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+ | <html> | ||
+ | |||
+ | <head> | ||
+ | <link rel="stylesheet" href="https://2018.igem.org/Template:NCKU_Tainan/css/hardware?action=raw&ctype=text/css"> | ||
+ | </head> | ||
+ | |||
+ | <body data-spy="scroll" data-target=".navbar-example"> | ||
+ | |||
+ | <!--Page_Content--> | ||
+ | <div class="container content"> | ||
+ | <h1 class="head">Results</h1> | ||
+ | <div class="navbar-example"> | ||
+ | <div class="row"> | ||
+ | <div class="col-2 side"> | ||
+ | <div id="sidelist" class="list-group"> | ||
+ | <a class="list-group-item list-group-item-action" href="#Overview">Overview</a> | ||
+ | <a class="list-group-item list-group-item-action" href="#Construction">Construction</a> | ||
+ | <a class="list-group-item list-group-item-action" href="#Total_solution">Total solution</a> | ||
+ | <a class="list-group-item list-group-item-action" href="#Bioreactor">Bioreactor</a> | ||
+ | <a class="list-group-item list-group-item-action" href="#Nutrient_tank">Nutrient tank</a> | ||
+ | <a class="list-group-item list-group-item-action" href="#Materials_required">Materials required</a> | ||
+ | <a class="list-group-item list-group-item-action" href="#"><i class="fa fa-arrow-up fa-1x" | ||
+ | aria-hidden="true"></i></a> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="col-10"> | ||
+ | <div data-spy="scroll" data-target="#sidelist" data-offset="0" class="scrollspy-example"> | ||
+ | <div class="container"> | ||
+ | |||
+ | |||
+ | <div id="Overview"> | ||
+ | </br></br></br></br> | ||
+ | <h3>Overview of the result</h3> | ||
+ | <ol> | ||
+ | <li>Construct each part and test the function of CA, and PRK.</li> | ||
+ | <li>Develop a new measurement approach to determine the carbon fixation ability of | ||
+ | each strain.</li> | ||
+ | <li>Estimate the carbon fixation amount with our experiment result.</li> | ||
+ | <li>Characterizing the pH sensing system.</li> | ||
+ | </ol> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | <div id="Construction"> | ||
+ | </br></br></br></br> | ||
+ | <h3>Construction and functional test </h3> | ||
+ | |||
+ | <div id="pt"> | ||
+ | <h8>PRK</h8> | ||
+ | <p class="hpcontent"> | ||
+ | Achievements:</br> | ||
+ | </p> | ||
+ | |||
+ | <ol> | ||
+ | <li>Construction and digestion of DNA gel shows that the size of it was | ||
+ | right</li> | ||
+ | |||
+ | <li>The SDS-PAGE of PRK showed that the expression of PRK in the expected | ||
+ | protein size</li> | ||
+ | |||
+ | <li>PRK toxicity test proves that the function of it varies when cloned | ||
+ | into | ||
+ | different plasmid</li> | ||
+ | </ol> | ||
+ | |||
+ | <p class="hpcontent"> | ||
+ | We constructed PRK fragments from IDT DNA synthesis. After PCR amplification, | ||
+ | PRK is then cloned into pSB1C3 and transformed into DH5α. SDS-PAGE ensured that | ||
+ | the protein expression was as expected. The results are shown below: | ||
+ | </p> | ||
+ | </div> | ||
+ | |||
+ | <img class="contentimg" src=" "> | ||
+ | |||
+ | <div id="pt"> | ||
+ | <p class="pcontent"> | ||
+ | Fig. 1: Confirmation of prk digestion.</br> | ||
+ | Fig. 2: Confirmation of PRK expression in DH5˚α. The expected protein size is | ||
+ | 37.7kDa. | ||
+ | </p> | ||
+ | </div> | ||
+ | |||
+ | <div id="pt"> | ||
+ | |||
+ | <p class="pcontent"> | ||
+ | We initially decided to test its function by HPLC to measure the amount of RuBP | ||
+ | inside the cell. Our instructors pointed out some difficulties in HPLC | ||
+ | measurement such as excessive noise signal in our sample.We therefore | ||
+ | determined to test its function with a toxicity test. The product of PRK-RuBP | ||
+ | cannot be metabolite by wild type E. coli. The accumulation of RuBP depletes | ||
+ | the sugar from the native pentose phosphate pathway. Lack of carbon source, the | ||
+ | growth of that strain may be repressed. We incubate the PRK expressing strain | ||
+ | and control stain that contains no plasmid in M9 medium and altered M9 medium | ||
+ | with 0.4% xylose as its sole carbon source. In normal M9 medium, glucose will | ||
+ | not be converted into RuBP. In altered M9 medium, xylose will go through the | ||
