Difference between revisions of "Team:Shiyan SY China/Project.html"

With increased agriculture activities around the world, it becomes a common practice to use pesticides to manage pest problem. Runoff can carry field pesticide into aquatic environment while wind can carry them to other fields, potentially affecting other species. Over time, repeated application increases pest resistance and facilitate the pest resurgence. Further, especially in China, toxic pesticide residues on green vegetable and fruits become a major public health problem.

With the increased environmental pollution and climate change, pest problem becomes a serious issue in agriculture. According to the statistical data from Chinese Academy of Agricultural Sciences, in recent years, over seventy pest species have been recorded on vegetables and fruits. If not controlled, these pests could lead to complete wipeout of various crops, such as vegetables, fruits and grains. Therefore， to deal with the pest issue, the farmers have to constantly spray pesticides, and even use the high-toxic pesticides which are forbidden by China. As a result, a majority of vegetables and fruits, containing over dosed pesticides which are way above the permitted limits, get onto the dining tables of thousands of families through channels such as farmer’s markets, supermarkets and roadside stalls.

According to the statistics from World Health Organization, there are at least 500,000 pesticide poison accidents per yearwith 115,000 death due to pesticide poisoning all over the world; furtherover 85% of cancer cases and 80 kinds of diseases are suspected to related to over-dosed pesticide usage. In many major cities in China, it is reported that over 47% vegetables and fruits on the market have over-dosed pesticide residues.

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.

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:

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 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).

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 32C, RNA translation process can be started. Its DNA sequence is as follows:

Ccgggcgcccttcgggggcccggcggagacgggcgccggaggtgtccgacgcctgctcgtccagtctttgctcagtggaggattactag

In this project, to secrete the opdA enzyme from inside the bacteria to outside, an ompA signal peptide is introduced into the construction. By adding this secreting peptide, OpdA is secreted outside the E. Coli, without its accumulating inside.

Signal peptide often refers to a N-terminal amino acid sequence which is used to direct the trans-membrane process in newly synthesized proteins. There is a segment of RNA area programming hydrophobic amino acid sequence which is generally behind the start codon. This amino acid sequence is called signal peptide sequence, which introduces protein into sub-cellular organelles which contain different membrane structures. If the function of this signal peptide is to secrete 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 functional inE. Coli. Therefore, we need to select an E. colisignal peptides to achieve our plan.

The ompA signal peptide used in this project is one of the E. coli secreting signal peptides and can lead secretion of its downstream protein outside of its host E. coli. The DNA sequence of this signal peptide is as follows:

Atgaaaaaaaccgctatcgcgatcgcagttgcactggctggtttcgctaccgttgcgcaggcc

In this project, we consider not only how to use genetically engineered bacteria to eliminate organophosphorus, but also after organophosphorus is eliminated, how we should deal with the rest genetically engineered bacteria. Genetically engineered bacteria, after all, come from E. coli. We don’t want them released into environment or remaining on the surface of fruits and vegetables. Thus we must design a method to eliminate them. Consequently, we decide to design a suicide gene into our genetically engineered bacteria, which is inactivated under normal condition and only activated under special environments to eliminate the bacteria and avoid “secondary pollution”.

To achieve the above goal, we select suicide gene ccdb. ccdB is a known toxin system, which exists in pathogenic E. coli F plasmid. The ccdB gene programs a toxin protein CcdB. On conditions where there is a lack of antitoxin, CcdB poisons gyrase inside cells to interfere with the DNAsynthesis and damage host cells. The ccdb sequence we use in this project is as follows:

Atgcagtttaaggtttacacctataaaagagagagccgttatcgtctgtttgtggatgtacagagtgatattattgacacgcccgggcgacggatggtgatccccctggccagtgcacgtctgctgtcagataaagtctcccgtgaactttacccggtggtgcatatcggggatgaaagctggcgcatgatgaccaccgatatggccagtgtgccggtctccgttatcggggaagaagtggctgatctcagccaccgcgaaaatgacatcaaaaacgccattaacctgatgttctggggaatataa

In the project we chose a RecA(SOS) promoter to control to suicide gene ccdB to express. When the bacteria receive ultraviolet lights, DNA injurｙ activate the RecA repairing system. The system can activate RecA(SOS) promoter to drive the downstream 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 induced promoter 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

