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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<a class="example-image-link" href="https://static.igem.org/mediawiki/2015/6/64/KU_Leuven_1-2-3_RD.jpg" | <a class="example-image-link" href="https://static.igem.org/mediawiki/2015/6/64/KU_Leuven_1-2-3_RD.jpg" | ||
data-lightbox="example-set" data-title="Method1Test"> | data-lightbox="example-set" data-title="Method1Test"> | ||
− | <img src="https://static.igem.org/mediawiki/2015/6/64/KU_Leuven_1-2-3_RD.jpg" height=' | + | <img src="https://static.igem.org/mediawiki/2015/6/64/KU_Leuven_1-2-3_RD.jpg" height='200px'></a> |
<a class="example-image-link" href="https://static.igem.org/mediawiki/2015/a/a0/KU_Leuven_5-6_RD.jpg" | <a class="example-image-link" href="https://static.igem.org/mediawiki/2015/a/a0/KU_Leuven_5-6_RD.jpg" | ||
data-lightbox="example-set" data-title="Method1Test"> | data-lightbox="example-set" data-title="Method1Test"> | ||
− | <img src="https://static.igem.org/mediawiki/2015/a/a0/KU_Leuven_5-6_RD.jpg" height=' | + | <img src="https://static.igem.org/mediawiki/2015/a/a0/KU_Leuven_5-6_RD.jpg" height='200px'></a> |
<h4> | <h4> | ||
<div id=figure14>Figure 14</div>Restriction digestion map using NotI and BglII for 1-2-3 and NcoI for 5-6</h4> | <div id=figure14>Figure 14</div>Restriction digestion map using NotI and BglII for 1-2-3 and NcoI for 5-6</h4> | ||
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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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<img class="example-image" src="https://static.igem.org/mediawiki/2015/d/d2/KU_Leuven_ResultOHHL1.jpeg" width="50%"></a> | <img class="example-image" src="https://static.igem.org/mediawiki/2015/d/d2/KU_Leuven_ResultOHHL1.jpeg" width="50%"></a> | ||
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− | <div id= | + | <div id=figure16>Figure 16</div> |
Culture tubes of inoculated <i>Chromobacterium violacein</i> CV026 with different amounts of OHHL. click to enlarge</h4> | Culture tubes of inoculated <i>Chromobacterium violacein</i> CV026 with different amounts of OHHL. click to enlarge</h4> | ||
</div> | </div> | ||
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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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<img class="example-image" src="https://static.igem.org/mediawiki/2015/8/84/KU_Leuven_ResultOHHL2.jpeg" width="50%"></a> | <img class="example-image" src="https://static.igem.org/mediawiki/2015/8/84/KU_Leuven_ResultOHHL2.jpeg" width="50%"></a> | ||
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− | <div id= | + | <div id=figure17>Figure 17</div> |
− | Violacein | + | Violacein was removed from the samples by centrifugation and resuspension in DMSO. Click to enlarge</h4> |
</div> | </div> | ||
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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 ( | + | <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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<img class="example-image" src="https://static.igem.org/mediawiki/2015/5/5f/KU_Leuven_ResultOHHL.jpg" width="50%"></a> | <img class="example-image" src="https://static.igem.org/mediawiki/2015/5/5f/KU_Leuven_ResultOHHL.jpg" width="50%"></a> | ||
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− | <div id= | + | <div id=figure18>Figure 18</div> |
96-well falcon microtiter plate containing the three technical replicates of the dilution series. Click to enlarge</h4> | 96-well falcon microtiter plate containing the three technical replicates of the dilution series. Click to enlarge</h4> | ||
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− | <p>First a broad concentration range was used to estimate the linear part. This range was made by a two-fold dilution series. When measuring the absorbance, LB medium was used as blank. Later the absorbance values of the blank were subtracted from the absorbance values of the standards. Then these values were divided by the absorbance values at 600 nm measured in the microtiterplate which gives an indication of the cell number. Eventually the values were corrected by setting the point with a concentration of 0 mM OHHL in the origin. These values were plotted in figure | + | <p>First a broad concentration range was used to estimate the linear part. This range was made by a two-fold dilution series. When measuring the absorbance, LB medium was used as blank. Later the absorbance values of the blank were subtracted from the absorbance values of the standards. Then these values were divided by the absorbance values at 600 nm measured in the microtiterplate which gives an indication of the cell number. Eventually the values were corrected by setting the point with a concentration of 0 mM OHHL in the origin. These values were plotted in figure 19. The concentrations 2.56 mM and 5.12 mM were left out because these values were not distinguishable from the blank. This can be explained because the OHHL is dissolved in DMSO which lowers the growth of <i>C. violaceum</i> CV026. Between the concentrations 0.64 mM and 1.28 mM, the curve is stagnating. This is probably due to saturation of the medium or the inhibitory effect of DMSO. In a next step, a more narrow range was investigated. |
