Difference between revisions of "Team:LN-Shiyan-China/Description"
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| + | <p>1) Micro-organisms degrades organophosphorus, and organophosphorus-degradation enzyme opdA | ||
| + | Faced with the stress of human pollution such as pesticides, nature itself has evolved many methods to deal with these problems. For example, many natural micro-organisms contain enzymes to degrade organophosphorus pesticides. Currently the micro-organisms which are capable to degrade organophosphorus pesticides include bacteria, fungus, actinomycete and alga. As the research goes further, people find that these degrading effects come from secreting an enzyme, which can hydrolyze phosphoester bonds, organophosphorus degradation enzyme. Because each organophosphorus pesticide has similar structure and protein sequence, one kind of organophosphorus degradation enzyme is capable todegrade multiple kinds of organophosphorus pesticides. Organophosphorus-degradation enzyme has been mostly recognized as the best method to eliminate pesticide residues currently. At present, many enzymes have been identified to be used to degrade organophosphate pesticides. Among these enzymes, the organophosphorus-degradation enzyme (opdA) which comes from Agrobacterium radiobacter P230 has wider targets and higher enzyme-catalyst efficiency. In recent years, the research on the structure and function of organophosphorus-degradation enzyme has gained promising progress,. Thus, it is possible to improve the properties of organophosphorus-degradation enzyme through genetic engineering and protein engineering method, which meet requirements of different applications. | ||
| + | 2) In this project, we will use organophosphorus-degradation enzyme opdA to eliminate residual organophosphorus pesticide on fruits and vegetables. | ||
| + | The organophosphorus-degradation enzyme (opdA) gene opdA （NCBI genbank：Accession: AY043245.2） programmed by Agrobacterium radiobacter contains 1,155 nucleic acids, programming 384 amino acid residues. The N-terminal of protein sequence is the signal peptide while the C-terminal is the degradation-enzyme sequence. The nucleic acid sequence and amino acid sequence are as follows: | ||
| + | Nucleotide sequence | ||
| + | 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 | ||
| + | Amino acid sequence | ||
| + | MQTRRDALKSAAAITLLGGLAGCASMARPIGTGDLINTVRGPIPVSEAGFTLTHEHICGSSAGFLRAWPEFFGSRKALAEKAVRGLRHARSAGVQTIVDVSTFDIGRDVRLLAEVSRAADVHIVAATGLWFDPPLSMRMRSVEELTQFFLREIQHGIEDTGIRAGIIKVATTGKATPFQELVLKAAARASLATGVPVTTHTSASQRDGEQQAAIFESEGLSPSRVCIGHSDDTDDLSYLTGLAARGYLVGLDRMPYSAIGLEGNASALALFGTRSWQTRALLIKALIDRGYKDRILVSHDWLFGFSSYVTNIMDVMDRINPDGMAFVPLRVIPFLREKGVPPETLAGVTVANPARFLSPTVRAVVTRSETSRPAAPIPRQDTER | ||
| + | DNA SEQUENCE | ||
| + | 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 | ||
| + | PROTEIN SEQUENCE | ||
| + | MQTRRDALKSAAAITLLGGLAGCASMARPIGTGDLINTVRGPIPVSEAGFTLTHEHICGSSAGFLRAWPEFFGSRKALAEKAVRGLRHARSAGVQTIVDVSTFDIGRDVRLLAEVSRAADVHIVAATGLWFDPPLSMRMRSVEELTQFFLREIQHGIEDTGIRAGIIKVATTGKATPFQELVLKAAARASLATGVPVTTHTSASQRDGEQQAAIFESEGLSPSRVCIGHSDDTDDLSYLTGLAARGYLVGLDRMPYSAIGLEGNASALALFGTRSWQTRALLIKALIDRGYKDRILVSHDWLFGFSSYVTNIMDVMDRINPDGMAFVPLRVIPFLREKGVPPETLAGVTVANPARFLSPTVRAVVTRSETSRPAAPIPRQDTER | ||
| + | 3) Genetically engineered E. Colibacteria | ||
| + | In this project, we will use E. Coli to construct genetically engineered bacteria which can secrete organophosphorus-degradation enzyme opdA protein, to eliminate the pesticides. | ||
| + | Genetically engineered bacteria are bacteria which can channel target gene into bacteria to express the genes and produce required protein. | ||
