Chloroplast Cell-Free Systems

The Chloroplast Cell-Free Systems - Design

New transplastomic plants are much needed, but transformation is labour- and resource-intensive. Achieving homoplasmy after successful transformation takes months, making the process time-consuming as well. While researching simplified and time-saving alternatives, we encountered cell-free systems. Cell-free systems are in-vitro tools that are used as prototyping platforms for metabolic networks and genetic constructs. Typically, they are based on crude cell extracts and contain the whole machinery needed for protein biosynthesis. These advances in cell-free systems offer exciting opportunities to fundamentally transform synthetic biology. It enables new approaches for model-driven design of synthetic gene networks, rapid and portable acquisition of targeted components, on-demand biomanufacturing, and building cells from scratch.

Figure 1: Thawed cell-free extracts ready for use in measurements.

For more information on the Design of our Project:

Read more
Figure 2: Botanical illustration of spinach.

Plants That We Worked With

Neither the idea of chloroplast engineering nor prototyping in a cell-free context is a novel approach. Lauren Clark is the lab manager of the Jewett lab at Northwestern University and is responsible for cell-free extracts of Nicotiana tabacum. Lauren Clark's research provides the backbone of our project.
With our project, we achieved our goal of successfully developing cell-free systems for chloroplasts of various agricultural crops and fully sequenced model plants.
Our decision fell on the model organisms Tobacco (Nicotiana tabacum), Spinach (Spinacia oleracea), as well as on Arabidopsis (Arabidopsis thaliana). We also decided to focus on highly relevant crops, such as Maize (Zea mays B73), Wheat (Triticum aestivum) and Rice (Oryza sativa), as well as other plants that play a major role in world nutrition like Soybean (Glycine max), Canola (Brassica napus) and Tomato (Solanum lycopersicum).
We chose the Pedunculate Oak as it is strongly represented in Marburg's forests and is to showcase the ability of our project to be applied for chassis rarely used in Synbio Projects.

For more information about the plants we used:

Read more

Learn more about our Measurement OpenPlast System!

Read more

Troubleshooting The Chloroplast Extracts

Protocols for the isolation and lysis of intact chloroplasts from the Jewett Lab at Northwestern University in Evanston served as a basis for the design of our project Openplast. However, since our idea was to prototype more chloroplast cell-free systems than Tobacco, including non-model organisms, it was inevitable to adopt modifications to the basic Tobacco protocol according to the needs of different species. The input we received from interviews that we conducted with experts in plant physiology and cell-free systems made us consider several factors for modifying the isolation and preparation procedure.

Figure 3: A density gradient used for the extraction of intact chloroplasts. In the lower band, intact chloroplasts are collected. In the upper band and above, broken chloroplasts and other cell debris are found.

Having environmental preservation in mind, we first considered factors that could negatively impact the ecosystem. Therefore, we tested whether the absence of environmentally harmful substances commonly used in isolation buffers, such as β-mercaptoethanol show effects on the expressions of our extracts. Moreover, with plastic being one of the most persistent pollutants on earth, it was also assessed whether there were any effects from the reuse of laboratory plasticware on the efficiency of our system.

We were motivated to make our methods available worldwide, including laboratories with limited budgets and self-funding iGEM teams. Therefore, it was in our greatest interest to test cost-effective alternatives to certain chemicals needed for the chloroplast isolation protocol. To achieve this goal, we explored substituting expensive resources needed for the creation of a chloroplast cell-free system with affordable alternatives.

Apart from environmental and funding constraints, we optimized the protocol to address the problems that arose from handling the plant material, such as high starch levels that disturbed the gradients or tested long-term storage possibilities. We examined different variations in methods such as the duration of dark incubation times, concentrations of centrifugation gradients, or storage temperatures.

To evaluate the efficiency of our system, the amount of chloroplast isolate and lysed extract yield was evaluated, but the final expression of luminescence was the deciding factor. Thus, optimizations were also made in the area of measurements. The variations of concentrations of essential factors used in translational buffers, as well as the variation of quantity of total reaction volume played a major role.

Find out more about the Troubleshooting:

Read more
Figure 4: Fluorescent image for chloroplasts of oak.

Biolistic Chloroplast Transformation of N. tabacum

Our cell-free chloroplast extract allows the comparison of genetic parts towards their effect on protein expression level without the need for prior transformation. This opens up the possibility of efficient and cheap high-throughput testing of different constructs and even the potential for multiplexing. However, results of this in vitro part analysis are only of value, if the produced results are comparable with the effect the part will have on protein production of actually transformed plants in vivo. To show that results in our cell-free systems are comparable to data gathered in vivo, we set out to transform the Tobacco chloroplast with some of our constructs. The goal was to measure the same set of genetic parts in vivo and in vitro to show that our system can give valuable insights into the living system. Therefore, we set out to transform N. tabacum chloroplasts.

For more information about the Tobacco chloroplast transformation:

Read more

Check out our in vivo transformation diary with plenty of pictures!

Read more

Also check out our in vivo Tobacco transformation gallery!

Read more