Overview

Background

Nitrogen takes up almost 4/5 in earth’s atmosphere. However, it cannot be assimilated as carbon dioxide by plants. Plant productivity has been circumvented in contemporary agriculture by this limitation. In order to break the limitation, chemical nitrogen fertilizers are applied with the invention of ammonia synthesis technology. The chemical reduction process of nitrogen consumes large amounts of energy and the reactive nitrogen released into the environment leads to greenhouse emissions, widespread eutrophication of aquatic ecosystems. (Rogers & Oldroyd, 2014) Moreover, the application nitrogen fertilizer, such as carbamide, ammonium nitrate, ammonium sulfate will also make the soil crust and hard to sow. (Alvarez, 2005) Chemical fertilizers will also lead to low agricultural productivity and malnutrition. (Rogers & Oldroyd, 2014) Biological fixation of nitrogen does not have these disadvantages and it is a promising eco-friendly way to replace the traditional industrial synthetic method.

Azotobacter is also called nitrogen-fixing bacteria, which plays an important role in symbiotic nitrogen fixation. Although some archaea can also fix nitrogen, they only make up a small percentage of biological nitrogen-fixing. Hence, most of the research works focus on azotobacter and the study it can be divided into three sub-areas (Shen & Jing, 2003), which are the study of the factors that affect the efficiency of nitrogen fixing of azotobacter, the study of enlarging azotobacter’s symbiosis range and the study of azotase.

The Prometheus program aims at improving the efficiency of nitrogen fixing via the first sub-area. Metals are key elements of all living organisms, including bacteria and plants (Silava & Williams, 1991) and they are an integral part 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 an integral part of protein ModA, which is 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.

A recombinant plasmid was transfer into E.coli Prometheus to make it express a special fusion protein, one side of which anchors on the outer membrane and the other side of which catches molybdate. The engineered E.coli Prometheus can bind molybdate and with the help of auxin tendency system design by Imperial College 2013, our Prometheus will head for the roots. A suicide system depending on time is also design by us for the reason of bio-safety. 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 bacteria having the ability of chemotaxis. E.coli has five kinds of chemoreceptors, which interact with factors of the flagella that leads to chemotaxis. But E.coli doesn’t have specific chemotaxis towards some TCA intermediates while Pseudomonas putida has some McpS, like McfR. We made a part BBa_K1405004 containing the sequence of McfR, which detects malate and succinate. Then we 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:

Our mathematical model was completed by the program simulating the process of E.coli’s chemotaxis towards root surface dynamically and quantitively. Although it has not been designed to be a real “software” with beautiful user interface, it can show a virtual peanut root with the concentration gradient of attractant and a bunch of bacteria’s motion updated per second.

The input items are the vertexes of the root surface (or other things), which can be got from laser scanning (as we did), the concentration on the surface, 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