetic biology. As part of these efforts, I helped foster new corporate partnerships and recruit new students by speaking at events both on and off campus. Students that I have taught and mentored in the research laboratory have become more involved in college activities, secured internships, and successfully transferred to four-year institutions. I am continuing to perfect my approach and develop new and innovative ways to sustain and exceed these positive outcomes.

Adjunct Professor

Suffolk Community College and Touro College School of Health Sciences
August 2013 to December 2013
Summary of Experience:
As a Adjunct Professor at two different Universities on Long Island, I had gained valuable teaching experience. I taught BIO150, Introduction to Modern Biology (a six-contact hour course that includes lecture and laboratory), at Suffolk Community College and I assist in a laboratory section of BIO211, Genetics, at Touro College. Each class had drastically different demands, class size, and gave me a perspective on how different teaching strategies can be effective in different situations.

Graduate Research Assistant

Karzai Laboratory Group, SUNY at Stony Brook
January 2008 - May 2013
Summary of Experience:
During my tenure working in the Karzai Laboratory Group, I worked on a variety of projects. The Karzai laboratory group studies different aspects of protein and translation quality control in E. coli. My projects all focused on various aspects of unproductive pauses in translation, also known as ribosome stalling. If you are unfamiliar with translation, you should read my introduction to the central dogma of molecular biology. Ribosome stalling causes a problem for the cell, and E. coli (and all other bacteria studied to date) resolves this problem through a process known as trans-translation. The ability to resolve the problems of ribosome stalling is known to be important for virulence in some pathogenic bacteria. Hence, studying this problem is not purely academic: the knowledge gained may one day lead to more effective treatments for bacterial infections.

Although some details concerning the resolution of ribosome stalling by trans-translation are known, there are many outstanding questions. Here are a few that I have personally investigated: In an attempt to fill the above gaps in knowledge, I took a variety of approaches. One involved the creation of a complex, fluorescence based in vivo reporter assay. In this assay, bacteria would fluoresce in a ribosome stalling, A-site cleavage, and tmRNA rescue dependent manner. Creation of this assay enables the following: Creation and optimization of this assay involved many rounds of cloning and site directed mutagenesis, as well as protein purification and analysis from the reporter strains. Library screening required iterative rounds of transformation, flow cytometry, P1 transduction, and conjugation.

In addition to the fluorescent assay, I employed other assays commonly used in the field, such as the lambda-tagging assay. The tagging assay of tmRNA function was utilized to analysis hybrid SmpB and tmRNA molecules. This work, which required both site directed mutagenesis, protein expression, and western blot analysis, comprises the bulk of a manuscript currently being prepared. Therefore, I am unable to provide additional details.

Teaching Assistant

Biochemistry Department, SUNY at Stony Brook
January 2008 - December 2008
Summary of Experience:
I was a teaching assistant during two semesters of my graduate career. First, as a TA for BIO 362, I lead recitations to correct any misunderstanding students had over the lecture material. Even though there were only half a dozen students attending my recitation on a regular basis, I enjoyed this role very much. The small size allowed me to interact with the students on an individual basis, and it was especially gratifying to see their reaction when they experienced a large (and positive) increase in their grade. On several occassions, usually the week before an exam, 30 or more students would attend the recitation. This has given me experience teaching in classrooms of varying sizes.

My second assignment was as a preparatory TA for an undergraduate biochemistry course. In this role, I had much less direct interaction with students, as most of the time was spent preparing materials to be used during the class hours. However, I prepared more than just reagents. This assignment gave me experience developing a new module for the course. This involved both designing and optimizing the module, as well as writing the introduction and protocols for the student's course handbook.

Rotation Research Assistant

Mackow, Furie, and Smith Laboratory Groups, SUNY at Stony Brook
April 2007 - December 2007
Summary of Experience:
As a rotation student, I was exposed to a variety of different projects, concepts, and techniques. My longest rotation, from April to September, took place in the Mackow laboratory group. This was actually performed on a volunteer basis before my official enrollment in the graduate program at SUNY Stony Brook. During this time, I led a team of several other summer undergraduates in an attempt to create a reverse genetics system for Rotavirus. This project led to a successful proof of principle result. Along the way, I developed skills in mammalian cell culture, microinjection, virology, and molecular cloning.

Although it was shorter, my rotation in the Furie laboratory group was also successful and eventually led to authorship on an article in Infection and Immunity (PMCID:2849404, see publications section for the abstract). This work involved using ELISA to measure cytokine production by HUVEC derived macrophages in response to purified GroEL from F. tularensis. This work allowed me to strenghen the mammalian cell culture skills I initially developed in the Mackow laboratory group.

