Overview

Background

Nitrogen takes up almost 4/5 in earth’s atmosphere, but it cannot be assimilated the similar way plants fix carbon dioxide. Plant productivity has thus been largely circumscribed, which is among major concerns in contemporary agriculture. To ensure plants' growth, chemical nitrogen fertilizers are widely applied with the advances in ammonia synthesis technology. The industrial chemical reduction of nitrogen is considerably energy-consuming, while also leading to greenhouse emissions, and widespread eutrophication of aquatic ecosystems. (Rogers & Oldroyd, 2014) Moreover, applying nitrogen fertilizer, such as carbamide, ammonium nitrate, ammonium sulfate will also destablize the soil micro-structure, and make it hard to sow. (Alvarez, 2005) Chemical fertilizers abuse would eventually lead to low agricultural productivity and malnutrition. (Rogers & Oldroyd, 2014) Biological fixation of nitrogen, on the other hand, is free of above drawbacks, and is a promising eco-friendly way to replace the traditional industrial chemical method.

Azotobacter, the nitrogen-fixing bacteria, plays an important role in symbiotic nitrogen fixation. Some archaea can also fix nitrogen, but they contribute little to overall global biological nitrogen fixation. Therefore, most of the research works are focusing on azotobacter, and can be further divided into three sub-areas (Shen & Jing, 2003): the study of factors affecting fixation efficiency, the study to enlarge azotobacter’s symbiosis range, and the study of azotase. Here, our Prometheus program aims to improve the fixation efficiency.

Metals are key elements of all living organisms, including bacteria and plants (Silava & Williams, 1991) and they are integral parts of 30-50% of a typical cell (Waldrom & Robinson, 2009). Metal, such as Fe, Zn, Cu, Ni involves in many important biological process, including the legume-specific stages of symbiotic nitrogen fixation. (Gonzalez-Guerrero et al, 2014) Molybdenum is also a pivotal part of protein ModA, an essential enzyme for azotobacter to fix nitrogen. The ModA protein was localized to the periplasmic space of the cell, and it was released following a gentle osmotic shock. The N-terminal sequence of ModA confirmed that a leader region of 24 amino acids was removed upon export from the cell. ModA gene product is essential for high affinity molybdate uptake by the cell.

Module 1: Armed with Molybdate

Schematic representation of cell surface display using INP. INPN domain and surface display with only the N-terminal anchoring domain. ModA is shown in orange.

We transferred plasmids into E.coli Prometheus to make it express a special fusion protein: one side of it, INPN, anchors on the outer membrane, and the other side, ModA, absorbs molybdate. The engineered E.coli Prometheus can thus recruit molybdate. With the help of auxin tendency system design by Imperial College 2013, our Prometheus would head for the roots. We also design a suicide clock system for bio-safety concerns. E.coli Prometheus will deliver Mo directly to the root of plants, so the heavy mental pollution of Mo will almost decrease to Zero according to our experiment results. This will also open a new chapter of biological fertilizer.

Module 2: Delivery System

The plants root exudates contain TCA intermediates that can attract chemotaxic bacteria. E.coli have five kinds of chemoreceptors, interacting with factors on flagella and inducing chemotaxis; but they don't have receptors for some specific TCA intermediates. In this respect, Pseudomonas putida can help, with its McpS, like McfR, which can detect malate and succinate. We made the part BBa_K1405004 to include the McfR sequence. We then detected the chemotaxis towards malate and succinate of both of our part BBa_K1405004 and BBa_K515102. At last, we designed a model to mimic the movement pattern and predict the efficiency of the Prometheus E.coli.

Modeling:

We complete our mathematical modeling with the program designed to dynamically and quantitatively simulate the behavior of chemotaxic E.coli towards root surface. Although we didn't make it a real "software" with beautiful user interface, it can still show us a virtual peanut root, based on laser scanning data, and with the concentration gradient of attractant. With this setting, it can simulate a bunch of bacteria's motion, updated every second.

The input items are: 1) the vertices of the root surface based on our laser scanning data, 2) the attractant concentration on the immediate root surface, 3) the spatial gradient function and the number of bacteria. The information of our program can be seen in our wiki (modeling part).

The output is the concrete information of each bacterium per sec in the set time (velocity, direction, position, the surrounding concentration of attractant and number of bacteria reaching one requirements). The result is to be correct and successful for it fit the theoretical curve and the data from experiment well (see our wiki).

It can be used to help a student or professor who needs the data of the concrete information of bacteria every second in a chemotaxis.One run of the program needs 2.5 times of the real time. However, it still deserves to use under an emergency in which is no time to prepare an experiment. It also can avoid the error led by an operation and get robust result which can be trusted.

The one for presentation can show the process above. However, for a fluent running of the program, it decrease the accuracy. It is suitable for a simple scientific presentation.

Kill Switch

We designed a kill switch to control "Prometheus" and to prevent potential contamination.

One difficulty we face is how to trigger the suicide progress spontaneously at a certain time. In the medium, the bacteria are easily controlled by adding or removing regulatory factors. However, after pouring E. coli into soil, it is hard for us to control. The suicide progress needs to be activated spontaneously. Moreover, the kill switch is supposed to be “off” for a certain time in the soil, so the bacteria will gain enough time to perform its function.

Considering the problems, toxin protein MazF is the best candidate for "Prometheus" to suicide with, as well as for us to restrict the bacterial number under reasonable level.

References

Gonzalez-Guerrero, M., Matthiadis, A., Saez, A. & Long, T. A. (2014) Fixating on metals: new insights into the role of metal s in nodulation and symbiotic nitrogen fixation. Plant Science, 45(5), 1-6.

Silva, J. J. R. F., & Williams, R. J. P. (2001). The biological chemistry of the elements: the inorganic chemistry of life. Oxford: London.

Rogers, C. & Oldroyd G. E. (2014) Synthetic biology approaches to engineering the nitrogen symbiosis in cereals. Journal of Experimental Botany, 65(8), 1939–1946.

Shen, S. H. & Jing, Y. X. (2003). The review of nitrogen fixation research and prospect in China [中国生物固氮研究现状和展望]. Chinese Science Bulletin, 48(6), 535-540.

Alvarez, R. (2005). A review of nitrogen fertilizer and conservation tillage effects on soil organic carbon storage. Soil Use and Management, 21(1), 38-52.

The Story of E.coli Prometheus

BNU-China