Difference between revisions of "Team:Shiyan SY China/Design"

Faced with the stress effects human pollution such as pesticides has on environment, nature has evolved many methods to deal with these problems. For example, many natural micro-organisms have the characteristic of degrading organophosphorus pesticides. Currently the micro-organisms which have been found being capable to degrade organophosphorus pesticides include bacteria, fungus, actinomycete and alga. As the research goes further, people find that these degrading bacteria degrade pesticides by secreting a kind of enzyme which can hydrolyze phosphaester bonds-organophosphorus-degradation enzyme. Because each organophosphorus pesticide has similar structure and is only different in substituent groups, one kind of organophosphorus-degradation enzyme can always degrade multiple kinds of organophosphorus pesticides. Organophosphorus-degradation enzyme has been widely acknowledged to be the most potential new method to eliminate pesticide residues currently. At present, many enzymes have been identified to be used to degrade organophosphate pesticides. Among these enzymes, organophosphorus-degradation enzyme (opdA) which comes from Agrobacterium radiobacter P230 of radioactive agrobactium tumefaciens genus has wider substrate and higher enzyme-catalyst efficiency. In recent years, the research on the structure and function of organophosphorus-degradation enzyme has gained relatively big development and comes into the molecule level, which makes it possible to improve the properties of organophosphorus-degradation enzyme through genetic engineering and protein engineering and invent organophosphorus-degradation enzyme products which meet requirements of different application fields.

The organophosphorus-degradation enzyme (opdA) gene opda （NCBI genbank：Accession: AY043245.2） programmed by Agrobacterium radiobacter bacteria contains 1 155 basic groups in total and programs 384 amino acid residues. The front end of protein sequence is signal peptide sequence while the back end is degradation-enzyme sequence. The nucleic acid sequence and amino acid sequence are as follows:

at gcaaacgaga agagatgcac ttaagtctgc ggccgcaata actctgctcg gcggcttggc tgggtgtgca agcatggccc gaccaatcgg tacaggcgat ctgattaata ctgttcgcgg ccccattcca gtttcggaag cgggcttcac actgacccat gagcatatct gcggcagttc ggcgggattc ctacgtgcgt ggccggagtt tttcggtagc cgcaaagctc tagcggaaaa ggctgtgaga ggattacgcc atgccagatc ggctggcgtg caaaccatcg tcgatgtgtc gactttcgat atcggtcgtg acgtccgttt attggccgaa gtttcgcggg ccgccgacgt gcatatcgtg gcggcgactg gcttatggtt cgacccgcca ctttcaatgc gaatgcgcag cgtcgaagaa ctgacccagt tcttcctgcg tgaaatccaa catggcatcg aagacaccgg tattagggcg ggcattatca aggtcgcgac cacagggaag gcgaccccct ttcaagagtt ggtgttaaag gcagccgcgc gggccagctt ggccaccggt gttccggtaa ccactcacac gtcagcaagt cagcgcgatg gcgagcagca ggcagccata tttgaatccg aaggtttgag cccctcacgg gtttgtatcg gtcacagcga tgatactgac gatttgagct acctaaccgg cctcgctgcg cgcggatacc tcgtcggttt agatcgcatg ccgtacagtg cgattggtct agaaggcaat gcgagtgcat tagcgctctt tggtactcgg tcgtggcaaa caagggctct cttgatcaag gcgctcatcg accgaggcta caaggatcga atcctcgtct cccatgactg gctgttcggg ttttcgagct atgtcacgaa catcatggac gtaatggatc gcataaaccc agatggaatg gccttcgtcc ctctgagagt gatcccattc ctacgagaga agggcgtccc gccggaaacg ctagcaggcg taaccgtggc caatcccgcg cggttcttgt caccgaccgt gcgggccgtc gtgacacgat ctgaaacttc ccgccctgcc gcgcctattc cccgtcaaga taccgaacga tga

MQTRRDALKSAAAITLLGGLAGCASMARPIGTGDLINTVRGPIPVSEAGFTLTHEHICGSSAGFLRAWPEFFGSRKALAEKAVRGLRHARSAGVQTIVDVSTFDIGRDVRLLAEVSRAADVHIVAATGLWFDPPLSMRMRSVEELTQFFLREIQHGIEDTGIRAGIIKVATTGKATPFQELVLKAAARASLATGVPVTTHTSASQRDGEQQAAIFESEGLSPSRVCIGHSDDTDDLSYLTGLAARGYLVGLDRMPYSAIGLEGNASALALFGTRSWQTRALLIKALIDRGYKDRILVSHDWLFGFSSYVTNIMDVMDRINPDGMAFVPLRVIPFLREKGVPPETLAGVTVANPARFLSPTVRAVVTRSETSRPAAPIPRQDTER