My final rotation before joining the Karzai laboratory group (in which my rotation seamlessly transitioned into my thesis work) was in the NMR laboratory of Dr. Steven Smith. During this rotation, I gained enough knowledge and experience with NMR (as well as HPLC and several other biochemistry techniques) to successful complete a series of experiments and make major contributions to a paper on RFDR NOESY, eventually published in the Journal of Magentic Resosnance (PMCID:2802820, see publications section for the abstract). This technique allows dipolar coupling to be reintroduced into a spinning, solid state NMR experiment. Using this technique, we were able to determine the relative positioning of peptides inserted into a lipid bilayer (as well as the position of the atoms in the lipids themselves).

Undergraduate Research Assistant / Intern

Leustek Laboratory Group, Rutgers University (Cook College / SEBS)
November 2003 - February 2007
Summary of Experience:
The Leustek laboratory group studied the metabolism and transport of amino acids in the model plant Arabidopsis thaliana. I started as a volunteer undergraduate research assistant, counting siliques to score an embryonic lethal phenotype in a heterozygote histidine auxotroph plant line. This work was followed by confirming the genotype in different plant lines using a PCR assay, which ultimately contributed to a published paper (in which I was acknowledged for technical assistance).

During the approximately four years I spent in the Leustek led, I also led an independent project to study proline metabolism, as well assisting a graduate student (Dr. Andre Hudson, now a professor at Rochester Institute of Technology.) in identifying the lysine biosynthesis pathway in A. thaliana. The proline metabolism project involved maintaining and treating several plant lines, scoring embryonic phenotypes, creating several expression plasmids through cloning, and introducing these plasmids into plants through co-incubation with Agrobacterium tumifacens. Dr. Hudson's work identified a novel enzyme responsible for a previously uncharacterized step in the lysine biosynthesis pathway in A. thaliana. Not only did this discovery complete the understanding of how plants synthesize this amino acid, it became apparent that this new pathway is present in other organisms, such as cyanobacteira and chlamydia. I contributed to this project by analyzing orthologues of the plant enzyme in several different bacteria, as well as optimizing a purification scheme for the enzyme from A. thaliana tissue. This allowed me to gain experience with both enzyme kinetics and protein purification.

Skill Sets, Talents and Qualifications

 Select any of the following to find out more information regarding the various skill sets, talents, and qualifications I have accumulated. Visit another section of the CV by using the menu at the top.

Communication Skills: Teaching / Pedagogy, Writing, Public Speaking

 Post-Graduate Teaching Experience: In most post-graduate career as first an adjunct professor and now as an Assistant Professor in the Biology Department at Kingsborough Community College, I have demonstrated teaching and communication skills in some of the following ways:



Graduate Teaching Experience: I also exercised my teaching and communication skills during my graduate career in a variety of ways, including:



Extracurricular Teaching Experience: In addition to exercising my communication skills as a assistant professor, adjunct professor, and graduate research assistant, I have also demonstrated these abilities in extracurricular activities such as:



Analytical and Critical Thinking Skills

 The research I conducted as a graduate student (in addition to other responsibilities) demanded strong analytical, objective critical thinking skills. My graduate education allowed me to apply and hone these skills in the following ways:



While pipetting and running gels generates the data (and perseverance to do it again and again), the driving intellectual force behind the research lies in analytical and objective thinking. In addition, I have exercised these same thinking skills in my favorite hobby. As a tournament chess player, I had to make objective assessments of different positions, and use analytical and strategic thinking skills within a time constraint in order to win. In addition to providing anonymous peer review service to journals (critiquing manuscripts prior to publication), I have also reviewed studies after publication and provide some of these on my blog, Just Me and Eubacteria. For example, see my review of a recent Science paper, Shi et al (2011).

Teamwork, Leadership and Organization Skills

Although most graduate research has a strong solitary component, there are some ways in which I have exercised teamwork abilities. In addition, managing several independent research projects over several years demanded strong organizational skills.



Virtually all of the notes I have kept while a member of the Karzai laboratory group were electronic. They were heavily cross-referenced, thanks to some clever use of Excel. Importantly, I could easily search through and retrieve old data and notes on demand, which was very useful when carrying out half a dozen different projects simulatenously.

