Team:Marburg/Human Practices/Integrated/Northwestern

Northwestern University

Developing our project, we noticed that cell-free systems have already been around for quite a while and even in the 1980s people started working on some of the first extracts from plant chloroplasts[1]. This now dates more than 40 years back, which is why we started to wonder why nowadays such systems are not very prominent in chloroplast research.
We set out to the academic field and asked experts in cell-free synthetic biology:

"What is the limiting factor for chloroplast extracts to become applicable in plant SynBio?"

We were happy to get the opportunity to talk to Michael Jewett and Lauren Gail Clark of the Jewett lab from Northwestern University in the state of Illinois, USA.
Michael Jewett is a Professor of Chemical and Biological Engineering and Director of the Center for Synthetic Biology at Northwestern University. His lab has done a lot of groundbreaking research on cell-free systems and is one of the most renowned in this field. Lauren Clark is Lab Manager and currently working on a cell-free protein biosynthesis platform based on chloroplast extracts, which makes her the most qualified person to answer our question.

Michael Jewett and Lauren Clark have been incredibly open in sharing their current research with us and could provide crucial insights about prominent issues and aspects to keep in mind when working on cell-free systems. We presented the current state of our project, talked about improvements we can work on to make our system more interesting for researchers and discussed future applications of our system and chloroplast synthetic biology at large.

Getting to the main question of the interview, the limiting factor for chloroplast cell-free systems, Lauren was quick to point us to the problem of endogenous transcription. She elaborated that systems have so far relied on transcription using the T7 promoter, which is known to give high expression levels. However, the key to running and optimizing whole genetic circuits in a chloroplast cell-free system lies in the use of endogenous promoters.
Transcriptional control of genetic circuits was always at the heart of synthetic biology and has been used extensively to realize complex circuit designs in bacterial systems[2]. Without getting this to work in a chloroplast cell-free system, it will barely be possible to prototype larger projects. Apart from the fact that a whole set of distinct regulatory elements would be needed to minimize potential recombination events when implementing larger pathways in the chloroplast, using the T7 promoter in vivo can be quite detrimental due to its high expression.
Furthermore, Michael Jewett further highlighted that in vitro systems of chloroplasts have so far not coupled transcription and translation, but rather delivered on either one of those processes.

Even though at this point we already managed to show a basic proof of concept of both transcription and translation in our chloroplast systems, our next goal became clear: we wanted to further improve the activity, so that we get high-level expression in our extracts using only endogenous parts from chloroplasts.

Our efforts paid off. Further optimizing our experimental procedures, like the lysis of isolated chloroplasts, as well as by screening various concentrations of magnesium and potassium in our reactions, we managed to develop chloroplast cell-free systems from that are fully capable of performing protein biosynthesis using endogenous transcription and translation! So far, we achieved this for extracts from tobacco, spinach and wheat chloroplasts (Figures 1 & 2), but we are optimistic that this can be expanded to other organisms.

To facilitate the creation of functional cell-free systems from chloroplasts of as many other plants as possible, we used machine learning to recognize a consensus sequence in the 16s promoter out of more than 6000 chloroplast genomes. Through this, we identified two distinct types between monocotyledonous and dicotyledonous plants and designed a synthetic hybrid-promoter that was constructed to be used as a universal promoter in chloroplasts.
To read more about these efforts sprouted from our discussion with Lauren Clark, check out our Modelling page!

Figure 1: Optimization of the magnesium concentration for endogenous transcription
Luminescence values are given as arbitrary units and the data is presented on a linear scale. The reaction was set up in a total volume of 10µl. Negative controls using only the plasmid DNA or the crude chloroplast extracts have been included respectively in order to verify the expression
Figure 2: Characterization of endogenous promoter
Luminescence values are given as arbitrary units and the data is presented on a linear scale. The reaction was set up in a total volume of 10µl. Negative controls using only the plasmid DNA or the crude chloroplast extracts have been included respectively in order to verify the expression

Building on our first promising results using endogenous transcription, we decided to further characterize the expression in our system and performed kinetic measurements over time, as well as titration of DNA concentration. Learn more about these results here!

We are incredibly thankful to Michael Jewett and Lauren Clark for taking their time to hold this fruitful discussion with us! The outcome is more than we could have hoped for at the beginning and without their invaluable input and support we would not have managed to achieve what we have now.

Sources

  1. Bard, J., Bourque, D. P., Hildebrand, M., & Zaitlin, D. (1985). In vitro Expression of Chloroplast Genes in Lysates of Higher Plant Chloroplasts. Proceedings of the National Academy of Sciences of the United States of America, 82(12), 3983–3987. http://www.jstor.org/stable/26014
  2. Nielsen AA, Der BS, Shin J, Vaidyanathan P, Paralanov V, Strychalski EA, Ross D, Densmore D, Voigt CA. Genetic circuit design automation. Science. 2016 Apr 1;352(6281):aac7341. doi: 10.1126/science.aac7341. PMID: 27034378.