at gcaaacgaga agagatgcac ttaagtctgc ggccgcaata actctgctcg gcggcttggc tgggtgtgca agcatggccc gaccaatcgg tacaggcgat ctgattaata ctgttcgcgg ccccattcca gtttcggaag cgggcttcac actgacccat gagcatatct gcggcagttc ggcgggattc ctacgtgcgt ggccggagtt tttcggtagc cgcaaagctc tagcggaaaa ggctgtgaga ggattacgcc atgccagatc ggctggcgtg caaaccatcg tcgatgtgtc gactttcgat atcggtcgtg acgtccgttt attggccgaa gtttcgcggg ccgccgacgt gcatatcgtg gcggcgactg gcttatggtt cgacccgcca ctttcaatgc gaatgcgcag cgtcgaagaa ctgacccagt tcttcctgcg tgaaatccaa catggcatcg aagacaccgg tattagggcg ggcattatca aggtcgcgac cacagggaag gcgaccccct ttcaagagtt ggtgttaaag gcagccgcgc gggccagctt ggccaccggt gttccggtaa ccactcacac gtcagcaagt cagcgcgatg gcgagcagca ggcagccata tttgaatccg aaggtttgag cccctcacgg gtttgtatcg gtcacagcga tgatactgac gatttgagct acctaaccgg cctcgctgcg cgcggatacc tcgtcggttt agatcgcatg ccgtacagtg cgattggtct agaaggcaat gcgagtgcat tagcgctctt tggtactcgg tcgtggcaaa caagggctct cttgatcaag hgcgctcatcg accgaggcta caaggatcga atcctcgtct cccatgactg gctgttcggg ttttcgagct atgtcacgaa catcatggac gtaatggatc gcataaaccc agatggaatg gccttcgtcc ctctgagagt gatcccattc ctacgagaga agggcgtccc gccggaaacg ctagcaggcg taaccgtggc caatcccgcg cggttcttgt caccgaccgt gcgggccgtc gtgacacgat ctgaaacttc ccgccctgcc gcgcctattc cccgtcaaga taccgaacga tga

MQTRRDALKSAAAITLLGGLAGCASMARPIGTGDLINTVRGPIPVSEAGFTLTHEHICGSSAGFLRAWPEFFGSRKALAEKAVRGLRHARSAGVQTIVDVSTFDIGRDVRLLAEVSRAADVHIVAATGLWFDPPLSMRMRSVEELTQFFLREIQHGIEDTGIRAGIIKVATTGKATPFQELVLKAAARASLATGVPVTTHTSASQRDGEQQAAIFESEGLSPSRVCIGHSDDTDDLSYLTGLAARGYLVGLDRMPYSAIGLEGNASALALFGTRSWQTRALLIKALIDRGYKDRILVSHDWLFGFSSYVTNIMDVMDRINPDGMAFVPLRVIPFLREKGVPPETLAGVTVANPARFLSPTVRAVVTRSETSRPAAPIPRQDTER

In this project, we will use colibacillus to construct genetically engineered bacteria which can secrete organophosphorus-degradation enzyme opdA protein, to achieve the biodegradation of organophosphorus residues and eliminate the pollution by pesticides.

Genetically engineered bacteria are bacteria which can channel target gene into bacteria to express the genes and produce required protein. These bacteria which are equipped with new inheritable characters given by humans are called genetically engineered bacteria.

Currently, the genetically engineered bacteria which are used most widely all over the world are still colibacillus because colibacillus have explicit genetic background, grow fast, have no resistance toward most antibiotics, are easy to be controlled in growth and activeness, are easy to be used in any production magnitude from laboratory production to industry production. (For example: scientists introduce human insulin gene into colibacillus cells and combine insulin gene with genetic materials of colibacillus. Human insulin gene directs colibacillus to product human insulin in colibacillus cells. As they reproduce, the insulin gene also gets transmitted down generation after generation and colibacillus of later generations can also produce insulin. The genetically engineered bacteria equipped with human insulin gene are put into large fermentor, which can provide them with appropriate conditions and nutrients, to be cultured manually. They can reproduce in large populations and produce large amount of human insulin. The colibacillus have become the “live factory” to produce insulin. In 1981, human insulin gene products were put into market and solved the problem of lack of insulin sources).

An RNA thermometer (or RNA thermosensor) is a temperature-sensitive non-coding RNA molecule which regulates gene expression. RNA thermometers often regulate genes required during either a heat shock or cold shock response. In general, RNA thermometers operate by changing their secondary structure in response to temperature fluctuations. This structural transition can then expose or occlude important regions of RNA such as a ribosome binding site, which then affects the translation rate of a nearby protein-coding gene.

