Sample Research Proposal on Expression of chlorophyll in E coli using synthetic biology

Expression of chlorophyll in E coli using synthetic biology

Just like the name photosynthesis suggests, the leaf uses energy from light to make the food it requires to conduct its internal processes. This research seeks to explore the successful expression of chlorophyll in E. coli so that it can contribute to the production of energy in form of a biosynthetic fuel. The simplified reaction for photosynthesis is represented by

6CO2 (g) + 6H2O (l) (+ light energy) → C6H12O6 (aq) + 6O2  (g)

Aims

The overall aims of the project include:

  • Exploration of ways expressing chlorophyll in E. coli
  • Manipulation of the chlorophyll in E- coli so as to lead to the production of energy as a biosynthetic fuel.

Supporting evidence from previous research

Photosynthesis is a complex process because it involves the use of organic raw materials to go through various stages and come out with energy as the end product. One of the initial raw materials is carbon dioxide, and it is mixed with other raw materials such as water to produce ATP and it is later on broken down through the carbon cycle to produce energy and carbon dioxide as one of the byproducts (Papageorgiu, 2007). For photosynthesis to take place in the leaf, chlorophyll must be present. The chlorophyll is found in the plastid, and it is synthesized in the early stages of leaf manufacture using compounds such as glutamate. The glutamate has to be activated into another form; glutamyl- transfer RNA (tRNA) which has α carboxyl group (Ilag & Kumar, 1994). The process has to go through stages and is catalyzed by glutamyl-tRNA synthetase. The tRNA’s ester bond is reduced to 1-semialdehyde (GSA) which is catalyzed by glutamyl-transfer RNA synthetase via NADPH. The resultant compound: GSA, is then converted to 5-aminolevulinic acid ALA, which is one of the major constituents, used in chlorophyll synthesis. Two molecules of ALA are required and put together to form porphobilinogen via a process catalyzed by porphoblinogen sythetase (ALA dehydratase) (Ashton, 2013). The final product is one of other three that are required to four a tetra figure. The tetra figure is then compacted via a catalyzed process by the enzyme porphobilinogen deaminase; then added to a cofactor resulting in a six figure structure (Feltman, 2013). A re-orientation takes place whereby the outer molecules are re-oriented to generate the molecule protoporphyrin IX. The first steps leading up to the synthesis of chlorophyll involve soluble proteins. The latter steps of this important process involve a higher number of hydrophobic proteins. The latter step involves integration of Magnesium ions, which makes chlorophyll synthesis unique from the manufacture of other tetrapyrroles (Masuda, 2013). Porphobilinogen is mixed with a cofactor to form an intermediate. The resultant hydroxymethylbilane is converted to uroporphyrinogen via a reaction that is catalyzed by urporphyrinogen III synthetase. This very important enzyme would have to be present for the chlorophyll synthesis process to be considered successful (Jensen, 1999). This is because the enzyme converts the inactive uroporphyrinogen I to uroporphyrinogen III via the inversion of existing pyrrole rings; half of which are isomer III compounds. The uroporphyrinogen III is then converted to protoporphyrinogen IX, a reaction that is catalyzed by coproporphyrinogen oxidase. The protoporphyrinogen IX is converted to protophyrin IX via a reaction that is catalyzed by protoporphyrinogen oxidase (Torella, et al, 2013).

All the 27 genes and 15 enzymes involved in the formation of chlorophyll are known. Therefore, this process; though time consuming and requiring very special attention and delicate care can be replicated under controlled laboratory conditions.

Background to project and Rationale

Known content from this area

Various studies have been carried out in the synthesis of E-coli in the laboratory, and the expression of chlorophyll. Synthesis of chlorophyll is complex because of the many enzymes and genes that are involved. However, all the genes and enzymes have been fully identified and therefore, they can be brought together and manipulated so that they are successfully expressed E-coli, for mass energy production.

Photosynthesis is a complex process because it involves the use of organic raw materials to go through various stages and come out with energy as the end product.

Supporting evidence from published research

According to Willows (2004), one of the greatest features that sets chlorophyll apart from other tetrapyrroles is an isocyclic ring in the fifth position of is structure, and magnesium. Other tetrapyrroles have vitamins and compounds such as heme in their structure. There are several types of chlorophyll but the major ones are, a and b. Chlorophylls absorb red light from the sunlight which gives them a green color. The ability of the chlorophyll to absorb sunlight is fundamental in ensuring that it is available for the photosynthesis process. Plants that are fully exposed to sunlight have more chlorophyll a and b in comparison to plants that are not fully exposed to sunlight. Instead, such plants have a high quantity of compounds referred to as light harvesting complexes. According to Willows (2006), algae and other types of bacteria that have photosynthetic systems have different types of chlorophylls that assist them in carrying out photosynthesis. The reason why this project would be successful is that it is possible to express chlorophyll in E- coli. For leaves, the genomes used in chlorophyll synthesis are well known and can therefore be manipulated in the laboratory with E- coli. E- coli is similar to α proteobacteria which makes use of carbohydrate compounds like succinate and glycine to synthesize ALA used in the initial steps of chlorophyll manufacture (Willows, 2004).

