SEED 2019

This past week I attended and presented at the Synthetic biology, Evolution, Engineering, and Design (SEED) conference 2019.

I presented a poster titled Controlling Cell-Free Gene Expression Level and Dynamics by Tuning Molecular Transport across Lipid Membranes, and shared my research in a 60 second rapid fire slide. This poster summarized our results of increasing membrane permeability to allow small molecules necessary for transcription and translation to support cell-free protein synthesis.

Graduation

I have finally graduated!

My family came to town to celebrate. We ate at Emilia in Market Square before the ceremony. The hooding ceremony was exciting. I was introduced as “Dr. Patrick Caveney hooded by Dr. Michael Simpson.” The hood has UT colors, orange and white, and a blue patch to show that my doctorate is in philosophy. Afterward we had drinks at the Library. I wore two different cufflinks, a microscope my sister got for me and an engraving of my initials that my grandmother got me.

On Friday we walked around campus and saw all the improvements that have been made. On Saturday my dad and I ran from Market Square to the UT agriculture campus gardens before the farmers market. Afterward we got coffee and tamales and walked around the farmers market. The same Saturday morning routine of my friends and I before they all graduated and moved away.

Dissertation

My finished dissertation, Steps toward cell-like systems: spatio-temporal control of shared molecular resources for cell-free gene expression has been accepted by the university! Below is the Abstract. The full dissertation can be downloaded here.

Abstract

The biosphere offers many promising economically and environmentally sustainable solutions to humanity's increasing energy demand such as biomass conversion, chemical production, and pharmaceutical fermentation. These solutions could close the carbon loop and improve manufacturing efficiencies, but they all depend on accurate control of protein expression. To avoid limitations to protein control present \textit{in vivo} such as membranes, homeostasis, and growth, artificial cell-like systems are being researched. These simplified systems are currently useful to study individual aspects of life such as regulating energy flux across membranes, responding to the environment, replication, and growth. These systems could be made more complex in the future to provide a simplified, engineered, cell-like platform for bioprocessing. Even at the single gene level, control of protein expression is hindered by resource sharing and bursting. To make proteins, genes require many reusable resources such as polymerase, ribosomes, and tRNAs which are shared among different genes. Resource sharing causes correlations in protein populations and limits steady state concentrations of competing genes. Bursty gene expression, periods of high expression, and thus high resource use, separated by periods of no expression, and thus no resource use, is a ubiquitous biological phenomenon that intimately links expression bursting and resource sharing. This dissertation investigates how gene expression bursting and variation is affected by expression resources being shared among genes and how the location of expression resources, either encapsulated or outside permeable lipid membranes, controls the level and the dynamics of cell-free protein expression. Cell-free protein synthesis systems, both crude and PURE, are used in combination with both physical PDMS barriers and defined lipid membranes to study the effects of shared and divided resource pools on gene expression bursting and protein production. Experimental results are supplemented with Gillespie simulations to add further insights. This work provides fundamental knowledge of protein expression and applied knowledge of the effects of resource sharing on cell-free gene expression bursting and variation in protein expression confined to cell-relevant volumes which are important steps toward artificial cell-like systems.

Membrane Transport

We submitted a paper titled Molecular Transport across Lipid Membranes Controls Cell-Free Expression Level and Dynamics to bioRxiv. This paper builds on the last paper that showed small resources needed for protein production can cross the membrane with light exposure. The purpose of this paper was to characterize how the location of resources (either inside the vesicle or outside the vesicle; Figure A) impacted the statics and dynamics of protein production inside vesicles (Figure B).

Abstract

Essential steps toward synthetic cell-like systems require controlled transport of molecular species across the boundary between encapsulated expression and the external environment. When molecular species (e.g. small ions, amino acids) required for expression (i.e. expression resources) may cross this boundary, this transport process plays an important role in gene expression dynamics and expression variability. Here we show how the location (encapsulated or external) of the expression resources controls the level and the dynamics of cell-free protein expression confined in permeable lipid vesicles. Regardless of the concentration of encapsulated resources, external resources were essential for protein production. Compared to resource poor external environments, plentiful external resources increased expression by ~7-fold, and rescued expression when internal resources were lacking. Intriguingly, the location of resources and the membrane transport properties dictated expression dynamics in a manner well predicted by a simple transport-expression model. These results suggest membrane engineering as a means for spatio-temporal control of gene expression in cell-free synthetic biology applications and demonstrate a flexible experimental platform to understand the interplay between membrane transport and expression in cellular systems.