Below is a schematic diagram of Thermometer RNA:

In this project, the Thermometer RNA we use has a sensitive temperature of 32℃, which means in environment above 32℃, RNA translation process can be started. Its DNA sequence is as follows:

Ccgggcgcccttcgggggcccggcggagacgggcgccggaggtgtccgacgcctgctcgtccagtctttgctcagtggaggattactag

In this project, opdA enzyme which is expressed by genetic engineering still cannot be directly used in degradation of organophosphorus pesticides because colibacillus genetically engineered bacteria can only express genetically engineered protein designed by humans in cell membranes, which will cause expressed opdA enzyme protein to accumulate in large amounts inside the genetically engineered bacteria and thus the protein cannot contact with external organophosphorus pesticides to accomplish the degradation process and poisons genetically engineered bacteria. Therefore, we need to come up with methods to transfer the expressed opdA protein outside the genetically engineered bacteria. We need the structure of signal peptide to help in this process.

Signal peptide often refers to N-end amino acid sequence which is used to direct the trans-membrane process (orientation) in newly compounded peptide chains. There is a segment of RNA area programming hydrophobi amino acid sequence which generally lies after initiation codon. This amino acid sequence is called signal peptide sequence, which takes charge of introducing protein into sub-cellular organelles which contain different membrane structures. If the function of this signal peptide is to introduce and secrete compounded protein outside the cell walls, this signal peptide is called secretion signal peptide.

As we know, on natural conditions, ompA which exists inside Agrobacterium radiobacter bacteria has its own signal peptide. However, this original signal peptide is only used by Agrobacterium radiobacter and is not necessarily useful in colibacillus genetically engineered bacteria. Therefore, we need to select some signal peptides which can be used by colibacillus, to achieve our plan.

The ompA signal peptide used in this project is one of the secretion signal peptides which are used most widely in colibacillus genetically engineered bacteria and can lead to expressing its downstream protein molecules to transfer across cell walls of host colibacillus and secrete target protein outside the host bacteria. The DNA sequence of this signal peptide is as follows:

Atgaaaaaaaccgctatcgcgatcgcagttgcactggctggtttcgctaccgttgcgcaggcc

In this project, we cannot only consider how to use genetically engineered bacteria to eliminate organophosphorus. Thus, after organophosphorus is eliminated, how should we deal with the rest genetically engineered bacteria? This is what we must pay attention to. Genetically engineered bacteria, after all, come from colibacillus. We don’t want to see it directly released into environment or continued to remain on the surface of fruits and vegetables, thus we must design a method or equipment to eliminate them. Consequently, we decide to design a suicide gene in our genetically engineered bacteria, which is not expressed commonly and only expresses suicide protein in special environments to eliminate the bacteria and avoid “secondary pollution” problems caused by genetically engineered bacteria.

In this project, we select suicide gene ccdb, ccdB is a currently known toxin system, which exists in pathogenic colibacillus F plasmid. The ccdB gene programs a kind of toxin protein CcdB. On conditions where there is a lack of antitoxin, CcdB poisons gyrase inside cells to interfere with the synthesis of DNA and damage host cells. The ccdb sequence we use in this project is as follows:

Atgcagtttaaggtttacacctataaaagagagagccgttatcgtctgtttgtggatgtacagagtgatattattgacacgcccgggcgacggatggtgatccccctggccagtgcacgtctgctgtcagataaagtctcccgtgaactttacccggtggtgcatatcggggatgaaagctggcgcatgatgaccaccgatatggccagtgtgccggtctccgttatcggggaagaagtggctgatctcagccaccgcgaaaatgacatcaaaaacgccattaacctgatgttctggggaatataa

In the project we chose a kind of RecA(SOS) promoter to control to suicide gene ccdB to express. It works because when the bacteria receive ultraviolet rays, DNA get injured so that the bacteria will guide RecA repairing protein’s expression. The protein can start RecA(SOS) promoter to drive the lower ccdB suicide gene’s transcription. In this way, we can control our engineering bacteria by eliminating them with ultraviolet rays to avoid secondary pollution.

The ccdb sequence we use in this project is as follows:

Aacaatttctacaaaacacttgatactgtatgagcatacagtataattgcttcaacagaacatattgactatccggtattacccggcatgacaggagtaaaaatggctatcgacgaaaacaaacagaaagcgttggcggcagcactgggccagattgagaaacaatttggtaaaggctccatcatgtaataa

(1)The strong initiation sequence used in this project referred to BB_J23106 from igem

(2)The RNA thermometer sequence used in this project referred to BB_K115017 from igem

(3)The organophosphorus-degradation enzyme opdA gene sequence used in this project referred to BB_K21509, BB_K215091 and BB_K1010008 from igem

(4)The repair enzyme inducement initiation codon RecA (SOS) sequence used in this project referred to BB_J22106 from igem

(5)The suicide gene ccdB sequence used in this project referred to BB_K145151 and BB_K1010007 from igem

(6)The ompA signal peptide sequence used in this project referred to sequence with the code AJ617284.1 from GenBank