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<img class="example-image" src="https://static.igem.org/mediawiki/2015/2/24/KU_Leuven_ResultOHHL4.jpeg" width="50%"></a> | <img class="example-image" src="https://static.igem.org/mediawiki/2015/2/24/KU_Leuven_ResultOHHL4.jpeg" width="50%"></a> | ||
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− | <div id= | + | <div id=figure19>Figure 19</div> |
First estimation of the OHHL standard curve. click to enlarge</h4> | First estimation of the OHHL standard curve. click to enlarge</h4> | ||
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− | <p>In figure | + | <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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<img class="example-image" src="https://static.igem.org/mediawiki/2015/8/89/KU_Leuven_ResultOHHL5.png" width="50%"></a> | <img class="example-image" src="https://static.igem.org/mediawiki/2015/8/89/KU_Leuven_ResultOHHL5.png" width="50%"></a> | ||
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− | <div id= | + | <div id=figure20>Figure 20</div> |
Standard curve ranging from 0 to 0.1 mM. The error bars represent the standard deviation between the technical replicates.</h4> | Standard curve ranging from 0 to 0.1 mM. The error bars represent the standard deviation between the technical replicates.</h4> | ||
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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> | ||
<p> | <p> | ||
− | 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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− | To make characterization easier, DNA for fusion proteins was designed and ordered in gBlock format. A His-tag was fused to LuxI and GFP to CheZ. Using PCR, the iGEM prefix and suffix were added. Cutting the PCR fragments with EcoRI and PstI was not favorable due to the non-existing extra nucleotides necessary for the enzymes to cut. Therefore, EagI, a restriction enzyme also cutting in the NotI site was used to clone the fragment in an empty pSB1C3 vector.<br/> | + | To make characterization easier, DNA for fusion proteins was designed and ordered in gBlock format. A His-tag was fused to LuxI and GFP to CheZ. These two new constructs are the basic BioBricks submitted. Using PCR, the iGEM prefix and suffix were added to this basic parts as well as to the parts containing a RBS. Cutting the PCR fragments with EcoRI and PstI was not favorable due to the non-existing extra nucleotides necessary for the enzymes to cut. Therefore, EagI, a restriction enzyme also cutting in the NotI site was used to clone the fragment in an empty pSB1C3 vector.<br/> |
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BBa_J23101 was transformed in <i>E. cloni</i> to multiply the amount of vector DNA. | BBa_J23101 was transformed in <i>E. cloni</i> to multiply the amount of vector DNA. | ||
− | After miniprepping, the | + | After miniprepping, the BioBrick was cut with EagI together with a phosphatase to overcome self-ligation.<br/> |
+ | T4 DNA ligase was used and a 1:2 vector-insert ratio was added. Since digestion by EagI does not allow directional cloning, multiple colonies were tested by colony PCR to check insert directionality (Figure 21). | ||
+ | <div class ="center"> | ||
+ | <div id="image21"> | ||
+ | <a class="example-image-link" href="https://static.igem.org/mediawiki/2015/3/38/KU_Leuven_BB.jpg" | ||
+ | data-lightbox="example-set" data-title="Method1Test"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/3/38/KU_Leuven_BB.jpg" width='50%'></a> | ||
+ | <h4> | ||
+ | <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> | ||
+ | <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). 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 23). </p> | ||
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+ | <div class ="center"> | ||
+ | <div id="image22"> | ||
+ | <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> | ||
+ | <br/> | ||
+ | |||
+ | <div class ="center"> | ||
+ | <div id="image23"> | ||
+ | <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> | ||
+ | <h4> | ||
+ | <div id=figure23>Figure 23</div>GFP is expressed in the cells. This confirms the correct construction of the BioBrick</h4></div></div> | ||
+ | <br/> | ||
+ | |||
+ | <p>Further characterization could be done by transforming the <i>cheZ</i> knockout Keio strain with this plasmid. These cells should then regain their possibility to swim.</p> | ||
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Latest revision as of 09:35, 20 October 2015
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Results
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