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+ | <div class="container content"> | ||
+ | <h1 class="head">Measurement</h1> | ||
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+ | <div class="col-2 side"> | ||
+ | <div id="sidelist" class="list-group"> | ||
+ | <a class="list-group-item list-group-item-action" href="#achievement">Achievement</a> | ||
+ | <a class="list-group-item list-group-item-action" href="#XUI">XUI</a> | ||
+ | <a class="list-group-item list-group-item-action" href="#Carbon_Fixation">Carbon Fixation</a> | ||
+ | <a class="list-group-item list-group-item-action" href="#Reference">Reference</a> | ||
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+ | <div id="achievement"> | ||
+ | <h3>Achievement</h3> | ||
+ | <div class="achievementborder"> | ||
+ | <ol> | ||
+ | <br> | ||
+ | <li class="bigli">Develop a new measurement approach to determine the carbon fixation ability of each strain </li> | ||
+ | <br> | ||
+ | <li class="bigli">Estimate the carbon fixation amount with our experiment result </li> | ||
+ | <br> | ||
+ | </ol> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="XUI"> | ||
+ | <h3>The Xylose Utilization Index</h3> | ||
+ | <p class="pcontent">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="pcontent">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="pcontent">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="pcontent">To define the XUI index, we firstly made two assumptions: </p> | ||
+ | <ol> | ||
+ | <li class="licontent">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> | ||
+ | <div class="centerimg"> | ||
+ | <img class="smallimg" src="fig1.png"> | ||
+ | <p class="smallp">Fig 1. 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> | ||
+ | </div> | ||
+ | <li class="licontent">The elemental formula of <i>E. coli</i> 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="pcontent">Combine these two assumptions, we can conclude that in a fixed O.D. 600 value, | ||
+ | the composite weight of carbon is also fixed. | ||
+ | Thus, O.D. 600 can be considered equivalent to carbon weight of the bacteria. | ||
+ | </p> | ||
+ | <p class="pcontent">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 <i>E. coli</i>, it only consumes xylose (the sole carbon source provided in our medium) | ||
+ | as its carbon source. Although some native <i>E. coli</i> pathway may utilize CO<sub>2</sub> | ||
+ | (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="gif" src=""> | ||
+ | <p class="pcontent">We utilize the Xylose Utilization Index to compare the carbon fixation efficiency of | ||
+ | each strain and prove the function of each system. | ||
+ | For the experiment result, please view the Result(hyperlink) page. | ||
+ | </p> | ||
+ | </div> | ||
+ | |||
+ | <div id="Carbon_Fixation"> | ||
+ | <h3>Carbon Fixation amount estimation</h3> | ||
+ | <p class="pcontent">To find out how much and how efficient genetically engineered <i>E. coli</i> can fix | ||
+ | carbon dioxide, we use the material balance concept to evaluate the heterotrophic CO<sub>2</sub> fixation process. | ||
+ | Consider a system composed of a single component, the general material balance can be written as | ||
+ | </p> | ||
+ | <p class="pcontent">{Input to the system} – {Output to the system} = {Accumulation in the system}</p> | ||
+ | <p class="pcontent"> | ||
+ | A system can be defined as an arbitrary portion of a process considered for analysis, | ||
+ | in which in this case, is an engineered carbon capturing <i>E. coli</i>. | ||
+ | </p> | ||
+ | <p class="pcontent">The engineered <i>E. coli</i> BL21(DE3) are cultured in M9 medium with formula adjusted | ||
+ | so that xylose is the sole carbon source. | ||
+ | The aforementioned M9 Medium contains 0.4% xylose and 1/1000 LB medium | ||