+ | native pathway and be converted into RuBP. Growth arrest of PRK strain should | ||
+ | be observed. | ||
+ | </p> | ||
+ | |||
+ | <p class="pcontent"> | ||
+ | We tested PRK in different strains. We first cloned prk into pSB1C3 and | ||
+ | transformed into BL21(DE3). After 12 hours, the strain without plasmid could | ||
+ | grow up to 1.4 O.D.600 in altered M9 xylose medium. The strain that contains | ||
+ | PRK can grew up to 0.75 O.D.600 in normal M9 medium either. In contrast, the | ||
+ | PRK strain that grew in altered M9 xylose medium showed no growth at all. The | ||
+ | result shows that PRK can suppress/inhibit the growth, which matches to our | ||
+ | expectation. | ||
+ | </p> | ||
+ | |||
+ | <img class="contentimg" src=" "> | ||
+ | |||
+ | <p class="pcontent"> | ||
+ | Fig. 3 The result of PRK test in BL21(DE3). The PRK expressing strain is | ||
+ | incubated | ||
+ | in both normal M9 medium and altered M9 xylose medium to compare with the | ||
+ | strain | ||
+ | without plasmid. The PRK expressing strain grown in altered M9 xylose showed | ||
+ | merely | ||
+ | no growth, which proves the function of PRK. | ||
+ | </p> | ||
+ | |||
+ | <p class="pcontent"> | ||
+ | Although the function of PRK have been confirmed, we would like to lower the | ||
+ | expression of it to minimize the growth arrest. We thus cloned the part into | ||
+ | pSB3K3, a low copy number plasmid to lower its protein expression. We then | ||
+ | compare | ||
+ | the growth under high and low copy number plasmid. We found out that pSB3K3 | ||
+ | shows a | ||
+ | little growth arrest comparing to the strain without plasmid. The growth of it | ||
+ | exceed that of PRK expressed in pSB1C3. We can regulate the expression of PRK | ||
+ | via | ||
+ | high or low copy number plasmid to optimize the growth and carbon fixation | ||
+ | efficiency of the bacteria. | ||
+ | </p> | ||
+ | |||
+ | <img class="contentimg" src=" "> | ||
+ | |||
+ | <p class="pcontent"> | ||
+ | Fig. 4 Compares the growth in M9 xylose medium of PR K expressing strain in | ||
+ | high | ||
+ | and low copy number plasmid. The low copy number plasmid, pSB3K3, shows a | ||
+ | little bit of growth retard compare to non-PRK expressing strain. However, the | ||
+ | toxicity is much less than high copy number expressing strain. | ||
+ | </p> | ||
+ | |||
+ | <p class="pcontent"> | ||
+ | We also transformed pSB3K3-prk into W3110 strain. W3110 is reported to have | ||
+ | higher pressure tolerance. The trend of the results is similar to that of the | ||
+ | BL21(DE3) but there is no statistically significant between the experiment and | ||
+ | the control group. We deduce that PRK can still function in W3110 since the | ||
+ | trend matches our expectation. As pSB3K3 is a low copy number plasmid, the | ||
+ | expression of protein may be lower than that of high copy number plasmid. The | ||
+ | pressure tolerance of W3110 strain may also lessen the toxicity influence by | ||
+ | PRK. | ||
+ | </p> | ||
+ | |||
+ | <img class="contentimg" src=" "> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | <div id="pt"> | ||
+ | <h8>CA</h8> | ||
+ | <p class="hpcontent"> | ||
+ | Achievements:</br> | ||
+ | </p> | ||
+ | |||
+ | <ol> | ||
+ | <li>Construct the ca and transform it into BL21(DE3)</li> | ||
+ | |||
+ | <li>Run the SDS-PAGE to confirm its expression</li> | ||
+ | |||
+ | <li>Measure the activity of CA enzyme</li> | ||
+ | </ol> | ||
+ | |||
+ | <p class="hpcontent"> | ||
+ | We cloned the DNA fragments into pSB1C3 plasmid after the gene is amplified | ||
+ | with PCR. We transform the plasmid into DH5-alpha and BL21(DE3). Next, we | ||
+ | confirm its protein expression with SDS-PAGE. | ||
+ | </p> | ||
+ | |||
+ | <img class="contentimg" src=" "> | ||
+ | |||
+ | <p class="hpcontent"> | ||
+ | Fig. 5 Confirmation of ca digestion</br> | ||
+ | |||
+ | Fig. 6 Confirmation of CA expression in BL21(DE3). The expected protein size is | ||
+ | 27.9kDa. | ||
+ | </p> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | |||
+ | |||
+ | <div id="pt"> | ||
+ | <p class="hpcontent"> | ||