Bio-informatic, Dry-lab, Programming and Computer related Skills

I have exercised the following bioinformatic skills and experience during my independent research



Through a creative use of formulas, I have adapted Microsoft Excel to serve a multitiude of functions. These include an interactive database system for managing information related to my research projects, to a way to perform mutual information analysis on multiple sequence alignments. If there was such a ranking, I would likely qualify for the black belt in Excel use. I have even used Excel in pursuit of non-academic interested: I created worksheets that convert FEN strings into a chessboard diagram, which I have used to facilitate the creation of a series of novel chess puzzles.

The bioinformatic analysis I performed nicely illustrates many of the computer skills I posses. Briefly, I took a bioinformatic approach to identify interacting pairs of residues between two molecules, SmpB and tmRNA. This involved using a statistical technique known as mutual information, which identifies sets of covarying residues (or whatever other element or data is subjected to it: for example, ) For more detail on mutual information, please refer to the introductory article I wrote for my blog.

In order to subject these molecules to mutual information analysis, I first needed two large, multiple sequence alignments for each molecule. I achieved this by using a combination of Applescript, to query the NCBI database to automatically retrive the necessary sequences, and Microsoft Excel, to format the queries and the sequences once obtained. Once the sequence alignments were formatted correctly, I again used Microsoft Excel to count the occurrence of residue pairs and calculate mutual information.

'Wet'-Laboratory Expertise and Technical Knowledge

 Molecular Cloning and Bacterial Genetics: I gained expertise with a variety of molecular biology, cloning, and bacterial genetic techniques through the course of conducting independent research during both my undergraduate and graduate research tenure. Techniques I am familiar with include, but are not limited to:



Exercising these skills was a major feature of my graduate research. I created dozens of mutant strains through both P1 transduction and a high throughput conjugation (similar to that found in the study by Babu et. al, 2011). I also, through cloning and mutagenesis procedures, created over 200 plasmids. The accomplishments I achieved with these techniques are dwarfed by the troubleshooting expertise I gained in this endeavor. As most aficionados are aware, cloning and mutagenesis are deceptive techniques that can prove to be quite tricky.



Protein Purification and Analysis: I gained expertise with with protein purification and analysis techniques through the course of conducting independent research during both my undergraduate and graduate research tenure. Techniques I am familiar with include, but are not limited to:



My most recent experience deploying these skills involve the following experiments (in reverse chronological order) Western blot analysis of multiple forms of a reporter protein. One of the assays I utilized requires the resolution and detection of proteins from a E. coli lysate that are only 0.5 kDa apart in size (resolving three proteins specifically, around 11.0, 11.5, and 12.0 in size). This has required optimization of a Tris Tricine gel system, which can be found in my protocols section (To follow).

As an undergraduate, I was tasked with developing a biochemical purification strategy to obtain an aminotransferase from Arabidopsis thaliana. This involved homogenization of plant tissue (liquid nitrogen, mortar and pestle), ammonium sulfate precipitation, size filtration, and finally either three-phase partitioning or hydrophobic interaction chromatography.



Additional Laboratory Expertise: During the course of a graduate career, the demands of several projects necessitates developing experience with a wide range of techniques, some of which are difficult to categorize together. In addition to the above skills in molecular genetics and protein purification, I also have experience with:



Publication and Presentation History

 Select any of the following to browse publications in which I am either listed as a first author, co-author, or simply acknowledged for other contributions. You will also find a record of oral and poster presentations I have given. Visit another section of the CV by using the menu at the top.

Authored Publications

 ACeS: Accessible Co-evolution Suite for facilitating co-evolution analysis between proteins and/or nucleic acids
Camenares, D. (2016)
Bioinformatics
Manuscript Submitted (12-20-16)


Abstract (Condensed):
This manuscript introduces a set of open-source, freely available and user friendly tools for conducting co-evolution analysis of proteins, nucleic acids, or both. Included in the manuscript are descriptions of several of the tools as well as data to demonstrate the utility of the tools. Determining co-evolution between proteins and/or nucleic acids is worthwhile; co-evolving residue pairs are predicted to form functionally important, sequence specific interactions. Identifying these interactions can lead to insights into molecule function and can facilitate engineering of orthogonal systems. In earlier studies, I sought such insights into a specialized RNA (tmRNA) and its interacting protein partner smpB. However, I found there were no available resources or tools that would enable a molecular biologist (with limited computational skills at the time) to perform co-evolution analysis. This has motivated me to learn programming languages and develop the software described in the manuscript. These tools are open-source and now freely available online: http://www.bioinformatics.org/aces/

My salient contribution:
I am solely responsible for this work, with the exception of some guidance and insight on the programming of the software tools by Christopher Camenares.


