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| - | {{Kyoto_Foreground|active_page=project}}
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| - | {{Kyoto_Background}}
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| - | {{Kyoto_WikiDesign}}
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| - | = '''Project Hunger''' =
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| - | Production of needless enzymes is a heavy burden especially when the resource is scarce. This can be reduced by using nitrogen regulatory proteins, NtrB and NtrC<sup>†</sup> which activate σ<sup>54</sup>promoter when the supply of nitrogen is not enough. NtrB and NtrC are coded in glnL and glnG, respectively.However, σ-54 promoter has never evaluated quantitatively. Here, we evaluated the relationships between expression under σ<sup>54</sup>promoter and that of these genes by the aid of RPU(a relative promoter unit).
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| - | We decided to use RPU which has many advantages for quantitative assay. We have used GFP fluorescence to measure RPU, but this time, we tried to calculate RPU another way for this reason:
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| - | *we know that it is a lot of trouble to calculate RPU using GFP fluorescence.
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| - | *we imagined that the concentration of the glutamine doesn’t keep because σ-54 promoter relates to the metabolism of glutamine.
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| - | This is because when we calculate RPU using GFP, we need measure the concentration of glutamine at two points in an exponential growth phase, but we have following problems.
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| - | *before an exponential growth phase, the concentration of glutamine changed.
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| - | *the concentration of glutamine is different between one point and another point.
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| - | We devise the new way of calculating RPU using the amount of mRNA. This way used following equation.
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| - | † NtrB and NtrC are otherwise called NR<sub>II</sub>, NR<sub>I</sub>
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| - | ところでこの遺伝子はもともとBioBrickに登録されていたものか、今回新たに作ったもののどちらなのでしょう
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| - | <br>http://partsregistry.org/wiki/index.php/Part:BBa_J64978
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| - | <br>などがもう登録されているようですが
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| - | <br>リンク先を見ましたがどうなんでしょうね とりあえずこの登録されているパーツのシーケンスは見ましたが今回我々が作ろうとしたglnLとは全然違うシーケンスでした 名に由来かは知りませんがE.coliのものじゃないでしょうねby Hashiya
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| - | == '''Introduction''' ==
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| - | For every living thing, needless biological activity is not efficient. Cells must be controlled so that enzymes are produced only when they are necessary. Ammonia is an essential nitrogen source for the bactria. When enteric bacteria are deprived of ammonia, they express glnA to produce glutamine synthetase(GS). Nitrogen is used in the reaction of
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| - | Glutamate + NH<sub>3</sub> + ADP → Glutamine + ADP + phosphate
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| - | GS
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| - | The expression of glnA is regulated by several proteins including NtrB, NtrC, Pii.
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| - | Fig. NtrCがσ54,RNAポリメラーゼ,DNAの関係図(窒素源あり/なし)
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| - | Fig. NtrBCの関係図
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| - | Fig. Ntr, Pii, GSの関係図
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| | == '''Method''' == | | == '''Method''' == |
| | We created two following constructions to measure gene expression depending on the concentration of glutamine. We used BBa_J23101 as a promoter for construction1,which is used as the criteria, and binding sites and σ-54 promoter for construction2, which is object to be measured. | | We created two following constructions to measure gene expression depending on the concentration of glutamine. We used BBa_J23101 as a promoter for construction1,which is used as the criteria, and binding sites and σ-54 promoter for construction2, which is object to be measured. |
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| | </style></html> | | </style></html> |
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| | <div><span class="eqn">[[File:Kyoto_kiga_eqn1.png|center]]</span><span class="no">(1)</span></div> | | <div><span class="eqn">[[File:Kyoto_kiga_eqn1.png|center]]</span><span class="no">(1)</span></div> |
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| | <div><span class="eqn">[[File:Kyoto_kiga_eqn8.png|center]]</span><span class="no">(8)</span></div> | | <div><span class="eqn">[[File:Kyoto_kiga_eqn8.png|center]]</span><span class="no">(8)</span></div> |
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| - | == '''Result''' ==
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| - | Gragh1
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| - | The data, which is not corrected by internal control
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| - | *Although the amount of RNA is different first, we think more than the twice difference is not accidental error of the experiment.
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| - | *After 15 minutes, expression level greatly changed, but from 0 to 10 min, expression level is steady state.
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| - | *We think TBP and PGK are unsuitable for internal control.
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| - | *The behaviors of GAPDH and actin is analogous.
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| - | Gragh2～5
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| - | The data, which is corrected by internal control
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| - | *when we applied internal control to GAPDH, the changes of actin are little(×1.0～1.2)
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| - | *when we applied internal control to actin, the changes of actin are little(×0.8～1.0)
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| - | *We concluded that GAPDH and actin are suitable for internal control.
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| - | == '''Discussion''' ==
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| - | == '''Reference''' ==
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We created two following constructions to measure gene expression depending on the concentration of glutamine. We used BBa_J23101 as a promoter for construction1,which is used as the criteria, and binding sites and σ-54 promoter for construction2, which is object to be measured.
We also measured the amount of mRNA to calculate RPU of the steady state for the each concentration of glutamine. First, we made following preliminary experiment to measure the length of times before steady state.
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.PNG)