Non eukaryotes such as proteobacteria are similar to E- coli and therefore, similar raw materials and conditions for chlorophyll synthesis can be copied to achieve similar final products. The various types of E-coli that can be manufactured in the laboratory are not disease causing and are therefore easy to work with.

Synthetic fuel production involves the use of raw materials, such as carbon monoxide and hydrogen to produce a liquid type of gas. Synthetic fuel is beneficial for use because it does not contain harmful products such as aromatics and sulfur compounds (Witkowski et al, 1999).

Background to the project

With an increase in globalization, there is bound to be an increase of population in the world today. An increase in population is bound to be matched by an increase in energy needs in the world. There is already uproar from health and environmental groups on the increased use of fuel as a source of energy (Harvey, Wright & Quintana, 2009). Use of carbon fuels continues to contribute to an increase in greenhouse gases, which in turn contribute to an increase in global warming. Alternative forms of sources of energy need to be aggressively sought and pursued so that future generations can live under healthy conditions that will have been maintained by the current generation. According to the Joint Bioenergy Institute (2011), organizations all over the world have already taken initiatives to pursue viable and alternative sources of energy that provide long term benefits in the energy industry.

Why this project is interesting and my commitment to doing it

I want to conduct this research so that I can contribute to the scientific and environmental friendly production of energy. So far, the enzymes that are used in the characterization of both chlorophyll and bacteria chlorophyll have been discovered through genetic analysis processes (Willows, 2003). One of the most important steps in the synthesis of Magnesium is the one that involves magnesium chelatase whose insertion after protoporhyrin IX is similar to that of Iron ions Fe2+. The Fe2+ insertion is catalyzed by ferrochelatase and does not involve any cofactors. Mg2+ insertion requires the use of ATP and several protein components specifically: Bchl, BchD and BchH. This is a requirement in the flora that manufacture bacteriochlorophylls with additional Ch1l, ChlD and Ch1H. The step that requires the insertion of Mg2+ might sound complex but the advantage is that the sequences of the genes required are well known. Initially, the studies that were conducted on R.capsulatus and R. sphaeraoides (Willows, 2003).

Methodology

Achievement of Objectives and Aims of the research

The aims and objectives of the study will be achieved practically via laboratory investigations. The process of transferring chlorophyll to coli is expected to be expensive since the laboratory equipment needed in the isolation and provision of favorable conditions is not found in simple laboratories. Besides the researcher, another laboratory employee should also be employed to assist in monitoring and processing of the biosynthetic fuel. The staff members that are employed to assist in maintenance of the said conditions require to have special and advanced training in the area if the project is to work and have the needed conditions maintained (Witkowski et al, 1999).

Flow diagram to show experimental approach

 

 

Milestones for the proposal

1-2 weeks Biosynthesis of E-coli

4 weeks- 8 weeks: Development of chlorophyll

2-4 weeks Expression of chlorophyll in E-coli

The timelines that are given have allowances so that there can be sufficient time to manipulate the genome of the organisms and view its expression in the final intended subject: e-coli.

Significance & Expected Outcomes

Planned outcomes and their impact

The researcher plans on being able to successfully express chlorophyll in E-coli so as to achieve a viable synthetic bio-fuel production.

The biosynthetic fuel produced is expected to cut down government expenditure by over 50% as a result of savings on fuel costs. The current expenditure on fuel is too high and the costs keep on fluctuating, which has a negative effect on the country’s and world industries (Stephen, Wong, Garcia, Keasling and Peralta-Yahya, 2014). The successful execution and completion of this project offers an alternative through which industries, companies and individuals can use more efficient and cheaper sources of energy for their important uses (Wyss Institute for Biologically Inspired Engineering at Harvard, 2013).

Significance of the research and its importance in addressing an important problem

This research is significant because it involves a renewable source of energy. If successful, the E-coli could be used for mass energy production. In the past, E-coli have been used in the production of insulin for diabetes, and it has been a large success. With an increase in the number of patients who have diabetes, it is important for the use of E-coli to continue being used. In the same manner, E-coli should be utilized for the production of biosynthetic energy (Sengodon, Steen & Hays 2013).

The biosynthetic fuel that is produced has a clear color because it does not contain impurities such as sulfur and other aromatics that contribute to an increase in greenhouse gases in the atmosphere. If large amounts of E-Coli can be manipulated so as to express chlorophyll, large companies can utilize the process for energy production which would also contribute to cutting of economic costs (Phelan, Sekurova,Keasling &  Zotchev, 2014). With increased globalization, nations such as Australia should ensure that it is in the forefront in being efficient as far as expenses and mass production are concerned. The nation wants to be at the top when being benchmarked against other countries. Energy use and efficiency is a top concern that should be looked into if a nation is to achieve high profits, reduce greenhouse gases emission and create employment through science and technology (Zepeck, 2005).

Innovativeness and novelty of project aims and concepts

My project aims and concepts are innovative and novel because they have not been used in the past. Expression of chlorophyll so that it can be synthesized has been though and talked about, but it has never been expressed in E-coli. If successful, this novel idea of expressing chlorophyll and E-coli could go a long way in ensuring production of large amounts of sustainable energy.

References

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Jensen, P., Gibson, L., Shephard, F., Smith, V. & Hunter, C.  (1999). Introduction of a new

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