Active and Accurate trans-Translation Requires Distinct Determinants in the C-Terminal Tail of SmpB and the mRNA-Like Domain of tmRNA
Camenares, D., Dulebohn D., Svetlanov, A., Karzai, AW., (2013)
Journal of Biological Chemistry
Oct 18, Vol 288 (No. 42) pp. 30527-42
Epub ahead of print Aug 28 2013
[PMID: 23986442; Free Full Text at JBC.org]


Abstract (Condensed):
Ribosome stalling in eubacteria can be resolved by the actions of tmRNA and SmpB through a process known as trans-translation. This rescue process relies on the ability of the ribosome to disengage the mRNA template upon which it had stalled, then accommodate the mRNA-like region of tmRNA, and finally select the translation resumption point within tmRNA. The mechanism by which this process occurs, in particular how SmpB and tmRNA interact during the template switching step, is not well understood. To gain insight into the determinants of this process, we assayed the ability of hybrid tmRNA and SmpB molecules to carry out trans-translation. .... Taken together, this data suggest that the engagement of the mRNA-like domain of tmRNA and the selection of a resumption point are distinct activities during trans-translation, and are influenced by separate determinants.

My salient contribution:
Together with Dr. Wali Karzai, I am responsible for all of the writing. I also performed the experiments in Figures 4 through 11. Dulebohn performed analysis of SmpB residues, Svetlanov created the initial hybrid constructs.
This article was chosen as 'Paper of the Week' when published in the Journal of Biological Chemistry! Read more about the honor, and find relevant links, at my science blog.


Development of a ribosome stalling, A-site cleavage, and trans-translation dependent in vivo fluorescent assay.
Camenares, D., Karzai, AW., (2013)
Unpublished; some details omitted


Abstract:
An in vivo assay for trans-translation activity was developed by introducing a stalling motif near the C-terminus of GFP. This stalling motif would prevent production of full length GFP. However, completion of translation via ribosome rescue by a modified tmRNA containing the missing C-terminal residues for GFP would restore fluorescence. The assay construct was tested and evaluated, and several different genome-wide screens were performed to identify factors that influence ribosome stalling.

My salient contribution:
Briefly, I was responsible for the construction of plasmids used in this study, optimization of the reporter system, and all of the other materials and experimental data (except for the mass spec. analysis). I am also responsible for a majority of the writing. AW Karzai is largely responsible for the conceptual design behind the assay.


GroEL and lipopolysaccharide from Francisella tularensis live vaccine strain synergistically activate human macrophages
Noah, CE., Malik, M., Bublitz, DC., Camenares, D., Sellati TJ, Benach, JL., Furie, MB., (2011)
Infection and Immunity
Vol 78 (4) pp. 1797-806. [PMC2849404]


Abstract:
Francisella tularensis, the causative agent of tularemia, interacts with host cells of innate immunity in an atypical manner. For most Gram-negative bacteria, the release of lipopolysaccharide (LPS) from their outer membranes stimulates an inflammatory response. When LPS from the attenuated live vaccine strain (LVS) or the highly virulent Schu S4 strain of F. tularensis was incubated with human umbilical vein endothelial cells, neither species of LPS induced expression of the adhesion molecule E-selectin or secretion of the chemokine CCL2. Moreover, a high concentration (10 μg/ml) of LVS or Schu S4 LPS was required to stimulate production of CCL2 by human monocyte-derived macrophages (huMDM). A screen for alternative proinflammatory factors of F. tularensis LVS identified the heat shock protein GroEL as a potential candidate. Recombinant LVS GroEL at a concentration of 10 μg/ml elicited secretion of CXCL8 and CCL2 by huMDM through a TLR4-dependent mechanism. When 1 μg of LVS GroEL/ml was added to an equivalent amount of LVS LPS, the two components synergistically activated the huMDM to produce CXCL8. Schu S4 GroEL was less stimulatory than LVS GroEL and showed a lesser degree of synergy when combined with Schu S4 LPS. These findings suggest that the intrinsically low proinflammatory activity of F. tularensis LPS may be increased in the infected human host through interactions with other components of the bacterium.

My salient contribution:
I performed replicates for some of the experiments already done by others (Figure 6, if my memory serves), and am solely responsible for Figure 7.


