Project.htm

        Nitrate (NO3-) and nitrite (NO2-) are chemical roots of nitric acid (HNO3) and nitrous acid (HNO2) respectively. They exist in the aquatic environment, the living organisms and artificial products, as pollutants, which can cause food poisoning, cancers or even death. It's important to detect and eliminate such chemicals with the efficient methods. Currently, several detection methods have already been in use but all are not entirely harmless. For example, granular Cd grains are recommended for the NO3- determination for fruits and vegetables because they are easy to make and recover, and can be used repeatedly. However, Cd cannot be recycled easily, and may cause additional pollution. So the goal of our project is to provide an efficient and a more environment friendly method of detecting nitrates/nitrites and we strive to design biosensors to avoid the disadvantages of traditional methods.
        Specific details of the pollution of the Nitrites and Nitrates:
        Nitrate (NO3-) and nitrite (NO2-) are Chemical roots of nitric acid (HNO3) and nitrous acid (HNO2). They exist in nature, especially in the gaseous water, surface water and groundwater, as well as plants and animals and food within, as environmental pollutants.
        There exist a lot of pollution sources of nitrate and nitrite in the environment, such as:
        1. Artificial fertilizers: ammonium nitrate, calcium nitrate, potassium nitrate, sodium nitrate and urea;
      2. Sewage, garbage and human and animal feces: according to tests, 1 liters of domestic sewage in the natural degradation process can produce 110 mg of nitrate; 1 kg of garbage and fecal compost under natural conditions can produce 492 mg of nitrate leaching decomposition;
      3. Large amounts of ammonia which food, fuel or oil refining factories emit in waste: after nature biological, chemical conversion cause the formation of nitrate into the environment;
      4. Cars, trains, ships, aircraft, boilers, civil furnace combustion of petroleum fuels, coal, natural gas: can produce large amounts of nitrogen oxides. Average burning one ton of coal, of 1 kiloliter oil and 10,000 cubic meters of natural gas can produce nitrogen dioxide gas, respectively 9.13 and 63 kg. These nitrogen dioxide gas can be formed after precipitation leaching nitrate descend to the ground and waters ;
        5. Food preservation and preservation: nitrate and nitrite are widely used in corrosion protection and preservation of meat and fish, so that the meat is red and aroma. Adding sodium nitrate per kilogram of meat in food (usually sodium nitrite) 5 mg or less, within a certain time will percept a good flesh color ; added 20 mg or more, and may exhibit the needed stability in the commercial color; added 50 mg of a special smell.
        These chemical fertilizers in irrigation, sewage, garbage and fecal, industrial nitrogenous waste, fuel combustion, emissions containing nitrogen gas and other pollutions, after precipitation leaching and decomposition in natural conditions, will form nitrate into the river, the lake and into the ground, resulting in nitrate pollution of surface water and groundwater. Infiltration such as sewage, sewage irrigation and unrestrained use of fertilizers sharp increase in groundwater nitrate content can be from a few mg / l to 400 mg / l or more (the national life of the nitrate content of drinking water health standards less than 88.6 mg / l to nitrogen of less than 20 mg / l); indiscriminate use of fertilizers, irrigation, nitrate pollution of water irrigation cause crops absorb large amounts of nitrates, such as over-fertilization spinach production per kilogram dry weight containing nitrite 3600 mg of salt. Moreover, pickled sauerkraut, vegetables after the lon        g-distance transport and long-term storage, as well as overnight cooked vegetables are all unhealthy. Because not only a large number of nitrate content increased, but also in the role of nitrate-reducing bacteria, nitrates are reduced to nitrites.
        Large amounts of nitrate and nitrite contain in drinking water, vegetables, grain, fish, meat, sauerkraut, overnight cooking, allow direct poisoning by human consumption. Nitrate in the human body can also be reduced to nitrite. Nitrite reacts in human blood; cause the formation of methemoglobin, so that the blood loss of oxygen functionality makes oxygen poisoning. The symptoms caused by it such as dizziness on light, palpitations, vomiting, lips cyanosis, severe cases, unconsciousness, convulsions, shortness of breath, can be life threatening if not rescued in time. Moreover, nitrite may react with secondary amines and from nitrosamines in the environment both in and out human bodies, which will become carcinogenic, teratogenic and mutagenic substances if reaches a certain dose, this will totally do great harm to human health.
        In order to prevent the hazard of nitrate and nitrite, in addition to achieve scientific and rational application of fertilizer, prohibit the use of sewage irrigation ,implement the manure treatment of sewage and garbage and other environmental protection measures to protect surface and ground water sources from nitrate and nitrite nitrate pollution, human should also keep a minimum to eat pickled, smoked, wax system of meat, canned food, stains sauerkraut, and salted vegetables; does not buy foods that were kept too long, lethargic or tertian; finish the food right after buying them; does not eat overnight cooked vegetables or keep tertian boiled vegetables; and remember not to confuse the industrial nitrite with normal salt for eating.

