Difference between revisions of "Team:KU Leuven/Research/Results"
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− | <p>In figure 20, our standard curve is plotted. A linear correlation between the absorbance and the concentration OHHL can be found. The variance of the technical replicates, visualised by the error bars, and the variance of the regression curve, shown by the R | + | <p>In figure 20, our standard curve is plotted. A linear correlation between the absorbance and the concentration OHHL can be found. The variance of the technical replicates, visualised by the error bars, and the variance of the regression curve, shown by the R<sup>2</sup> value, can be explained by pipetting and measuring errors. Also, working with biological cells generates a background noise. This standard curve could give an estimation of bacterial AHL production. But it is important to keep in mind that there is background noise. Please note that we only had two attemps to perform this experiment, the first time the broad range was investigated, the second time the more narrow range was investigated. Optimisation of this curve can be done by making more biological and technical replicas. |
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+ | <p>In comparison to HPLC, the chosen method would be less time consuming without the need of specialized equipment. Due to a lack of time, we were not able to complete the plasmid assembly and therefore we could not quantify the amount of leucine produced by the designed bacteria. But we did an attempt to test the quantification method by making the standard curve. </p> | ||
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− | The standard curve from 0 to 100 µM did not give | + | The standard curve from 0 to 100 µM did not give clear signals, so the working method needs optimisation. Reasons for this result could be the use of different enzyms than mentioned in the article. Because the enzymes originate from other organisms than mentioned in Kugimiya and Fukada (2015), it is possible that the enzymes have another efficiency and as a consequence need another ratio substrate over enzyme. Additionally, we did not have the same equipment as described in the article. We had to manually pipet the luminol solution while in equipment described in the acticle this happens autimatically. Probably there was too much time between adding the luminol solution and measuring. |
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− | + | <p>Please note that we were only able to do one attempt on this experiment. | |
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<p>The correct colonies were selected, miniprepped and sent for sequencing. <br/> | <p>The correct colonies were selected, miniprepped and sent for sequencing. <br/> | ||
− | To characterize the CheZ-GFP BioBrick, the fragment containing a RBS was cloned directly after a strong promotor (BBa_J23101).<br/> | + | To characterize the CheZ-GFP BioBrick, the fragment containing a RBS was cloned directly after a strong promotor (BBa_J23101). Figure 22 shows the gel right before ligation.<br/> |
− | The colonies were checked by restriction mapping using BcuI and PstI (results not shown). The DNA sequence was also confirmed by DNA sequencing. Results can be provided by email. The presence of colonies expressing GFP proves that the plasmid was designed and cloned correctly (Figure | + | The colonies were checked by restriction mapping using BcuI and PstI (results not shown). The DNA sequence was also confirmed by DNA sequencing. Results can be provided by email. The presence of colonies expressing GFP proves that the plasmid was designed and cloned correctly (Figure 23). </p> |
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+ | <a class="example-image-link" href="https://2015.igem.org/File:KU_Leuven_GelPurification.jpeg" | ||
+ | data-lightbox="example-set" data-title="Method1Test"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/2/22/KU_Leuven_GelPurification.jpeg" width='50%'></a> | ||
+ | <h4> | ||
+ | <div id=figure22>Figure 22</div>Gel after purification. Lanes 2-5: insert (1400bp). Lane 6: linearized vector. Lanes 7-10 : insert. Lane 11: linearized vector</h4></div></div> | ||
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data-lightbox="example-set" data-title="Method1Test"> | data-lightbox="example-set" data-title="Method1Test"> | ||
<img src="https://static.igem.org/mediawiki/2015/3/34/KU_Leuven_Fluorescent.jpeg" width='50%'></a> | <img src="https://static.igem.org/mediawiki/2015/3/34/KU_Leuven_Fluorescent.jpeg" width='50%'></a> | ||
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− | <div id= | + | <div id=figure23>Figure 23</div>GFP is expressed in the cells. This confirms the correct construction of the BioBrick</h4></div></div> |
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Latest revision as of 09:35, 20 October 2015
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Results
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