1.Get 1-3ml bacteria solution, centrifuge the solution for 30 seconds with the speed of 10000rpm and collect bacteria cells

2.Pour away supernate culture medium and make bacteria cells float in 250μl solution S1, blow and beat evenly

3.Add 250μl solution S2, mix the total solution upside down gently, put them for no more than 5 minutes under room temperature

4.Add 350μl solution S3, mix the total solution upside down gently and slowly, centrifuge the solution for 12 minutes with the speed of 12000rpm

5.Draw supernate carefully and add the supernate into nucleic acid combination cylinder, put it quietly under room temperature for 1 minute

6.Centrifuge the above for 30 seconds with the speed of 12000rpm, and pour away effluent fluid

7.Add 500μl cleaning mixture W1 into nucleic acid combination cylinder, and centrifuge it for 30 seconds with the speed of 12000rpm, pour away the effluent fluid

8.Add 750μl cleaning mixture W2 into nucleic acid combination cylinder, and centrifuge it for 30 seconds with the speed of 12000rpm,pour away the effluent fluid

9.Repeat above steps again

10.Centrifuge for 1 minute with the speed of 12000rpm, and thoroughly get rid of residual W2

11.Put nucleic acid combination cylinder into new 1.5ml centrifuge tube, add 50-100μl, and put under room temperature for 1 minute

12.Centrifuge for 1 minute with the speed of 12000rpm, and collect plasmid solution

1. PCR reaction system: (25μl)

10 X buffer 2.5μl

Upstream primer 1μl

Downstream primer 1μl

dNTP 2μl

Module plasmid DNA 1μl

Taq enzyme 0.2μl

Sterile super-nature water added the volume to 25μl

2. PCR reaction conditions:

Denaturation for 5 minutes under 95℃;

Denaturation for 30 seconds under 95℃;

Annealing for 30 seconds under 55℃;

Extension under 72℃, extension time is determined by different length of sequence;

Return to (2), and carry out 29 circulations of above steps;

Extension for 10 minutes under 72℃, storage under 4℃

1.Under ultraviolet light, carefully cut off agar block which contains target DNA, put it into 1.5ml centrifuge tube;

2.Add two-times volume of sol solution A (add 200μl sol solution A into each 100mg agar block), heat it in water under 80℃ until the block is completely dissolved, mix evenly and cool to room temperature;

3.Put dissolved solution into gel recycle centrifuge tube, and centrifuge for 30 seconds with the speed of 10000rpm, then the DNA is attached to cylinder;

4.Add 450μl cleaning solution B into centrifuge tube, and centrifuge for 1 minute with the speed of 12000rpm, pour away the solution inside the tube;

5.Wash again using solution B;

6.Put absorption cylinder into a clean EP tube, add 30μl eluent C into the center of absorption membrane in centrifuge tube;

7.Put it quietly under 37℃ for 5 minutes, and centrifuge for 2 minutes with the speed of 12000rpm, and thus purified DNA is eluted into the solution.

Use corresponding nucleic acid endonuclease to carry out double enzyme digestion on PCR gene product or plasmid DNA segment which is purified by above digestive gel recycle kit, the digestive products are recycled and stored for future use after purified by purification kit. Enzyme digestion conditions are as follows:

10xH Buffer 5μl

Endonuclease 1 2μl

Endonuclease 2 2μl

Plasmid DNA less than 1μg

Sterile super-pure water added to the volume of 50μl

Double enzyme digestion reaction condition: 37℃, 3-4 hours

Connect the segment gene product obtained from above double enzyme digestion and plasmid DNA segment on the corresponding expression carrier which is also processed by the same enzyme respectively, the connection system is as follows:

10 x T4 connection buffer 2μl

Linear plasmid DNA 3μl

Endonuclease 2 2μl

Connection segment DNA the amount of molecules are 3-10 times more than plasmid DNA

T4 DNA ligase 1μl

Sterile super-pure water added to the volume of 20μl

Connection conditions: under 16℃ for 4 hours

(1)Get single colony of colibacillus (or the ratio of bacteria solution and culture medium is 1:1000), inoculate it to 1000ml LB culture medium, culture under 37℃ for 4 hours with the speed of 200rpm.

(2)Get 50ml centrifuge tube, pour cultured bacteria solution into the tube and put the tube into ice to cool for 10 minutes, and centrifuge for 10 minutes with the speed of 4000rpm.