| + | Currently, the mostly used genetically engineered bacteria all over the world are still E. Coli. E. Coli have explicit genetic background, fast growth rate, limited antibiotics resistance, Thus, E. Coli, are easy to be used in any production magnitude from laboratory to industry production. (For example: scientists introduced human insulin gene into E. Coli genome. E. Coli can express functional human insulin protein, which is used as a medicine for diabetes treatment. Human insulin production from E. coli has been applied to industry and this method is also widely used in biotech industry for other drug purposes In 1981, human insulin gene products were put into market and solved the problem of lack of insulin sources). | ||
| + | 4) Lac operator | ||
| + | The E. Coli wouldn’t produce things that doesn’t belong to itself, the lac operator takes charge of the controlling of the operation of the configuration in E. Coli to be specific the opdA enzyme. | ||
| + | 5) Histindine tag | ||
| + | The aim of eradicating the organophorphorus residual in vegetables is to help eliminate the potential harm that organophorphorus may done to human’s physical health. Considering the harm that E. Coli itself has, this year’s aim is to purify the opdA enzyme from the E. Coli. In order to extract the opdA out of the bacterium, we use histidine tag to label the opdA enzyme, because the nickle in the resin can form strong molecular bonds with histidine thus grab the opdA which connect with his-tag as well. So when the E. Coli with opdA enzyme pass through resin with nickle, the opdA will cling to the resin and the rest of the E coli will follow the solution be excluded from the resin. | ||
| + | The next step to replace the opdA enzyme with his-tag on resin is to add over-dose of histidine, since the nickle in resin can form molecular bonds with histidine, those bonds that histidine+opdA is relatively weak comparing with the bonds between nickle and histidine. In regard of this, the over-dosed histidine will replace the opdA, thus exclude the opdA from the resin. When the opdA is excluded, we can readily obtain the finished product of the enzyme. | ||
Revision as of 05:49, 5 October 2016
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1) Micro-organisms degrades organophosphorus, and organophosphorus-degradation enzyme opdA Faced with the stress of human pollution such as pesticides, nature itself has evolved many methods to deal with these problems. For example, many natural micro-organisms contain enzymes to degrade organophosphorus pesticides. Currently the micro-organisms which are capable to degrade organophosphorus pesticides include bacteria, fungus, actinomycete and alga. As the research goes further, people find that these degrading effects come from secreting an enzyme, which can hydrolyze phosphoester bonds, organophosphorus degradation enzyme. Because each organophosphorus pesticide has similar structure and protein sequence, one kind of organophosphorus degradation enzyme is capable todegrade multiple kinds of organophosphorus pesticides. Organophosphorus-degradation enzyme has been mostly recognized as the best method to eliminate pesticide residues currently. At present, many enzymes have been identified to be used to degrade organophosphate pesticides. Among these enzymes, the organophosphorus-degradation enzyme (opdA) which comes from Agrobacterium radiobacter P230 has wider targets and higher enzyme-catalyst efficiency. In recent years, the research on the structure and function of organophosphorus-degradation enzyme has gained promising progress,. Thus, it is possible to improve the properties of organophosphorus-degradation enzyme through genetic engineering and protein engineering method, which meet requirements of different applications. 