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

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.Collect bacteria from　1-3ml bacteria solution, centrifuge 30” at 10,000rpm

2.Pour away supernatant and resuspend in 250μl S1 solution

3.Add 250μl S2 solution, mix the solution gently, incubate at room temperature (RT) for no more than 5’

4.Add 350μl S3 solution, mix the solution gently, centrifuge 12’at 12,000rpm

5.Transfer supernatant into nucleic acid reaction column, incubate at RT for 1’

6.Centrifuge the column 30” at 12,000rpm, and trash the elute

7.Add 500μl W1 washing buffer into column, and centrifuge 30” at 12,000rpm, trash the elute

8.Add 750μl W2 washing buffer into column, and centrifuge 30” at 12,000rpm, trash the elute

9.Repeat step 8 once more

10.Centrifuge 1’ at 12,000rpm, and completely remove residual W2

11.Transfer column into new 1.5ml centrifuge tube, add 50-100μl water, and incubate at RT for 1’

12.Centrifuge 1’ at 12,000rpm, and collect the elute

1. PCR reaction system: (25μl)

10 X Taq buffer 2.5μl

5’ primer 1μl

3’ primer 1μl

dNTP 2μl

Template 1μl

Taq 0.2μl

ddH2O added up to 25μl

2. PCR reaction conditions:

Denature for 5’ at 95°C;

Denature for30” at 95°C℃;

Anneal for 30” at 55°C℃;

Extension at 72C,℃, extension time is determined by the length of extension sequence;

Return to (2), and cycle 29 times;

Extension for 10’ at 72°C, store at 4°C;

1.Under UV light, carefully cut agarose block containing target DNA, put it into 1.5ml centrifuge tube;

2.Add two-times volume of solution A (add 200μl solution A into each 100mg agarose gel), heat it in heat block at 80°C until the gel is completely dissolved, mix evenly and cool to room temperature;

3.Put dissolved gel solution into gel recycle column, and centrifuge 30” at 10,000rpm;

4.Add 450μl washing solution B into column, and centrifuge 1’ at 12,000rpm, trash the elute;

5.Repeat step 4 once more;

6.Transfer column into a clean EP tube, add 30μl elution buffer C into the column;

7.Incubate at 37°C for 5’, centrifuge 2’ at 12,000rpm, and collect the eluted DNA solution;

Double enzymatic digestion on PCR product or plasmid DNA, the digestive products are purified and stored for future use. Enzymatic digestion conditions are as follows:

10xH Buffer 5μl

Enzyme 1 2μl

Enzyme 2 2μl

Template (plasmid or PCR product) less than 1μg

ddH2O added upto 50μl

37°C, 3-4 hours.

Ligation of linear plasmid and inserted fragment double digested with same pair of enzymes, the ligation protocol is as follows:

10 x T4 ligase buffer 2μl

Linear plasmid 3μl

Inserted fragment 3-10 times Mol of plasmid

T4 DNA ligase 1μl

ddH2OSterile super-pure water added upto 20μl

16°C , 4 hours.

(1)Inoculate a single colony of E. coli (or the ratio of E. coli solution and culture medium is 1:1000) in to 1000ml LB culture medium, culture at37°C for 4 hours at 200rpm.

(2)Collect E. coli into 50ml centrifuge tube, cool the tube on ice for 10’, and centrifuge for 10’ at 4,000rpm.

(3)Remove the supernatant completely, re-suspend the E. coli cells with pre-cooled 0.1M calcium chloride solution , do not use oscillator, pool resuspend E. coli solution into one centrifuge tube.

(4)Centrifuge for 10’ at 4,000rpm.

(5)Remove the supernatant completely, re-suspend the E. coli cells using 10ml pre-cooled 0.1M calcium chloride solution, add glycerin to a final concentration of 10%, and split them into 100 µl/tube and store them at -80°C.

(1)Thaw 0.1ml competent cells on ice;

(2)Add 10μl ligation product, and incubate on ice for 30’;

(3)Heat shock at 42°Cfor 90”, and immediately incubate it back on ice for 2’;

(4)Add 0.8ml LB culture, and slowly shake at 37°C for 60’;

(5)Pipet ~100-200 μl solution onto LB pate coated with certain antibiotic (eg. 45μg/ml chlorampenicol), plate evenly;

(6)Place the plate upside down, 37° Covernig,The next day, select positive single cloning colonies;

The project designed a biology module which integrates multiple bio-function components. This module can make the genetically engineered bacteria induced by temperature to produce organophosphorus-degradation enzyme opdA to eliminate the organophosphorus pesticides pollution in the environment. Later the bacteria is killed by activated suicide gene under UV light, which avoids the secondary pollution posed by bacteria.