+ | (the carbon proportion of LB medium can be ignored). | ||
+ | By applying the law of conservation of mass, which states that mass may neither be created nor destroyed, | ||
+ | the material balance for carbon in an engineered <i>E. coli</i> may simply be written as | ||
+ | </p> | ||
+ | <p class="pcontent">{C<sub>co<sub>2</sub> in</sub>} + {C<sub>xylose</sub>} - {C<sub>CO<sub>2</sub> out</sub>} - {C<sub>waste</sub>} = {C<sub>biomass</sub>}......(1)</p> | ||
+ | <p class="pcontent">Considering the difficulties in measuring carbon in <i>E. coli</i> metabolic waste | ||
+ | and that C<sub>waste</sub> would be positive, the equation reduces to | ||
+ | </p> | ||
+ | <p class="pcontent">{C<sub>co<sub>2</sub> in</sub>} - {C<sub>CO<sub>2</sub></sub> out} ≥ {C<sub>biomass</sub>} - {C<sub>xylose</sub>}......(2)</p> | ||
+ | <p class="pcontent">Let {C<sub>co<sub>2</sub> net</sub>} = {C<sub>co<sub>2</sub> in</sub>} - {C<sub>CO<sub>2</sub> out</sub>}, equation (2) further simplifies to </p> | ||
+ | <p class="pcontent">{C<sub>co<sub>2</sub> net</sub>} ≥ {C<sub>biomass</sub>} - {C<sub>xylose</sub>}......(3)</p> | ||
+ | <p class="pcontent">If C<sub>waste</sub> is very small and negligible, | ||
+ | we can obtain the net amount of carbon dioxide fixed over time. If, on the contrary, | ||
+ | Cwaste cannot be neglected, equation (3) allows us to estimate the minimum net amount of carbon dioxide fixed. | ||
+ | </p> | ||
+ | <p>C<sub>biomass</sub> can be calculate by multiplying O.D. 600 to DCW and mass percent of carbon in <i>E. coli</i> biomass. | ||
+ | The O.D. 600 of engineered <i>E. coli</i> is measured after a 12-hour cultivation and the result obtained is 0.45O.D. . | ||
+ | Yin Li et al. reported that dry cell weight (DCW) of <i>E. coli</i> is $${0.35g \over {L \cdot O.D. 600}}$$ , determined by experiment. | ||
+ | <i>E. coli</i> biomass contains 48% of carbon by mass | ||
+ | </p> | ||
+ | <p class="pcontent">C<sub>biomass</sub> = 0.45 × 0.35 × 48% = 0.0756g/L</p> | ||
+ | <p class="pcontent">On the other hand, C<sub>xylose</sub> can be calculated by multiplying the amount of xylose consumed | ||
+ | per unit volume of broth to the mass percent of carbon in xylose. | ||
+ | Xylose consumption is calculated by using a DNS kit that measures the concentration of reducing sugar | ||
+ | and the result obtained is 0.172324g of xylose consumed per litre of M9 medium. | ||
+ | Carbon weight percentage of xylose is 40%.</p> | ||
+ | <p class="pcontent">C<sub>xylose</sub> = 0.172324 × 40% = 0.0689296 g/L</p> | ||
+ | <p class="pcontent">By equation (3)</p> | ||
+ | <p class="pcontent">C<sub>co<sub>2</sub> net</sub> = 0.0756 - 0.0689296 = 0.0066704 g/L</p> | ||
+ | <p class="pcontent">Since the <i>E. coli</i> has been cultured for 12 hours, we can calculate the rate of carbon fixation by</p> | ||
+ | <p class="pcontnet">$${Rate \ of \ carbon \ fixation = {C_{co_{2} \ net} \over 12} = {0.0066704 \over 12} = {0.5558{g \over {L \cdot hr}}}}$$</p> | ||
+ | <p class="pcontent">To find out how much carbon in biomass comes from the carbon in CO<sub>2</sub> captured by the heterotrophic microbes, | ||
+ | divide the net amount of carbon fixed by the mass percent of carbon in biomass. | ||
+ | </p> | ||
+ | <p class="pcontent">$${{Ratio \ of \ carbon \ in \ CO_2 \ fixed \ to \ carbon \ in \ biomass} = {0.0066704 \over 0.0756} = 8.82 \%}$$</p> | ||
+ | </div> | ||
+ | |||
+ | <div id="Reference"> | ||
+ | <h3>Reference</h3> | ||
+ | <ol> | ||
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+ | <li class="smallp"></li> | ||
+ | <li class="smallp"></li> | ||
+ | <li class="smallp"></li> | ||
+ | <li class="smallp"></li> | ||
+ | </ol> | ||
+ | </div> | ||
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Revision as of 17:46, 10 October 2018