        Introduction:
        Our team is going to develop novel modular biosensors that enable cost-effective and on-site detection of nitrates/nitrites by using an Escherichia coli (E.coli) chassis. To introduce the whole process of our project, we split our works into 3 parts.
        Design
        Firstly, we aim to build a part containing the nitrates/nitrites sensing promoter and the most visible and observable reporter.
        This can also be called a development from previous work of Edinburgh 2009. Their aim was to enable E.coli to report the existence of nitrates/nitrites with green fluorescent protein (GFP) and the PyeaR promoter (the sensor of the nitrates/nitrites they created), but we found several drawbacks of their work. Firstly, the GFP reporter within the bacteria is not efficient enough to respond to the inducer so only dim green fluorescence can be detected. Also, this biosensor cannot be recycled for repeated usage because it does not have an efficient way to turn off the reporting process after the detection. Thus, our team aims to improve the biosensor by taking addition of a more remarkable and observable reporter. From the parts registry, we eventually find a possible reporter, the CrtEBI, that, when synthesized, will lead the E. coli to release a specific pigment, lycopene, as the visible color to determine the existence of nitrates/nitrites. Simultaneously, we will incorporate another reporter, the banana odor enzyme (ATF1) generator, which enables our biosensor to produce an aroma of bananas with the existence of nitrates/nitrites. Therefore, our biosensor containing dual reporters may ensure a high accuracy in the detection of nitrates and nitrites.
        Secondly, our team planned to build another part that is functional to eliminate the effect given by part 1.
        The first step that we took in this plan was finding a reporter in the registry that can cause the degradation of the pigment. More specifically, the proteins produced by this appropriate reporter are useful to degrade the pigment, so that after easily adding certain promoter to control it, this part 2 that we were willing to build could restore the bacteria to the original state. But the fact was crucial. We could not find this reporter at any sources that we were capable to reach. Based on this result, we started step 2, another solution that might enabled us to finish our design. According to the Web sources of the Peking University 2010 (the creator of the CrtEBI), we learned that the pigment, lycopene would be naturally degrade by bacteria in an appropriate acidic environment or under high temperature. Thus we aimed to find any reporter or protein that could produce heat or acid. Although there were some of the proteins in this range, we could not choose them because of the indeterminacy of their experiment, and we failed again as a result. But in the final step, we eventually thought of a more effective and simple method of this part. We planned to find a part that can reduce the expression of the proteins. According to the description of the CrtEBI, which produces enzymes that convert colorless farnesyl pyrophosphate to red lycopene, the only thing we need to do in this part is to enervate the enzymes. Although the pigment still would not disappear, the bacteria would stop producing the pigment and thus it is convenient for us to eliminate the remaining pigment by changing PH or temperature because of the part 2 that we made.
        Thirdly, our team aimed to enhance our biobricks by designing a color gradient system which can mark the various concentrations of the nitrates/nitrites.
        The overall design of the color gradient system is to repeatedly add different color (nestification) to reveal the different molarities of the nitrites and nitrates. Based on the work by Peking University, we found several different colors with the same function as the CrtEBI. Such as the BBa_K274002,BBa_K274001.
        To give a simple example, If we linked the PyeaR promoter , the color reporter 1 (we assume that it is red) and a protein reporter ( we assume that it is TetR reporter) together as the first part, then linked the color reporter 2 ( assumed that it is green) and a TetR negative promoter together as the second part, we could probably observe that the color change from green to grey (as the red color was added) when the concentration of nitrites and nitrates was increasing.
        To finish this part 3, the most important work for us is to create an appropriate model as measurement of the input, the nitrites and nitrates, and the output, the variable colors.
        MORE DETAILS
        Promoter:
        We choose the the PyeaR promoter because it is the only suitable one as the essence of our project obviously. It can be found easily on the parts registry: BBa_K216005 Unlike other E. coli promoters responding to nitrate and nitrite, this promoter is not repressed under aerobic conditions. It was built based on the work of Edinburgh 2009.
        Reporter:
        The CrtEBI which can cause the bacteria to produce the red pigment, lycopene is suitable for our team to replace the conventional part, GFP. It can be also found in parts registry: BBa_K274100
        Inside the reporter, together, enzymes CrtE, CrtB and CrtI convert colourless farnesyl pyrophosphate to red lycopene (via intermediates geranylgeranyl pyroiphosphate and phytoene). The red lycopene pigment can then be used as a colored signal output, e.g. for biosensors.
        This design has been created with a list of other different colorful designs. For example, a related Composite Biobrick is Part BBa_K274200 (CrtEBIY with rbs), which "goes on" one step after lycopene, converting red lycopene to orange beta-carotene pigment.
        The banana odor enzyme (ATF1) generator was a standard reporter given by MIT 2006. Many other projects has used this design as a remarkable reporter. It can be found in the parts registry: BBa_J45199