High-resolution 1H MAS RFDR NMR of biological membranes
Aucoin, D., Camenares, D., Zhao, X., Jung, J., Smith, SO., (2009)
Journal of Magnetic Resonance
Vol 197 (1) pp. 77-86. [PMC2802820]


Abstract:
The combination of magic angle spinning (MAS) with the high-resolution 1H NOESY NMR experiment is an established method for measuring through-space 1H…1H dipolar couplings in biological membranes. The segmental motion of the lipid acyl chains along with the overall rotational diffusion of the lipids provides sufficient motion to average the 1H dipolar interaction to within the range where MAS can be effective. One drawback of the approach is the relatively long NOESY mixing times needed for relaxation processes to generate significant crosspeak intensity. In order to drive magnetization transfer more rapidly, we use solid-state radiofrequency driven dipolar recoupling (RFDR) pulses during the mixing time. We compare the 1H MAS NOESY experiment with a 1H MAS RFDR experiment on dimyristoylphosphocholine, a bilayer forming lipid, and show that the 1H MAS RFDR experiment provides considerably faster magnetization exchange than the standard 1H MAS NOESY experiment. We apply the method to model compounds containing basic and aromatic amino acids bound to membrane bilayers to illustrate the ability to locate the position of aromatic groups that have penetrated to below the level of the lipid headgroups.

My salient contribution:
I took command (as primary graduate student) of the project following a leave of absence from Jung, J. Some experiments where repeated to control for artifacts, and I processed data (most of which was already) collected to generate figure 5. I contributed to the writing and design of some figures (especially figures 3-8).

Published Acknowledgements

 Co-Evolution of Multipartite Interactions Bwtween an Extended tmRNA Tag and a Robust Lon Protease in Mycoplasma
Zhiyun, G., Karzai, AW., (2009)
Molecular Microbiology
Vol 75 (25) pp. 1083-1099. [PMC2806816]


Abstract:
Messenger RNAs that lack in-frame stop codons promote ribosome stalling and accumulation of aberrant and potentially harmful polypeptides. The SmpB-tmRNA quality control system has evolved to solve problems associated with nonstop mRNAs, by rescuing stalled ribosomes and directing the addition of a peptide tag to the C-termini of the associated proteins, marking them for proteolysis. In E. coli, the ClpXP system is the major contributor to disposal of tmRNA tagged proteins. We have shown that the AAA+ Lon protease can also degrade tmRNA tagged proteins, but with much lower efficiency. Here, we present a unique case of enhanced recognition and degradation of an extended Mycoplasma pneumoniae (MP) tmRNA tag by the MP-Lon protease. We demonstrate that MP-Lon can efficiently and selectively degrade MP-tmRNA tagged proteins. Most significantly, our studies reveal that the larger (27 amino acid long) MP-tmRNA tag contains multiple discrete signaling motifs for efficient recognition and rapid degradation by Lon. We propose that higher affinity multipartite interactions between MP-Lon and the extended MP-tmRNA tag have co-evolved from pre-existing weaker interactions, as exhibited by Lon in E. coli, to better fulfill the function of MP-Lon as the sole soluble cytoplasmic protease responsible for the degradation of tmRNA tagged proteins.

My salient contribution:
I provided a critical reading and review of the manuscript before submission.


Genetic Dissection of Histidine Biosynthesis in Arabidopsis
Muralla, R., Sweeney, C., Stepansky, A., Leustek, T., Meinke, D., (2007)
Plant Physiology
Vol 144 (2) pp. 890-903. [PMC1914156]


Abstract:
The biosynthesis of histidine (His) in microorganisms, long studied through the isolation and characterization of auxotrophic mutants, has emerged as a paradigm for the regulation of metabolism and gene expression. Much less is known about His biosynthesis in flowering plants. One limiting factor has been the absence of large collections of informative auxotrophs. We describe here the results of a systematic screen for His auxotrophs of Arabidopsis (Arabidopsis thaliana). Ten insertion mutants disrupted in four different biosynthetic genes (HISN2, HISN3, HISN4, HISN6A) were identified through a combination of forward and reverse genetics and were shown to exhibit an embryo-defective phenotype that could be rescued by watering heterozygous plants with His. Male transmission of the mutant allele was in several cases reduced. Knockouts of two redundant genes (HISN1B and HISN5A) had no visible phenotype. Another mutant blocked in the final step of His biosynthesis (hisn8) and a double mutant altered in the redundant first step of the pathway (hisn1a hisn1b) exhibited a combination of gametophytic and embryonic lethality in heterozygotes. Homozygous mutant seedlings and callus tissue produced from rescued seeds appeared normal when grown in the presence of His but typically senesced after continued growth in the absence of His. These knockout mutants document the importance of His biosynthesis for plant growth and development, provide valuable insights into amino acid transport and source-sink relationships during seed development, and represent a significant addition to the limited collection of well-characterized auxotrophs in flowering plants.

