Difference between revisions of "Team:KU Leuven/Research/Results"
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− | The Centre of Microbial and Plant Genetics (KU Leuven) provided us with three <i>E. coli</i> K-12 strains with each one representing the | + | The Centre of Microbial and Plant Genetics (KU Leuven) provided us with three <i>E. coli</i> K-12 strains with each one representing the knock-out for the genes <i>tar</i>, <i>tsr</i> or <i>cheZ</i>. The kanamycin cassette of the <i>tar</i> knock-out strain was removed by the enzyme flippase on pCP20. This excision was checked by PCR. The original knock-out strain of <i>tar</i> was used as a positive control giving a band at 1232 bp on gel. If the cassette is removed, a band at 438 bp is visible. Ten colonies were tested and all have lost their cassette (Figure 1).</p> |
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− | <p>The PCP20 plasmid contains a temperature sensitive origin of replication. To remove this plasmid, the colonies were grown overnight at 42°C. PCP20 is resistant to ampicillin | + | <p>The PCP20 plasmid contains a temperature sensitive origin of replication. To remove this plasmid, the colonies were grown overnight at 42°C. PCP20 is resistant to ampicillin - this characteristic is useful to verify the removal of the plasmid. Single colonies were streaked on one LB plate with and one without ampicillin. Figure 2 proves that the PCP20 plasmid is removed in all mutant cells.</p> |
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− | <p>We | + | <p>We received the lysate from Oscar Torres. The donor strains (ΔcheZ and Δtsr) were infected with this lysate. In figure 3, the plaques, as a result of the infection, are visible. Some of the plaques will contain DNA of ΔcheZ and Δtsr due the sloppy packaging mechanism of the phage P1.</p> |
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− | <p>The lysate was plated out on LB plates as control. No colonies are visible in figure 4, this means that | + | <p>The lysate was plated out on LB plates as control. No colonies are visible in figure 4, this means that the lysate is not contaminated by cells.</p> |
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− | <p>The plaques of the acceptor strains were extracted and different amounts of lysate were used to infect our donor strain (Δtar). The | + | <p>The plaques of the acceptor strains were extracted and different amounts of lysate were used to infect our donor strain (Δtar). The resulted cells were plated out on kanamycin plates to select the right colonies (Figure 4).</p> |
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− | <p> | + | <p>The Tar knock-out cells without the kanamycin cassette were also plated out on kanamycin as a control. In figure 5 is visible that there is no growth. |
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− | In the first step, the <i>Chromobacterium violacein</i> CV026 was grown with different concentrations of OHHL. The <i>C. violacein</i> CV026 was added to the mixtures at an OD of 1.11. | + | In the first step, the <i>Chromobacterium violacein</i> CV026 was grown together with different concentrations of OHHL. The <i>C. violacein</i> CV026 was added to the mixtures at an OD of 1.11. The cells were grown for 24 hours in air-lid culture tubes at 30 °C in a shaking incubator (200 rpm). In Figure 16, it is clearly visible that a violet pigment is produced. </p> |
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− | <p> First the OD of our cultures | + | <p> First the OD of our cultures was measured in a cuvette (1 cm). Then the violacein was isolated from the cells by centrifugation, resuspension in DMSO and a second centrifugation step (Figure 17). |
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<div id=figure17>Figure 17</div> | <div id=figure17>Figure 17</div> | ||
− | Violacein | + | Violacein was removed from the samples by centrifugation and resuspension in DMSO. Click to enlarge</h4> |
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− | <p>After isolating violacein from our samples, 200 µL was pipetted into a 96-well falcon microtiter plate and measured at 585 nm. In total, three technical replicates were measured to estimate the pipetting and measuring error (Figure 18). | + | <p>After isolating violacein from our samples, 200 µL was pipetted into a 96-well falcon microtiter plate and the absorbance was measured at 585 nm. In total, three technical replicates were measured to estimate the pipetting and measuring error (Figure 18). |
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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. |
− | < | + | </p> |
− | + | <p>Please note that we were only able to do one attempt on this experiment. | |
− | + | </p> | |
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− | <h2> | + | <h2>BioBricks construction</h2> |
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<img src="https://static.igem.org/mediawiki/2015/3/38/KU_Leuven_BB.jpg" width='50%'></a> | <img src="https://static.igem.org/mediawiki/2015/3/38/KU_Leuven_BB.jpg" width='50%'></a> | ||
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− | <div id=figure21>Figure 21</div></h4></div></div> | + | <div id=figure21>Figure 21</div>Gel to check directionality. A band around 1400bp using the insert-reverse primer pair (up) show the gene in correct orientation (forward), a band around 1600bp using the insert-forward primer (down) show the gene in reversed orientation. </h4></div></div> |
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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 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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<div id="image22"> | <div id="image22"> | ||
− | <a class="example-image-link" href="https:// | + | <a class="example-image-link" href="https://2015.igem.org/File:KU_Leuven_GelPurification.jpeg" |
data-lightbox="example-set" data-title="Method1Test"> | data-lightbox="example-set" data-title="Method1Test"> | ||
− | <img src="https://static.igem.org/mediawiki/2015/3/ | + | <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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+ | <a class="example-image-link" href="https://static.igem.org/mediawiki/2015/3/34/KU_Leuven_Fluorescent.jpeg" | ||
+ | data-lightbox="example-set" data-title="Method1Test"> | ||
+ | <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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