The project using synthetic biology successfully assembled this biology module, and transformed into E. coli, which can proliferate enormously.

Evaluate the function of the engineered E. coli. Whether the engineered E. coli can eliminate the pesticide? Whether UV or sunlight can induce the self-death of the engineered E. coli? If it works, what the best working condition?

Optimize the growth conditions for E. coli which contain this biology module, and determine the best culture conditions for manufacture scale growth.

On the basis of this biology module, the project further extend its functions. For example, integrate other degradation enzyme systems for other pesticides or other organic pollutants, or combine different enzymes to degrade multiple pollutants.

Failure experience (difficulties we met) and our solutions:

1)Because the linear plasmid provided by IGEM had a low purity, using the plasmid construction method recommended by IGEM protocol caused large quantities of false positives in the process and interfered greatly to select positive clones. Additionally, because the linear plasmid provided by IGEM cannot reproduce, the quantities used in experiment and times of experiment repeatswere severely restricted.

Solution: Counseled with other IGEM, we gave up the linear plasmid provided by IGEM and turned to J04450 plasmid to carry out plasmid extraction, and recieved large amounts of J04450 . By carrying out EcoRI and PstI double digestion on and cutting off mRFP, and replacing with our biology module, we finally successfully constructed plasmid which contained the biology module we designed.

2)Ligation of multiple segments, our biology module contains four different segments, 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, ligating four segments to 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 utilized the enzyme digestion site pre-designed at each ends of each segment, to achieved the mutual connection among segments without vector. Then we used PCR technology without repeated ligation, transformations, clone selection processes to gain final F1+2+3+4 segments and clone them to pSB1C3 plasmid at a single step.

Bacteria strain BL21 (DE3) of colibacillus genetically engineered bacteria which contains our target plasmid goes through extended culture in our LB liquid substrate for sixteen hours under the temperature of 30°C, then centrifuge the culture for five minutes with the speed of 5000rpm under the temperature of 4°C and use sterile MSM (mineral base medium) to wash and sediment the bacteria, then use fresh MSM culture substrate to dilute the bacteria. Inoculate the bacteria to MSM culture substrate which contains 10μg/mL chlorpyrifos or methyl-parathion (the final concentration of bacteria is 106 CFU/mL), and culture it with the speed of 160rpm under the temperature of 37°C while shaking it. Regularly get samples from the solution and measure the concentration of organophosphorus pesticides in the solution. Measure the OD600 of the culture to indicate the growth of the bacteria, use the culture solution which is not inoculated as a blank control while using the colibacillus inoculated with the bacteria strain BL21 (DE3) which does not contain target plasmid as a reference control. Adopt gas chromatography to measure the concentration of organophosphorus pesticides in the culture.

Extraction of organophosphorus pesticides: Get 5mL MSM solution which is inoculated and cultured as above, add 25mL acetonitrile into it and shake it violently for five minutes, then add 2.5g sodium chloride into it and shake it violently, place the solution quietly for 30 minutes then laminarize it, get 1mL organic phase in the upper layer and dry it with pressure blowing concentrator, add 1mL acetone into it to dissolve above dry organic phase again.

Gas chromatography detecting conditions: Use Shimadzu 2014C gas chromatographic analyzer, equip it with fire photometric detector (FPD) and quartz capillary chromatographic column (length of column is 30 meters, inner diameter of column is 0.53mm, thickness of membrane layer is 1μm, RESTEX, USA), flow speed of nitrogen is lmL/min while the flow speeds of air and hydrogen are respectively 81.8mL/min and 3.2mL/min. The temperatures of vaporizing chamber, column and detector are respectively 250°C, 150°C and 250°C. The initial temperature of column is 150°C and should be kept for three minutes. Then the temperature of column should be heated to 250°C using the speed of 8°C/min and the final temperature should be kept for eight minutes. The quantity of input sample is 10μL.

Later, different change influences culture temperatures, PH values and inoculation quantity have on degrading organophosphorus can be set. Among these conditions, the organophosphorus pesticides are not degraded at the temperature below 32°C while they begin to be degraded at the temperature of over 32°C. The degradation effects generally must be good at neutral PH values. Generally, the more the inoculation quantity is, the better the degrading effects are.