+ | We then ran the activity test of CA. In our bypass pathway, the function of CA | ||
+ | is to | ||
+ | convert proton and bicarbonate into water and carbon dioxide. During the | ||
+ | reaction, the | ||
+ | concentration of proton can be easily observed by the pH meter. By measuring | ||
+ | the time | ||
+ | interval between the pH shift and compare with the sample without CA, we can | ||
+ | determine | ||
+ | the activity of that specific enzyme. The enzyme activity of our CA is 21.8 | ||
+ | unit/liter. | ||
+ | To confirm the contribution of the CA to the whole pathway, we also ran the | ||
+ | total | ||
+ | solution which will be described in the following content. | ||
+ | </p> | ||
+ | </div> | ||
+ | |||
+ | <div id="pt"> | ||
+ | <h8>Rubisco</h8> | ||
+ | <p class="hpcontent"> | ||
+ | Achievements:</br> | ||
+ | </p> | ||
+ | |||
+ | <ol> | ||
+ | <li>Construction and digestion of DNA gel shows that the size of it was right</li> | ||
+ | |||
+ | <li>The SDS-PAGE of PRK showed that the expression of PRK in the expected size</li> | ||
+ | </ol> | ||
+ | |||
+ | <p class="hpcontent"> | ||
+ | We constructed PRK fragments from IDT DNA synthesis. After PCR amplification of | ||
+ | the three subunits, rubisco is then cloned into pSB1C3 and transformed into | ||
+ | DH5α. SDS-PAGE ensured that the protein expression was as expected. The results | ||
+ | are shown below: | ||
+ | </p> | ||
+ | |||
+ | <img class="contentimg" src=" "> | ||
+ | |||
+ | <p class="hpcontent"> | ||
+ | Fig. 7 Confirmation of rbcL digestion.</br> | ||
+ | |||
+ | Fig. 8 Confirmation of rbcL expression in DH5˚α. The expected protein size is | ||
+ | 52.37kDa. | ||
+ | </p> | ||
+ | |||
+ | <p class="hpcontent"> | ||
+ | After mining a lot of information from the publications, we found out a method | ||
+ | to determine the activity of rubisco by thin-layer chromatographic has been | ||
+ | reported. However, due to time concern, we are not capable of measuring the | ||
+ | enzyme activity of Rubisco with this method. We finally confirm its function | ||
+ | from the results of total solution test. | ||
+ | </p> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | |||
+ | <div id="Total_solution"> | ||
+ | </br></br></br></br> | ||
+ | <h3>Total solution</h3> | ||
+ | |||
+ | <div id="pt"> | ||
+ | <h8>Rubisco</h8> | ||
+ | <p class="hpcontent"> | ||
+ | Achievements:</br> | ||
+ | </p> | ||
+ | |||
+ | <ol> | ||
+ | <li>Develop an index to evaluate the carbon fixation ability of each | ||
+ | constructed strain</li> | ||
+ | |||
+ | <li>Confirm the importance of Rubisco enzyme in bypass pathway</li> | ||
+ | |||
+ | <li>Check the growth and carbon fixation enhancement of CA enzyme</li> | ||
+ | |||
+ | <li>Compare the carbon fixation rate of W3110 and BL21(DE3) E. coli strains</li> | ||
+ | |||
+ | <li>Compare different CO2 incubation environment</li> | ||
+ | </ol> | ||
+ | |||
+ | <h8>Overview</h8> | ||
+ | <p class="hpcontent"> | ||
+ | In the total solution experiment, we strive to measure the carbon fixation | ||
+ | amount of each sample. After reading numerous publications, we found out that | ||
+ | previous researches determine the efficiency of carbon fixation via measuring | ||
+ | the decrease of carbon dioxide concentration in the closed system or measure | ||
+ | the weight | ||
+ | percentage of C14 radioisotope in the dry cell. However, due to biosafety | ||
+ | constrain of our lab, we can barely use the radioisotope. Measuring the | ||
+ | decrease of carbon dioxide concentration in the closed system is also | ||
+ | impractical for us since we have too much test samples. A new method to measure | ||
+ | multiple samples in the short period of time is developed by our team. We are | ||
+ | able to evaluate the fixation efficiency of each sample with optical density | ||
+ | O.D. 600 and xylose consumption. We have measure various construction to prove | ||
+ | that the enzyme of our construction is necessary for carbon fixation. | ||
+ | </p> | ||
+ | |||
+ | <p class="hpcontent"> | ||
+ | The test samples below were incubated in an altered M9 medium which substitute | ||