        Outcome If we can finish the entire plan hopefully, we are willing to have such these result.
       By inserting the first system that we built into the bacteria, we will receive a sensor which, after absorb the nitrites or nitrates, can efficiently produce the remarkable symbols such as pigments and banana odor.
        By inserting the second system into the bacteria, we can let the bacteria be capable to clean all the products it produced previously, so that the target of our experiment can be reused.
        By inserting the final system of our project, we can create a sensor which will produce a color gradient after approaching to the nitrites and nitrates. From this goal we are willing to reach, we can observe the visual change of the bacteria easily, and simultaneously receive a beautiful and cheerful ending of our whole project in the IGEM competition.
SZMS-iGEM Protocol
0. First of all, be prepared for the plates.(Resistance selection medium ,sterilization)
Part One Kitting and Incubating( LEAST 2+12 hours)
1. Find parts we want from 384 panel in the refrigerator. The parts we found and decided to use were 1-18C (BBa_J23100) and 2-2I (BBa_J23111), which were not used.
2. Inside the plate there were DNA powder, we used tips of trace extractor to puncture aluminum foil, and draw 10μL double-distilled(DD) water with the trace extractor. dissolve DNA into solution. suck the solution again, put into the Polypropylene centrifuge tube (before this we should always write its tab on)
---------------------------------------------------15 minutes waiting---------------------------------------------------
3. Get the competent cell from the -73C fridge with the commending of our mentors.
4. Draw the DNA solution into the tube of E.coli (by trace extraction) 2μL out of 10μL, and put it inside the ice box for 30 minutes.
--------------------------------------------------30 minutes waiting----------------------------------------------------
5. Turn on the Thermomixer comfort 10min before using.
6. Put E.coli tube into Thermomixer comfort for 42 Celsius degree and 90 seconds “Hot Shock”.
-------------------------------------------------90 seconds waiting------------------------------------------------------
7. Put E.coli back into the ice-box for 5 minutes.
---------------------------------------------------5 minutes waiting-----------------------------------------------------
8. Add 0.5mL LB (Luria-Bertani medium) into E.coli tube.
9. Get 4 culture medium after the tubes were shaken in Thermostatic oscillation incubator.
10. Use spreader to put E.coli on the plate (inside super clean bench and remember to grill the spreader on the alcohol burner before using).
11. Take the plates with E.coli outside and start germiculturing them in the Constant temperature incubator (37 Celsius degree).

Part Two Selecting and Retaining(LEAST 1+12 hours)
12. Take the plates out of the Constant temperature incubator.
13. Take the test tube from the super clean bench.
14. Add 0.5mL LB and 200X of antibiotic into the tubes.
15. Select the strain used in the experiment from the plate (the one which is integral and independent with lucid edge will be the proper choice), and put it into the tubes with tips of trace extractor.
16. Clean the super clean incubator, put the plates back into the Constant temperature incubator.
17. Put the tubes into the shaker and shake under the temperature of 37 Celsius degree.

Part Three Measuring(LEAST 2 hours)
18. Drop one μL of buffer on the probe as a blank control group.
19. Clean the probe and drop one μL of DNA solution on it.
20. Start the procedure.
21. Note the concentration showed on the screen. ※The concentration we noted was 260/280: 1.93, 44.2ng/μL The ideal data of concentration 260/280 is 1.8-2.0 Protein pollution if lower than 1.8 and ion pollution when higher than 2.0

Part Four Plasmid Collecting(LEAST 3 hours)
22. Select the strain (As 15)
23. Retain the backups of the bacteria (Into the Constant temperature incubator)
24. Add 500μL of equilibrium liquid BL into the tubes.
25. Put the tubes into the centrifugal machine to be centrifuged for 1min in 12000 rpm (Remember to arrange the tubes symmetrically).
26. At the same time of executing 25, take 1.5mL of bacteria liquid and centrifuge in the same situation as 25.
27. Outwell the effluent and add the turbid liquid (contains RNase and enzyme RNA)
28. Add P2 and dissociate.
29. Add P3 and precipitate the protein.
30. Centrifuge the solutions in 12000rpm for 10 minutes, and absorb the supernatant (contains DNA).
31. Repeat 30 but change the time to 40-60 seconds.
32. Outwell the effluent and add anhydrous ethanol, centrifuge the tubes for 1 minute in 12000rpm, outwell the effluent. (Repeat this step for one time)
33. Elution the solution (add the eluent and wash the tube with buffer).
34. Measure the concentration of DNA (Do as Part Three)
※The concentration data we got was 2.07