Figure: Resource location (internal or external) affects gene expression behavior. (A) Gene expression is affected by the encapsulated (orange circular sector) and external (red triangle) molecular resources. (B) The gene expression transient is the sum of two components: one controlled by the internal resource concentration and one controlled by external resource concentration. The expression transient due to external resources should experience a delay (labeled here as expression lag) related to the membrane transport properties.

Full text is here.

Patrick M. Caveney, Rosemary M. Dabbs, William T. McClintic, C. Patrick Collier, and Michael L. Simpson. Molecular Transport across Lipid Membranes Controls Cell-Free Expression Level and Dynamics bioRxiv 606863 doi: https://doi.org/10.1101/606863

Membrane Permeability

We have submitted a paper to biorxiv (pronounced bio archive) titled Controlling Cell- Free Gene Expression Behavior by Tuning Membrane Transport Properties. The goal of this paper was to increase the membrane permeability of vesicles to increase cell-free protein expression inside the vesicles. Previous papers have shown that ~10% of vesicles are permeable to small molecules necessary for making proteins (Figure A). We used light exposure to increase the permeability of the membranes so all the vesicles are permeable to small molecules necessary for making protein (Figure B).


Abstract

Controlled transport of molecules across boundaries for energy exchange, sensing, and communication is an essential step toward cell-like synthetic systems. This communication between the gene expression compartment and the external environment requires reaction chambers that are permeable to molecular species that influence expression. In lipid vesicle reaction chambers, species that support expression – from small ions to amino acids – may diffuse across membranes and amplify protein production. However, vesicle-to-vesicle variation in membrane permeability may lead to low total expression and high variability in this expression. We demonstrate a simple optical treatment method that greatly reduces the variability in membrane permeability. When transport across the membrane was essential for expression, this optical treatment increased mean expression level by ~6-fold and reduced expression variability by nearly two orders of magnitude. These results demonstrate membrane engineering may enable essential steps toward cell-like synthetic systems. The experimental platform described here provides a means of understanding controlled transport motifs in individual cells and groups of cells working cooperatively through cell-to-cell molecular signaling.

Figure: Engineering membranes for uniform protein production. (A) (Top) A population of vesicles where only a few vesicles are permeable to resources essential for expression (red triangles) in the outer solution and thus able to make protein (black diamonds and green background). (Bottom) The result is a highly skewed protein population distribution where most vesicles make no protein and a few make large amounts of protein. (B) A process to make more vesicles permeable to protein expression resources (Top) would result in a more uniform protein population distribution (Bottom).

The full text is here.

Patrick M. Caveney, Rosemary M. Dabbs, William T. McClintic, S. Elizabeth Norred, C. Patrick Collier, and Michael L. Simpson. Controlling Cell- Free Gene Expression Behavior by Tuning Membrane Transport Properties biorxiv 604454; doi: https://doi.org/10.1101/604454

Dissertation Defense

I passed my dissertation defense! My presentation is here. It starts with the importance of biological solutions to increasing energy needs. Then dives into how these biological energy solutions depend on accurate control of protein expression. I covered important background topics including noise in protein expression, gene expression bursting, resource sharing, cell-free protein expression, microfluidics, and artificial vesicles. Finally, the purpose of my dissertation is to investigate how gene expression bursting and variation is affected by expression resources being shared among genes. I specifically answer:

  1. How are gene expression characteristics altered by the size of resource pools?

  2. Can membrane permeability be tuned to enhance gene expression?

  3. How are the dynamics of gene expression affected by resource transport across permeabilized membranes?

My parents and grandmother were there to support me. Afterward we celebrated with dinner at J.C. Holdway in downtown Knoxville.

Gordon Conference

I was in Ventura, California from January 8 - 13, 2017 to present a poster titled Resource Sharing Controls Gene Expression Bursting at the Stochastic Physics in Biology Gordon Research Conference: Landscapes, Stochastic Dynamics and Heterogeneity in Biology. The research is now published open access at ACS Synthetic Biology. The Gordon Conferences are unique. Everyone signs an NDA and no photos are allowed, so very new results are presented and discussed. The conference topics are very specific so the audience is small and mostly the world experts in the field. Because the conferences are small, discussion is encouraged from everyone, even grad students. Finally the conferences are in remote locations and there is an early afternoon break every day where people are encouraged to hang out. During this time I played soccer, tennis, biked and ran with professors, post docs, and other grad students. It was a great way to get to know people in a less academic, less intimidating way. I was very fortunate to meet Dr. Daniel Gillespie and his wife Carol Gillespie. He created the Gillespie algorithm which I use to make stochastic simulations of my experimental system. He spoke about how the algorithm was created and how he received great skepticism when he first presented it. 