+ | glucose to xylose. 1/1000 of LB medium was added to support some rare elements. | ||
+ | Since the concentration of LB medium is too low, it doesn’t contribute the | ||
+ | carbon source of the bacteria. | ||
+ | </p> | ||
+ | |||
+ | <p class="hpcontent"> | ||
+ | We defined a new index, Xylose Utilization Index, to describe the potential of | ||
+ | carbon fixation. We can compare this index of each strain to find out the | ||
+ | strain that has highest capacity of carbon fixing. | ||
+ | </p> | ||
+ | |||
+ | <p class="hpcontent"> | ||
+ | To define the XUI index, we firstly made two assumptions: | ||
+ | </p> | ||
+ | |||
+ | <ol> | ||
+ | <li>O.D. 600 of the sample has linear relationship to dry cell weight | ||
+ | (biomass). Optical density is frequently used as a means of describing the | ||
+ | cell density in the broth. We measured the dry cell weight of samples in | ||
+ | different O.D. value and discovered that it has linear relationship. We | ||
+ | conclude that we can utilize O.D. value to estimate the dry cell weight. 1 | ||
+ | 0.D. of BL21(DE3) strain per litter yields the dry cell weight of 0.8 gram.</li> | ||
+ | </ol> | ||
+ | |||
+ | <img class="contentimg" src=" "> | ||
+ | |||
+ | <p class="hpcontent"> | ||
+ | Fig. 9 shows the dry cell weight of BL21(DE3) incubated in altered M9 xylose | ||
+ | medium. | ||
+ | A linear relationship between O.D. and dry cell weight is observed. | ||
+ | </p> | ||
+ | |||
+ | <ol start="2"> | ||
+ | <li>The elemental formula of E. coli should be fixed or varies within a | ||
+ | small range. Although there may exist slightly different in different | ||
+ | growth condition, we assume that such error can be ignore during the | ||
+ | following calculation.</li> | ||
+ | </ol> | ||
+ | |||
+ | <p class="hpcontent"> | ||
+ | After these two assumptions, the Xylose Consumption Index is designed to | ||
+ | evaluate | ||
+ | the carbon fixation ability of each strain. The definition of the index is | ||
+ | xylose | ||
+ | consumption over O.D. 600. O.D. 600 measurement can be viewed as the weight of | ||
+ | carbon of the bacteria. The index shows the ratio of xylose consumption per | ||
+ | biomass. For wild type E. coli, it only consumes xylose (the sole carbon source | ||
+ | provided in our medium) as its carbon source. Although some native E. coli | ||
+ | pathway | ||
+ | may utilize CO2 (such as lipid synthesis), the amount is too small to consider. | ||
+ | As | ||
+ | for engineered strain, carbon dioxide can be utilized as it’s carbon source. By | ||
+ | producing same amount of carbon biomass, it requires less xylose. We can thus | ||
+ | compare the XUI of each strain to determine the possible strain that fix | ||
+ | carbon. | ||
+ | The less the XUI in the sample, the more possibility that it fix carbon. | ||
+ | </p> | ||
+ | |||
+ | <img class="contentimg" src=" "> | ||
+ | |||
+ | <p class="hpcontent"> | ||
+ | We use the Dinitrosalicylic Acid (DNS) reducing sugar assay to measure the | ||
+ | xylose | ||
+ | concentration in the medium. Under base solution, DNS will turn to brown color | ||
+ | while reacting with reductive sugar in high temperature. In the specific | ||
+ | temperature range, the color will have linear relationship with the reductive | ||
+ | sugar | ||
+ | concentration. We can thus measure the xylose concentration at O.D.540. | ||
+ | </p> | ||
+ | |||
+ | <img class="contentimg" src=" "> | ||
+ | |||
+ | <p class="hpcontent"> | ||
+ | Fig. 10 Shows the calibration line of DNS assay kit. | ||
+ | </p> | ||
+ | |||
+ | <p class="hpcontent"> | ||
+ | Before measuring the XUI, we observe the growth curve of each strain. We found | ||
+ | out | ||
+ | that W3110(L5T7) constructed strain cannot grow in altered M9 solution. | ||
+ | W3110(L5T7) | ||
+ | is a newly constructed strain, we are not quite certain its characteristic. We | ||
+ | eliminate this strain from the following experiment. BL21(DE3) and W3110 | ||
+ | constructed strains show little growth after 24 hours. | ||
+ | </p> | ||
+ | |||
+ | |||
+ | |||
+ | <img class="contentimg" src=" "> | ||
+ | |||