2) In this project, we will use organophosphorus-degradation enzyme opdA to eliminate residual organophosphorus pesticide on fruits and vegetables. The organophosphorus-degradation enzyme (opdA) gene opdA （NCBI genbank：Accession: AY043245.2） programmed by Agrobacterium radiobacter contains 1,155 nucleic acids, programming 384 amino acid residues. The N-terminal of protein sequence is the signal peptide while the C-terminal is the degradation-enzyme sequence. The nucleic acid sequence and amino acid sequence are as follows: Nucleotide sequence 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 Amino acid sequence MQTRRDALKSAAAITLLGGLAGCASMARPIGTGDLINTVRGPIPVSEAGFTLTHEHICGSSAGFLRAWPEFFGSRKALAEKAVRGLRHARSAGVQTIVDVSTFDIGRDVRLLAEVSRAADVHIVAATGLWFDPPLSMRMRSVEELTQFFLREIQHGIEDTGIRAGIIKVATTGKATPFQELVLKAAARASLATGVPVTTHTSASQRDGEQQAAIFESEGLSPSRVCIGHSDDTDDLSYLTGLAARGYLVGLDRMPYSAIGLEGNASALALFGTRSWQTRALLIKALIDRGYKDRILVSHDWLFGFSSYVTNIMDVMDRINPDGMAFVPLRVIPFLREKGVPPETLAGVTVANPARFLSPTVRAVVTRSETSRPAAPIPRQDTER DNA SEQUENCE 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 PROTEIN SEQUENCE MQTRRDALKSAAAITLLGGLAGCASMARPIGTGDLINTVRGPIPVSEAGFTLTHEHICGSSAGFLRAWPEFFGSRKALAEKAVRGLRHARSAGVQTIVDVSTFDIGRDVRLLAEVSRAADVHIVAATGLWFDPPLSMRMRSVEELTQFFLREIQHGIEDTGIRAGIIKVATTGKATPFQELVLKAAARASLATGVPVTTHTSASQRDGEQQAAIFESEGLSPSRVCIGHSDDTDDLSYLTGLAARGYLVGLDRMPYSAIGLEGNASALALFGTRSWQTRALLIKALIDRGYKDRILVSHDWLFGFSSYVTNIMDVMDRINPDGMAFVPLRVIPFLREKGVPPETLAGVTVANPARFLSPTVRAVVTRSETSRPAAPIPRQDTER 3) Genetically engineered E. Colibacteria In this project, we will use E. Coli to construct genetically engineered bacteria which can secrete organophosphorus-degradation enzyme opdA protein, to eliminate the pesticides. Genetically engineered bacteria are bacteria which can channel target gene into bacteria to express the genes and produce required protein. Currently, the mostly used genetically engineered bacteria all over the world are still E. Coli. E. Coli have explicit genetic background, fast growth rate, limited antibiotics resistance, Thus, E. Coli, are easy to be used in any production magnitude from laboratory to industry production. (For example: scientists introduced human insulin gene into E. Coli genome. E. Coli can express functional human insulin protein, which is used as a medicine for diabetes treatment. Human insulin production from E. coli has been applied to industry and this method is also widely used in biotech industry for other drug purposes In 1981, human insulin gene products were put into market and solved the problem of lack of insulin sources). 4) Lac operator The E. Coli wouldn’t produce things that doesn’t belong to itself, the lac operator takes charge of the controlling of the operation of the configuration in E. Coli to be specific the opdA enzyme. 5) Histindine tag The aim of eradicating the organophorphorus residual in vegetables is to help eliminate the potential harm that organophorphorus may done to human’s physical health. Considering the harm that E. Coli itself has, this year’s aim is to purify the opdA enzyme from the E. Coli. In order to extract the opdA out of the bacterium, we use histidine tag to label the opdA enzyme, because the nickle in the resin can form strong molecular bonds with histidine thus grab the opdA which connect with his-tag as well. So when the E. Coli with opdA enzyme pass through resin with nickle, the opdA will cling to the resin and the rest of the E coli will follow the solution be excluded from the resin. The next step to replace the opdA enzyme with his-tag on resin is to add over-dose of histidine, since the nickle in resin can form molecular bonds with histidine, those bonds that histidine+opdA is relatively weak comparing with the bonds between nickle and histidine. In regard of this, the over-dosed histidine will replace the opdA, thus exclude the opdA from the resin. When the opdA is excluded, we can readily obtain the finished product of the enzyme.