Resource Sharing

My first first-author paper, Resource Sharing Controls Gene Expression Bursting, has been published [PDF]! 

Genes express proteins in bursts of activity with periods of no activity between bursts. Burst dynamics are characterized by a burst size (duration of a burst) and burst frequency (number of bursts per time). During a burst the gene draws on a limited pool of reusable resource. Little is known about the relationship between burst dynamics and resource sharing. Here we made cell-sized reaction chambers (both PDMS plastic and POPC lipid vesicles) and observed bursting dynamics as the size of the resource pools was varied. When the size of the resource pool was increased, the number of protein made increased. This increase in protein was achieved by increasing the burst size not burst frequency. This may be due to the fact that the 100 different molecules needed to make protein became localized. Localized components suggest large transcriptional burst sizes are correlated with large translational burst sizes. This correlation is confirmed with in vivo E.coli data. Our results demonstrate the link between bursting dynamics and resource sharing.

 
 

Caveney, Patrick M., S. Elizabeth Norred, Charles W. Chin, Jonathan B. Boreyko, Brandon S. Razooky, Scott T. Retterer, C. Patrick Collier, and Michael L. Simpson. "Resource Sharing Controls Gene Expression Bursting." ACS Synthetic Biology (2016).

SEED 2016

I presented a poster titled “Effects of Resource Sharing on Gene Expression Dynamics” at the 2016 Synthetic Biology: Engineering, Evolution & Design (SEED) Conference in Chicago.

Dr. Julius Lucks, Associate Professor at Northwestern University, won the ACS Synthetic Biology Young Investigator Award and presented “Uncovering How RNA Molecules ‘Make Decisions’ On the Fly: Towards Understanding and Engineering CoTranscriptional RNA Folding." This was an amazing lecture about visualizing RNA folding as it is being transcribed. He described their SHAPE-Seq technology to protect RNA from degradation and measure the reactivity with nucleotide resolution. This allowed them to see hairpin loops fold and unfold as the RNA was transcribed.

Watters, Kyle E., et al. "Cotranscriptional folding of a riboswitch at nucleotide resolution." Nature structural & molecular biology 23.12 (2016): 1124.

SEED 2015

I presented a poster titled “Noise in Confined Cell-free Reactions” at the 2015 Synthetic Biology: Engineering, Evolution & Design (SEED) Conference in Boston. We then toured Ginkgo Bioworks. Their labs were beautiful. Every sample is barcoded, and every time a sample is manipulated a record is made. What a wonderful thing!

This was my first time in Boston. I walked around MIT and saw the finish line for the Boston Marathon.

JOVE

March 2015 we open-source published Sealable Femtoliter Chamber Arrays for Cell-free Biology, our experimental setup, in the Journal of Visualized Experiments to allow other researchers to confine reactions in femtoliter chambers. We use this technique to observe cell-free protein synthesis.

SynBioBeta 2014

This week I attended SynBioBeta 2014. This is a biotech/synthetic biology industry conference. I saw presentations from many new biotech startups. This is a rapidly growing space. Many companies were focusing on the foundations needed for a biotech revolution: DNA sequencing, DNA writing (Cambrian Genomics), easy genetic manipulations in organisms (Ginkgo Bioworks). One of my favorite parts was a tour of Cambrian Genomics led by founder Austen Heinz. Austen and his company were inspiring because they were pushing the edge on a shoestring budget. He talked about modifying all kinds of organisms often in playful, but useful ways. He had an almost “we can do this fun thing so why not” attitude. This reminded me of the attitudes of the founders of early computer companies, and it was exciting to see the same attitude in biology.

I also met with researchers at the Gladstone Institute who are frequent collaborators of our group.

This was my first time in San Francisco. The city had a very cool atmosphere and it seemed like everyone was focused on building their own company and releasing the next product.

Caltech Workshop

This week I attended a cell-free synthetic biology workshop at Dr. Richard Murray's lab at Caltech. We learned how they make cell-free extracts for their experiments and made a batch ourselves. Cell-free extracts start with whole cells (in most cases E.coli), remove the cell membranes, centrifuge out genome, and dialyze to remove salts. What is left is a pared down set of proteins that are used to make proteins from any desired gene. Salts, nucleotides, amino acids, and energy molecules are added back to sustain protein synthesis. The goal of their work is to make cell-free systems more predictable and reliable. They mostly use cell-free extract to test genetic circuits before putting them in cells.

Their protocol is published in the Journal of Visualized Experiments (JOVE). Sun, Zachary Z., et al. "Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology.Journal of visualized experiments: JoVE 79 (2013).