Oral and Poster Presentations

Awards and Honors

 Below is a list of different awards and honors I have earned during my scientific and academic career.

Outreach and Activities

 The following is a list of a variety of activities and outreach efforts I have engaged in, or any other activities, honors, experiences, or qualifications which don't quiet fit into any other sections.

Teaching Philosophy

 Life science disciplines are changing rapidly, increasing both in complexity and in their impact on society. This is particularly true for the fields of biochemistry, molecular biology, and synthetic biology, which have the ability to revolutionize diverse industries such as manufacturing, biofuel production, and bioremediation. Concomitant with the increased importance of this science in society is a demand for students and a workforce with a strong background in STEM. This demand includes a need for both the knowledge and technical skills, as well as an ability to communicate and discuss the science. I am dedicated to addressing this challenge, particularly in a way that gives students a strong foundation in biochemistry and allows them to place the science in a real-world context. Fundamentally motivating my approach is the belief that education should be empowering, allowing students to benefit their lives, advance their careers, and transform their communities.

Despite the complexity of life science material, I seek to equip students with the understanding of the connections in the material needed to solve problems. Towards this end I utilize active learning exercises and assignments such as writing and reviewing lab reports, writing scientific press releases, analyzing case studies, or performing bioinformatic analysis. Thanks to these exercises, students become more engaged with the material. They perform better when they see the potential rewards that comes with mastery of life sciences, gaining confidence they carry beyond the classroom.

A significant portion of own my science education took place in a research laboratory. The task of solving a research problem is a great context to test and expand the knowledge and skills of any student. As a research mentor, I have acted as a guide, counsel, and teacher to several students engaged in their own independent research projects. In addition, I have led the development of a team based project at Kingsborough as part of the International Genetically Engineered Machine competition (iGEM). This team approach provides additional benefits by integrating science, engineering, and communication in a competitive but fun contest.

In 2016, the iGEM team sought to address the problem of wastewater pollution in nearby Jamaica Bay by genetically engineering a bacterium to more efficiently metabolize nitrogenous waste molecules (Visit the team Wiki site for more information). Students had to integrate their knowledge of chemistry, molecular biology, and metabolism with ecology, public perception, and engineering issues inherent in a modern wastewater treatment plant. Successful completion of the project involved more than just lab work, and included a tour of a local wastewater treatment facility. Together, this produced an engaging, comprehensive educational experience for the students that was focused on an important real-world problem. I am continuing as mentor for the iGEM team in 2017, and seeking to expand the program to other schools. The approach from our team is a model to be emulated, not only for future iGEM teams but in the classroom and across the curriculum as well. By working together, students not only build teamwork skills but also engage in peer-to-peer learning, educating each other on different aspects of the project and challenges. This prepares students for a future where collaboration is increasingly important, especially on projects that bring together expertise from different specialized disciplines.

Research Interests and Current Projects

 My research interests have been, and continued to be, motivated by a passion for the emerging field of synthetic biology. This field promises to deliver innovative and powerful solutions to many different problems and challenges in both the modern and developing world. I am interested in both advancing the field and capabilities of the synthetic biologist, as well as developing solutions to specific questions.

Synthetic biology relies on the ability of a scientist or engineer to manipulate microorganisms in a predictable way. Thus, a complete understanding of how gene expression is regulated in organisms such as bacteria is critical to the successful design of novel cellular behavior. Regarding this challenge, I am interested in discovering new insights into the behavior and mechanisms of the ribosome, the cell’s central processor of information. This includes examining interactions between the ribosome and other components of translation, as well as engineering novel ways in which gene expression can be regulated at the key step of translation. Central to my research is the use of an underutilized bioinformatic approach to determine co-evolving residue pairs, which may represent heretofore site-specific interactions important for molecular function.

Select from the list below for more detailed descriptions of my research goals. These are highlights, and my research activities are not limited to this set.

Synthetic Biology

 The emerging discipline of synthetic biology holds tremendous potential for both basic research and bio-engineering. In some ways, synthetic biology is a more ambitious expression of the same impulse behind most contemporary biotechnology; engineering entire pathways instead of single proteins and enzymes. However, this field goes beyond most traditional efforts at engineering bacteria towards a useful end both in scope and approach. Notable examples of synthetic biology in recent years include bacteria programmed to invade tumor cells (the work of Voigt and colleagues) as well as the production of microbial derived artemisinin (the work of Keasling and colleagues, which required importing an entire biochemical pathway into the microbe). In addition to these impressive feats of bio-engineering, synthetic biology also gives researchers a way to better understand the design principles by which normal cells function. After all, it is much easier to understand how a system works once you have succeed in constructing one of similar complexity.