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First of all, we use the PCR to proliferate the plasmid provided by IGEM. PCR is a kind of mechanism that proliferate the target DNA segment beyond the capacity of the nature, the velocity of the proliferation of the PCR mechanism can be up to 100,000 generations per day, thus provide us with plenty of plasmid for the experiment. Then the module is used with the purified water, 10*buffer, dNTP, F and R primer to synthesize the target opdA enzyme’s DNA. Then the result solution was put into the PCR again, waiting for later purification by using the reagent----Gel extraction kit. When waiting for the PCR with the solution we made earlier, the medium for the coli was made by mixing the soybean powder with water, the result 1 liter of solution is then equally divided into four parts because of the limitation of the container which has the maximum capacity of 250ml. Two of them are simply made by mixing water and soybean powder, while the rest two were added agar thus created two solid medium. Because of the rigid requirement of the coli and avoid contamination, the for containers with the medium are put into an autoclave to eliminate the potential contamination in the container. The next step is preparing for the separation gel by mixing the agarose and water, result in the solution with the volume of 30ml. The solution is then put into the oven to mix the agarose and the water entirely. After taking it out of the oven and cooling the solution, the dye which make the DNA appear when exciting light was lit upon the gel is put into the solution. The solution is put into the model with a comb, in order for the gel to concrete. With the previously made separation gel, the DNA model is put in to the hole left by the comb and the model is put into another hole. The electrophoresis is then used to extract the target DNA segment in the solution. The resulting gel has many stripes and one of them contains the target DNA segment. In order to purify the DNA segment, the stripe need to be neatly cut out of the gel. Then the Gel extraction kit is used to purify the DNA by following the instructions in the Gel extraction kit.
Judges like to read your wiki and know exactly what you have achieved. This is how you should think about these sections; from the point of view of the judge evaluating you at the end of the year. First lets start with the three basic components of genetic engineering: the restriction endonuclease, phosphodiester bond and the carrier in terms of the plasmid in colibacillcus. In our experiment, we used EcoR1 and spe1 as our restriction endonuclease to cut the target DNA fragment from pet32a, the fragment on pet32a is used to replace the strong promoter, RNA thermometer and ompA from last year’s project. The substitution for those three components are T7 promoter, lac operator and finally the histidine tag. With this improvement, we are able to extract the opdA enzyme directly from the E. coli. It is agreed that the E. coli is to some extent harmful to human body, so by extracting the enzyme we can readily prevent the potential harm of the E. coli. Since the histidine tag can help extract the enzyme from E. coli. The very first procedure of the project is to cut off the target DNA sequence from the model plasmid we selected, which is pet32a. We separately used EcoR1 and Spe1 to cut the sequence. Then we used PCR to proliferate the sequence. At the same time the original sequence from last year’s plasmid is cut off from the model. T4 phosphodiester bond functions as the connecting between the DNA segment and the model so the previously extracted target sequence is able to connect with the model provided by IGEM. The pivotal procedure of the whole experiment is to transport the plasmid into E. Coli and use E. Coli as an carrier to exponentially proliferate the plasmid. First we set the E. Coli in a low temperature refrigerator which is approximately -80℃, at that temperature makes the E. Coli be in a state of sensitivity which means its membrane is vulnerable. When it is about time to transport the plasmid into the E. Coli, we took the frozen E. Coli out of the refrigerator, it immediately rises its temperature to 0℃. When the plasmid is ready, the model E. Coli is put in 42℃ environment so because of the heat affect the volume of the whole bacterium and increase the size of the membrane thus small holes began appear on the membrane, letting the plasmid in to the E. Coli. When all the aforementioned procedures are done, the E. Coli are on ice again, making the holes on the membrane close. Finally, we put the culture dish with the E. Coli plus the plasmid in to 37℃ environment letting the E. Coli continues to grow.
References
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Inspiration
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