(3)Pour away the bacteria solution thoroughly, re-suspend the bacteria cells using 0.1M calcium chloride solution which is pre-cooled to 0℃, shake to make the bacteria float, do not use oscillator, combine each tube of bacteria solution into one centrifuge tube.

(4)Centrifuge for 10 minutes with a speed of 4000rpm.

(5)Pour away the bacteria solution thoroughly, re-suspend the bacteria cells using 10ml of 0.1M calcium chloride solution which is pre-cooled to 0℃,add glycerin with a final concentration of 10%, and divide them into 100 microlitres per tube and store them under -80℃.

(1)Get 0.1ml competent cells, and put them on ice to naturally and thoroughly melt;

(2)Add about 10μl recombinant expression carrier connection products, and bath them in ice for 30 minutes;

(3)Put them in water bath under 42℃, and heat shock for exactly 90 seconds, and immediately bath them in ice for 2 minutes;

(4)Add 0.8ml LB culture medium, and slowly shake under 37℃ for 60 minutes;

(5)Draw appropriate amount of bacteria solution and coat LB plate which contains appropriate amount of insulin (45μg/ml chlorampenicol), and put the plate under room temperature until the solution is absorbed;

(5)Draw appropriate amount of bacteria solution and coat LB plate which contains appropriate amount of insulin (45μg/ml chlorampenicol), and put the plate under room temperature until the solution is absorbed;

(6)Place the plate upside down, overnight culture under 37℃.The next day, select positive single cloning colonies.

The project designed a biology module which integrates multiple bio-functional components. This module can make the genetically engineered bacteria induced by temperature to produce organophosphorus-degradation enzyme opdA in order to eliminate the organophosphorus pesticides pollution in the environment and activate suicide gene under ultraviolet light, which avoids the secondary pollution problems posed by genetically engineered bacteria.

The project used methods of both synthetic biology and genetic engineering and successfully assembled this biology module outside externally, and transferred it to colibacillus to construct genetically engineered bacteria which can reproduce.

Explore and optimize the growth and function culture conditions for genetically engineered bacteria which contain this biology module, and determine the best bacteria and growth conditions to be used to collect the scale of bacteria used in this method.

Evaluate the processing effects the genetically engineered bacteria which contain this biology module have on organophosphorus pesticides pollution in natural environments, such as the surfaces of vegetables and fruits which are polluted by organophosphorus pesticides or the soils and waters which are polluted by organophosphorus pesticides, and the field elimination effects on organophosphorus pesticides.

On the basis of this biology module, the project further developed the module functions, for example, functional degradation enzyme systems of other pesticides or other organic pollutants can be introduced into this biology module on trial to achieve the degradation of other kinds of pesticides or organic pollutants or combined degradation of multiple pollutants to solve the problems of complicated pollutants.

Failure experience (difficulties we met) and our solutions:

1)Because the linear plasmid IGEM provided in distribution had a low purity, using the plasmid construction method recommended by IGEM protocol will cause large quantities of false positive cases in experiment results and interfere greatly with following positive cloning selection. Additionally, because the linear plasmid IGEM provided in distribution cannot reproduce, the quantities used in experiment and times of experiment repetitions were severely restricted.

Solution: Referring to the solutions by other teams before, we gave up using the linear plasmid IGEM provided in distribution and turned to using J04450 plasmid to carry out plasmid expansion and extraction, and gained large amounts of J04450 plasmid structures. By carrying out double enzyme digestion on EcoRI of J04450 and PstI and cutting off segments of mRFP, and replacing with our biology module, we finally successfully constructed plasmid which contained the biology module we designed.

2)Connection of multiple segments, our biology module contains four different sequences, which are F1, F2, F3 and F4 with a total length of over 1800bp. Given current DNA synthetic technology, it is inefficient and takes a long time to synthesize a length of over 1800bp. Moreover, connecting four segments in order to carrier plasmid pSB1C3 also requires four repetitive cloning transformation processes, which takes a lot of time and resources.

Solution: We designed methods of separately synthesizing four segments, which greatly increased synthesizing speed and saved time. Then, we used the enzyme digestion locus pre-designed at the two ends of each segment, and firstly achieved the mutual connection externally. PCR technology without repetitive connections, transformations, cloning selection processes made it possible for us to gain final F1+2+3+4 segments and clone them to pSB1C3 plasmid at a time.