Team:SKLMT-China/Project Overview
Motivation
Soil, you may feel very common and even a little inconspicuous, but as a resource that is difficult to regenerate, its importance is irreplaceable and self-evident. As the main area of human activities on the surface of the earth and natural filtration devices, land naturally bears most of the pollutants. In China, more than one-tenth of the cultivated land is polluted. Soil pollution has seriously affected the sustainable development of food safety and agriculture, and has become a major obstacle to solving the world food problem. From January 1, 2019, China will officially implement the Law on Prevention and Control of Soil Pollution, and the issue of soil pollution is receiving more and more attention.
According to China's demonstration project, the average cost of conventional restoration methods for cultivated land restoration is 75 million yuan/km2, and the minimum cost is 45 million yuan/km2. The repair cycle is based on 15 years, and it is conservatively estimated to be governed by 2020. The capital requirement for repairing 6667 km2 of cultivated land is 66.7 billion yuan.
Conventional repair methods are costly, so the application of synthetic biology methods gives microbes good degradation characteristics, making bioremediation an economical and effective option. However, in the existing research, E. coli is the main research substrate. The large intestine is an animal-inhabited bacteria that is difficult to proliferate in a natural environment with varying conditions. Therefore, we intend to transform a new microorganism as a site for environmental restoration, Pseudomonas fluorescens pf-5.
Here are the reasons why we chose P. fluorescens pf-5
First, it belongs to the genus Pseudomonas.The members of the genus demonstrate a great deal of metabolic diversity and consequently are able to colonize a wide range of niches. These give it an incredible ability of bioremediation. P. Pf-5 has this unparalleled advantage in heterologous expression.
Second, many of the representatives of the genus Pseudomonas are pathogenic, such as Pseudomonas aeruginosa, while P. pf-5 is not pathogenic to mammals and has passed the acute toxicity test of the Ministry of Agriculture of China. It can be released commercially or directly into the field. Moreover, it is a plant-promoting bacterium that combines with green plants to further enhance its bioremediation ability.
Finally, P. pf-5 is a model strain with a clear genetic background for genetic modification.
Promoter library
When compared to platform organisms such as Escherichia coli, the toolkit for engineering P. fluorescens isunderdeveloped. Heterologousgene expressionin particularis problematic.It is vital that a flexible set of molecular genetic tools be available. Inotherorganisms, tuningprotein expressioncan beveryimportant forachievingincreasedyieldsfrom engineeredpathways, but thereareno well-characterizedpromoterlibraries in P. fluorescenstoenable rational tuningof proteinexpression.
To improve the P. pf-5 toolkit, we organised a library of native promoters (n=25). We used Luciferase Reporter Assay Kit to characterize the strength of each promoter. Promoter strength prediction in pf-5 was then achieved by mathematical modeling.
Nicotine degradation engineered bacteria
In addition to the formation of the promoter library, we also constructed a nicotine-degrading engineered bacteria using pf-5 as the chassis.
These days everyone knows what smoking cigarettes does to our bodies, but the knowledge of what smoking does to the Earth is not as common. The pollution caused by cigarettes does not stop in our bodies or the air; it also affects the land we live on and the water that we drink.
Cigarettes contain over 4000 chemicals, like countless toxic tea bags, which are exhaled and released into the air and the atmosphere.Nicotineis one of the most harmful ingredients in cigarette and is the biggest resistance to smoking cessation.
In 1994, the US Environmental Protection Agency defined nicotine as "toxic hazardous waste."
The 2013 review suggests that the lower limit causing fatal outcomes is 500–1000 mg of ingested nicotine, corresponding to 6.5–13 mg/kg orally. An accidental ingestion of only 6 mg may be lethal to children.
Besides toxic to humans, nicotine is also very soluble in water. This makes nicotine easy to seep into soils and groundwater and cause serious pollution.
Moreover, nicotine relased in the environment can damage the normal soil microbial communitty and affects the survival of pollinators, such as bees. These can have far-reaching effects on the ecological environment.
There are several bacteria can degrade nicotine which include Pseudomonas Putita S16. we decided to clone the nicotine degradation gene cluster (~30kb) and express it in our P.pf-5 chassis.
Because if the entire metabolic pathway is introduced into Pseudomonas fluorescens, it is a huge burden on the expression vector, and the product of the first half of the pathway is 2,5-DHP, which can be used as a precursor of various drugs and can be placed in the environment. Turns waste nicotine into treasure.
Furthermore, we used promoter with different strength to control the expression of key enzyme, NicA2.
Red/ET Recombineering
Driven by the needs of functional genomics, DNA engineering by homologous recombination in Escherichia coli has emerged as a major addition to existing technologies. Two alternative approaches, RecA-dependent engineering and ET recombination, allow a wide variety of DNA modifications, including somewhich are virtually impossible by conventional methods. These approaches donot rely on the presence of suitable restriction sites and can be used to insert,delete or substitute DNA sequences at any desired position on a targetmolecule. Furthermore, ET recombination can be used for direct subcloningand cloning of DNA sequences from complex mixtures, including bacterialartificial chromosomes and genomic DNA preparations. The strategiesreviewed in this article are applicable to modification of DNA molecules of anysize, including very large ones, and present powerful new avenues for DNAmanipulation in general.
In conclusion, we constructed a promoter library in Pseudomonas fluorescens pf-5 and used a mathematical model and the resulting data to achieve promoter strength prediction. In order to further verify that our promoter can function well in the fluorescent chassis, we constructed a nicotine-degrading engineered strain and used different strength promoters to regulate the expression of key enzymes to achieve optimal degradation efficiency. In addition, we have introduced a new method of gene recombination for iGEM, Red/ET recombination system, which can more easily realize direct cloning of large fragments, multi-fragment assembly and seamless modification of DNA sequences.