I am interested in both developing a better understanding of molecular biology and how to engineer a living system, as well as ways in which such knowledge can be employed to solve real world problems. My passion for the field is manifest in several ways, including my blog Just Me and Eubacteria, which focuses heavily on synthetic biology.

The emerging discipline of synthetic biology holds tremendous potential, both for basic and applied research. Realizing the benefits of this new approach to biology demands an enormous capacity for both DNA sequencing and synthesis. DNA sequencing technology has improved by leaps and bounds, generally following or exceeding Moor's Law. This is in part due to a combination of scaling existing technology as well as the development of second and third generation sequencing technology, which improve the efficiency and capacity of sequencing by orders of magnitude. Unfortunately, similar groundbreaking technological developments in the realm of DNA synthesis have been lagging. Despite the limitations of current technology, there are several bright spots on the horizon. One that is particularly promising is DNA laser printing, which actually harnesses the power of next generation sequencing technology. However, much more work needs to be done to meet the synthesis demands of a growing field of synthetic biologists.

In addition to overcoming the technical challenges involved with DNA synthesis, I am very excited about research that explores how gene expression and cell behaviors can be manipulated. The design and construction of sophisticated cellular behaviors by synthetic biologists pushes the boundaries of current knowledge of how gene expression is regulated. Synthetic biologists benefit from an ever increasing toolkit of gene expression 'subroutines' (different motifs or ways in which gene regulation is used to produce particular behaviors), which can then be combined in ways to produce finely tuned cell responses. Beyond discovery of new gene expression dynamics from traditional research, synthetic biologists have both discovered and demonstrated diverse ways in which gene expression and cell behavior can be controlled. Two recent examples include the successful construction of an analog genetic circuit (by the Sarpeshkar group at MIT) and how spatial-temporal control of gene expression can form patterns (research from the You laboratory group at Duke University). Expansion of the 'toolkit' used by synthetic biologists, both in terms of parts and in different types of expression programs, will be critical to fulfilling the potential of the field. This set of gene regulation programs and strategies used by synthetic biologists can emulate the diversity found in nature and beyond.

Co-Evolution

 To date, our capability to sequence the biological information from living things has outpaced our ability to interpet this data. There is a wealth of sequence information now available, detailing both the diversity and unity of all life on a molecular level. Unity is observed in the form of sequences that are conserved in many different living things. For example, amino acid sequences that correspond to functionally critical parts of the resulting protein will be conserved and unchanged in many organisms. This conservation has been used successfully to narrow the focus of scientific investigations into these proteins: they act to highlight the functionally important parts of some proteins. Often, portions that appear unconserved are ignored or not considered a priority for analysis.

Many proteins and RNA molecules make functionally important contacts with other macromolecules for which sequence information is available. In some cases, the residues on each of these factors may have changed, but done so concurrently, to preserve these important functional interactions. This will lead these two parts of each molecule (or two parts of the same molecule) to appear to co-evolve: they will not be conserved individually, but as pairs they will more commonly be found together. Identifical of these coevolved pairs can be done through sequence analysis, in particular by using the mutual information technique (described below). Prediction of these coevolved pairs can identify novel roles for residues which have been ignored due to their lower levels of conservation. Furthermore, these residues pairs are likely making a sequence-specific functional interaction, identification of which may open up new possibilities for re-engineering these interactions.


What is Mutual Information? How can it be used?
I have written about mutual information elsewhere (see my introduction to mutual information article at my blog for a more complete description). There are different variations to how this technique is employed, but in essence this takes a large set of data and looks for coincidence or co-occurence between different parts of the set. In the diagram to the right, H(X) is the probability of result X occuring for event H. Other probabilities are listed accordingly; mutual information is a calculation to determine the effect one result has on another (the intersection on the venn diagram). In all cases, this approach produces a set of predicted inter- and intra-molecular interactions for the input sequences.

Mutual Information of a data set can be found in several ways, from efficient Java programs to simplistic Excel spreadsheet calculations. When using this technique to determine coevolution, a large multiple sequence alignment of one or more proteins or factors must be generated. However, it is not the only, or even the latest, way of measuring co-evolution - other methods, such as PSICOV and DCA have additional advantages over MI calculations.