Part Five Digestion(LEAST 3 hours)
35. Choose the buffer (2-3 μL 10X)
36. Add 14μL DD water.
37. Add 2μL buffer.
38. Add 2μL DNA.
39. Add 2μL enzyme (contained in the cone sugar).
40. Shake the tubes and put them into the Constant temperature incubator to germiculture under 30 Celsius degree.
--------------------------------------------------------2 hour waiting------------------------------------------------------

Part Six Glue Preparation(LEAST 2.5 hours)
41. Decide the size of Gum care and Comb.
42. Weigh the agarose. (The proportion can be 1.0% or 1.5%, depends on the experimental need and the size of glue).
43. Solute the agarose with TAE.
44. Heat the solution and cool it before using.
45. Pour the solution into the Gum care and cool for 30 minutes.
---------------------------------------------------30 minutes waiting----------------------------------------------------
46. Put the Gum care into the electrophoresis tank.
47. Drop the colored DNA into the groove on the glue and switch on the electricity. (Remember to put the glue on the side of cathode.)
---------------------------------------------------30 minutes waiting----------------------------------------------------

48. Color the glue with EB (EB is REAAAAAALLY DANGEROUS, be careful!)
49. Scan the glue.

Part Seven recycle and extract DNA(LEAST 3 hours)
50.follow the kit protocol on the book.

Part Eight Linking(LEAST 3 hours)
51. Get the EP tubes and make marks on them.(Better the name of DNA)
52. Get the glue made in Part Six and put it under the UV-light(remember to wear the safety goggles).
※2I: 1.110
GFP: 1.082
18C: 1,.080
GP: 1.086
53. Cut the glue with the parts we need.
※The weight
Total:
18C: 1.370
GFP: 1.230
2I: 1.244
6P: 1.345
Glue:
18C: 290mg*3 870
GFP: 148mg*3 444
2I: 134mg*3 402
6P: 259mg*3 777
※the weight of glue: solvent should be 1:3, the solvent is Buffer QG
54. Shake the tubes every 2-3 minutes for the duration of 10 minutes under the temperature of 50-60 Celsius degree in order to help dissolving.
55. Add isopropanol (as heavy as the glue), transfer the solution into the tubes, centrifuge for 1 minute, outwell the effluent.
56. Add PE, centrifuge for 1 minute, outwell the effluent.
57. Centrifuge for 1 minute again.
58. Add 50 μL DD water into the tubes.
59. Centrifuge the tubes again.
60. Get the water and DNA ligase.
※10μL in total
GFP: 2μL, buffer 1μL, enzyme 1μL
6P: 2μL, buffer 1μL, enzyme 1μL
2I: 2μL, buffer 1μL, enzyme 1μL
18C: 2μL, buffer 1μL, enzyme 1μL
Polish the solution with DD water.
61. Add those elements showed above(enzyme at last).
--------------------------------------------------------1 hour waiting------------------------------------------------------
Part Nine Transferring.(the same as Part One,LEAST 2+12 hours)
62. Get the competent cell from the -73C fridge with the commending of our mentors.
63. Draw the DNA solution into the tube of E.coli (by trace extraction) 2μL out of 10μL, and put it inside the ice box for 30 minutes.
--------------------------------------------------30 minutes waiting----------------------------------------------------
64. Turn on the Thermomixer comfort 10min before using.
65. Put E.coli tube into Thermomixer comfort for 42 Celsius degree and 90 seconds “Hot Shock”.
-------------------------------------------------90 seconds waiting------------------------------------------------------
66. Put E.coli back into the ice-box for 5 minutes.
---------------------------------------------------5 minutes waiting-----------------------------------------------------
67. Add 0.5mL LB (Luria-Bertani medium) into E.coli tube.
68. Get 4 culture medium after the tubes were shaken in Thermostatic oscillation incubator.
69. Use spreader to put E.coli on the plate (inside super clean bench and remember to grill the spreader on the alcohol burner before using).
70. Take the plates with E.coli outside and start germiculturing them in the Constant temperature incubator (37 Celsius degree).

Part Ten Check if we success or not
71. Take pictures, collect and arrange data, summary.