+ | <p class="hpcontent"> | ||
+ | Fig. 11 shows the growth of W3110(L5T7), BL21(DE3), W3110 incubated in normal | ||
+ | incubator for 24 hours. The growth of W3110(L5T7) is not obvious while other | ||
+ | strains shows growth after 24hours. | ||
+ | </p> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | |||
+ | <div id="Bioreactor"> | ||
+ | </br></br></br></br> | ||
+ | <h3>Bioreactor</h3> | ||
+ | <div id="pt"> | ||
+ | <p class="pcontent">We developed a closed bioreactor system and implemented online | ||
+ | monitoring system | ||
+ | which can live monitoring several environmental parameters. Here is the detail | ||
+ | of our bioreactor.</p> | ||
+ | </div> | ||
+ | |||
+ | <img class="contentimg" src=""> | ||
+ | |||
+ | <div id="pt"> | ||
+ | <p class="pcontent">Gas inlet port are located on the bioreactor’s lower part while | ||
+ | outlet port are | ||
+ | located on the bioreactor’s upper lid. As low concentration CO2 enters the | ||
+ | bioreactor, it flows | ||
+ | through the diffuser refiner and dissolves in the buffered medium to form acid. | ||
+ | A pH sensor and | ||
+ | temperature sensor is installed to monitor the bioreactor tank for further | ||
+ | control implementation. | ||
+ | Besides, the CO2 concentration level of exhaust gas is monitored by a CO2 gas | ||
+ | sensor, which is | ||
+ | mounted on the upper lid. These sensor’s output is connected to an Arduino | ||
+ | analog input and sensor | ||
+ | readings are displayed on a serial LCD which is attached on the lid of | ||
+ | bioreactor. The data is then | ||
+ | uploaded in real time to a web server via WiFi by using Arduino WiFi Shield.</br></p> | ||
+ | </div> | ||
+ | </br> | ||
+ | <h8>DIY Stirrer</h8></br> | ||
+ | |||
+ | <img class="contentimg" src="https://static.igem.org/mediawiki/2018/6/69/T--NCKU_Tainan--Capture.PNG"> | ||
+ | |||
+ | <div id="pt"> | ||
+ | <p class="pcontent">To prevent sedimentation of cells at the bottom of bioreactor, | ||
+ | we build our own | ||
+ | 3D printed slow speed magnetic stirrer which permits gentle mixing of | ||
+ | microcarrier cell cultures. | ||
+ | The 3D printed magnet bed is designed specifically for two magnets and can be | ||
+ | fitted on the DC | ||
+ | motor. The stirrer works by using a DC motor to spin two magnets with opposite | ||
+ | polarity, which | ||
+ | could create a magnetic field in the bioreactor and cause the stir bar to spin | ||
+ | and mix the | ||
+ | contents. For controlling the speed of the DC motor, we use Arduino and L298N | ||
+ | to control the input | ||
+ | voltage to the motor by using PWM signal.</p> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div id="Nutrient_tank"> | ||
+ | </br></br></br></br> | ||
+ | <h3>Nutrient tank</h3> | ||
+ | <div id="pt"> | ||
+ | <p class="pcontent">Besides, we also implemented fed-batch culture system in our | ||
+ | design. Nutrients are fed to the bioreactor during cultivation to prevent | ||
+ | nutrient depletion. The nutrients are pumped into the growth chamber at a rate | ||
+ | proportional to the growth factor of the culture, which is determined | ||
+ | experimentally through the doubling time of the particular bacterial strain.</p> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | <div id="Materials_required"> | ||
+ | </br></br></br></br> | ||
+ | <h3>Materials required</h3> | ||
+ | <div id="pt"> | ||
+ | <ul> | ||
+ | <li>Acrylic Sheet</li> | ||
+ | <li>Arduino UNO</li> | ||
+ | <li>Power Supply</li> | ||
+ | <li>Batteries</li> | ||
+ | <li>Rotameter</li> | ||
+ | <li>pH meter</li> | ||
+ | <li>Thermometer(DS18B20)</li> | ||
+ | <li>CO2 sensor(MG811)</li> | ||
+ | <li>Wi-fi sensor(ESP8266 NodeMcu)</li> | ||
+ | <li>Geared DC Motor</li> | ||
+ | <li>Tubes</li> | ||
+ | <li>Magnets</li> | ||
+ | <li>3D Printed Structure</li> | ||
+ | <li>Nuts and Screws</li> | ||
+ | <li>Wires</li> | ||
+ | <li>Pumps</li> | ||
+ | </ul> | ||
+ | </br></br></br></br> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
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Revision as of 13:51, 12 October 2018
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