Bacterial Translation and Ribosome Rescue

 Manipulation and engineering of bacteria requires a through understanding of how genes are regulated and expressed in these cells. Central to this regulation is the process of translation, which is the ultimate step in the flow of genetic information from DNA to protein. The step of translation is in some ways the very essence of life. It is the rate of this process which appears to limit and/or influence the growth rate of most organisms. Furthermore, it is the a process unique to life itself. While viruses exist that can carry out DNA replication and RNA transcription, as well as a variety of other enzymatic activities, all are thought to require the translation capacity of their host for survival.

While great strides have been made during the past few decades in understanding how translation occurs in bacteria, there still remains some open questions. The broadest and most important of which regard the mechanistic abilities of the ribosome itself and how signals are integrated at the ribosome to regulate translational programs in the cell. Researchers are constantly finding new factors which integrate cellular functions at the ribosome or otherwise modulate the rate of translation. One example of this is the association of RNAse R with the E. coli ribosome to promote degradation of defective mRNAs. Yet another example involves modification of the ribosome itself by MazF to alter the set of mRNAs that can be translation. Finally, the ribosome has been known to undertake unusual maneuvers in special circumstances. A great example of this is the template switching action promoted by tmRNA and SmpB during trans-translation. The mechanism by which the ribosome accomplishes this feat is still unknown, although a clearer picture is beginning to emerge.

Answering the questions above is not simply an academic matter. A better understanding of the ribosome, especially how it integrates signals and performs unusual tasks, allows for better experimental manipulation of translation. This is true both in the case of designing synthetic organisms as well as killing dangerous natural bacteria. For example, it is known that the tmRNA and SmpB ribosome rescue system is required in certain bacteria not for survival but for pathogenesis. Development of inhibitors to trans-translation could potentially serve as potent antibiotics with limited side effects. A better understanding of how template switching during this process occurs, or an assay dependent on trans-translation, is necessary to develop or identify such compounds.

Saline Agriculture and Bio-Desalination

 Marine ecosystems are complex, with a wide range of life adapted to plethora of different environmental conditions. The universal presence of sodium is a significant challenged faced by all marine life, including the microbial communities that are critical for the cycling of various nutrients in the ecosystem. Indeed, the salinity of ocean water is an obstacle for terrestrial organisms as well, necessitating either desalination for human consumption and irrigation of most crops, or the evolution of salt tolerance in plant life found in coastal habitats (Patel, 2016; Reef & Lovelock, 2014). A greater understanding of how changing salinity effects evolution of marine microbial communities will better inform models of marine ecosystem dynamics. In addition, such insights into how organisms adapt to salinity may facilitate the development of novel desalination technologies.

Salt Water and its Importance
Apart from water molecules, sodium chloride is the most abundant substance in the ocean by mass. Like water, sodium ions are vital for some organisms and have a significant impact on all living things. Many organisms utilize differences in intracellular and extracellular sodium concentrations and require at least trace amounts. Indeed, the function of our nervous system hinges upon the import and export of sodium and other ions across the membrane of a neuron, and is likewise sensitive to changes of internal ion concentration. Still other organisms, such as Halobacterium species, require copious amounts salt for growth and actively manipulate sodium to nucleate the growth of salt crystals (Jaakkola, Ravantti, Oksanen, & Bamford, 2016).

The presence of sodium chloride in the environment also presents an osmotic challenge to the vitality of the cells of any organism. This is a challenge constantly faced by all marine life, including ecologically important species of bacteria. This same osmotic challenge prevents the use of plentiful ocean water for human consumption or agricultural applications. Thus, the relative scarcity of freshwater is a significant issue for many communities, one which is predicted to become more critical in the future with a rapidly changing climate and population growth. Although desalination of ocean water provides one solution to this problem, traditional methods to remove salt require significant investments in infrastructure and energy and may not be available to impoverished regions (Taheri, Razmjou, Szekely, Hou, & Ghezelbash, 2016).

The importance of salinity levels is underscored by attempts to measure it on a global scale, such as the Soil Moisture and Ocean Salinity (SMOS) satellite mission (Barré, Duesmann, & Kerr, 2008). While ubiquitous globally, a variety of factors leads to an uneven distribution of sodium chloride amounts across different environments and regions. For example, the hydrological cycle, which describes the evaporation of ocean water and subsequent rainfall, helps to determine the salinity of different bodies of water. Evaporation leads to a local increase in salinity while rainfall, either over land or over the ocean, will lead to a local decrease in salinity. Indeed, changes to the global climate has led to an acceleration of the hydrological cycle, and subsequently an increased disparity in sodium distribution (Reul et al., 2014). Other human activities, such as desalination or mining, can affect the overall distribution of salt in some regions.