Difference between revisions of "Team:KU Leuven/Research/Results"

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main

Research

Interlab

Modeling

Outreach

Future

Team

Notebook

Results

Double Knockouts

The Centre of Microbial and Plant Genetics (KU Leuven) provided us with three E. coli K-12 strains with each one representing the knock-out for the genes tar, tsr or cheZ. The kanamycin cassette of the tar knock-out strain was removed by the enzyme flippase on pCP20. This excision was checked by PCR. The original knock-out strain of tar 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).

Figure 1
Excision of the kanamycin cassette of the knock-out of tar. As a positive control, the original knock-out of tar was used which should show a band at 1232 basepairs. If the kanamycin cassette is removed, a band at 438 bp will be visible. Besides, a 1 kb Plus DNA ladder of GeneRuler was used.

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.

Figure 2
Test to prove that the ampicillin resistant PCP20 plasmid is removed. The left plate contains ampicillin, the right plate contains no antibiotic.

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.

Figure 3
Plaques after infection of our donor strains (ΔcheZ and Δtsr).

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.

Figure 4
Control to check if the lysate of ΔcheZ and Δtsr is not contaminated with cells.

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).

Figure 5
Infection of the acceptor strain (Δtar) by lysate originating from donor strains (ΔcheZ and Δtsr)

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.

Figure 6
Tar knock-out cells without the kanamycin cassette were plated out on kanamycin plates as control.

Different colonies were screened to confirm the knock-out in cheZ. If the cassette is not there, a band should show at 863 bp. If the cassette is still there, there should be a band at 581 bp. As a positive control we used a knockout Tar strain which does not contain the kanamycin cassette anymore.

Figure 7
PCR to check that cheZ is knocked out in Δtar. The positive control is a knock-out in tar which lost the plasmid.

To confirm that the knock-out in tsr was successful, a PCR was performed. A knock-out in tsr should give a band at 1304 bp while the original gene should give a band at 1964 bp. As positive control, a knock-out in tar which lost the kanamycin cassette was used.

Figure 8
PCR to check that tsr is knocked out in Δtar. The positive control is a knock-out in tar which lost the plasmid.

Especially in the new constructed ΔtarΔcheZ, the chance exists that the knock-out in tar is undone due the sloppy packaging mechanism of the phage P1. Therefore, the knock-out in tar is checked again by PCR (see figure 9). The first positive control (c1) is a tar knock-out who has lost the kanamycin cassette. As a second control (c2), a cheZ knock-out strain was used. The gel of figure 8 shows that all our ΔtarΔtsr strains are ok, while only two of the ΔtarΔcheZ are still ok.

Figure 9
PCR to check the knock-out in tar after the P1 transduction

Finally, the all the genes of the operon containing cheZ were checked by PCR (see figure 10). As positive control the tar knock-out which does not contain the plasmid anymore was used, but each time with the primers corresponding to the checked gene. The negative control contains the mastermix but without template.

Figure 10
PCR to check the operon of ΔtarΔcheZ

Motility Test

The cheZ knock-outs are not able to swim anymore. Therefor we performed a phenotypical motility test. The result is visible in figure 11.

Figure 11
Motility test of our cheZ knock-out

Gibson Assembly Method

Method 1
The first Gibson assembly was performed using only gBlocks without a vector. Since the T5 exonuclease cleaves nucleotides from 5’ to 3’, an extra fill-in step was added after the 1 hour incubation at 50°C. Appropriate primers were added together with Phusion DNA polymerase and T4 DNA ligase to recreate the blunt insert fragment. The ligated fragments were checked by PCR and both reactions showed a band at the expected height (Figure 12). The 1-2-3 reaction however showed a lot of aspecific bands. The 5-6 fragment was stored untill further use and the 1-2-3 fragment was then digested to be directionally cloned in a pSB1C3 backbone and transformed in electrocompetent E. cloni cells.

Figure 12
PCR check of assembled gBlocks.

Positive colonies of 1-2-3 were further checked by colony PCR. Of this check, only one colony seemed to have the right insert (Figure 13). Restriction digestion was however negative (Figure not shown).

Figure 13
Colony PCR of 1-2-3 assembled pSB1C3 plasmids

Method 2
Because our method was aberrant compared to the IDT protocol, a vector with correct overhangs was PCR amplified. This vector was used in a one-reaction Gibson assembly. After transformation, liquid cultures were grown from which the plasmid DNA was isolated. Restriction mapping was performed (Figure 14) and showed presence of only pUC19.

Figure 14
Restriction digestion map using NotI and BglII for 1-2-3 and NcoI for 5-6

Method 3
To get rid of the template pUC, the PCR amplified backbone was digested with DpnI. This restriction enzyme only cuts methylated DNA and is thus inactive on PCR amplified pUC and synthesised gBlocks.

Even after digestion with DpnI, pUC19 kept appearing (Figure 15). The correct plasmid was gel purified and again transformed in E. cloni. After miniprep, gel analysis was negative for the assembled plasmid and positive for pUC19 only.

Figure 15
Miniprepped positive pSB1C3-1-2-3 (lane a-b), pUC-1-2-3 (lane c-k) and pUC-5-6 (lane l-r) clones

Method 4
To optimize the vector PCR, pUC19 was first linearized using the unique restriction enzyme XbaI. Again, the PCR product was digested with DpnI. This plasmid was also transformed as a negative control and did not show any positive colonies. The plates that should contain the insert did show positive colonies. Gel analysis of the positive colonies again showed only a positive band for pUC19. Even though it appeared that the assembly worked (shown by PCR and gel separation) and that the template was completely degraded, the pUC19 vector reappears almost after every purification. In the future, plasmid insert load should be reduced to make the plasmid more stable over time.

OHHL detection

In the first step, the Chromobacterium violacein CV026 was grown together with different concentrations of OHHL. The C. violacein 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.

Figure 16
Culture tubes of inoculated Chromobacterium violacein CV026 with different amounts of OHHL. click to enlarge

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).

Figure 17
Violacein was removed from the samples by centrifugation and resuspension in DMSO. Click to enlarge

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).

Figure 18
96-well falcon microtiter plate containing the three technical replicates of the dilution series. Click to enlarge

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 C. violaceum 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.

Figure 19
First estimation of the OHHL standard curve. click to enlarge

In the second experiment, a range between 0.01 and 0.10 mM was investigated. The difference with the previous experiment is that the OD was first measured in 1 cm cuvettes and that the violacein was afterwards isolated. In this way, the plate reader does not contain cells. This optimisation is done, because we noticed in the previous experiment that violacein was produced more quickly in a culture tube than in a microtiterplate, probably due to the amount of supplied oxygen. Another reason for this working method is because estimating the amount of cells is more standardised by using cuvettes of 1 cm than using a microtiterplate.

Table 1 contains the processed values of the absorbance measurements. The processing is similar to the previous experiment. First the absorbance of DMSO was subtracted from the absorbance of the standards. Thereafter, these values were divided by the OD measured in 1 cm cuvettes. And finally, these values were corrected by putting the standard with a concentration of 0 mM OHHL in the origin.

Table 1: Absorbance at 585 nm divided by the OD

Concentration (mM)
	0.10	0.09	0.08	0.07	0.06	0.05	0.04	0.03	0.02	0.01	0.00
T1 (corrected)	0.126	0.072	0.110	0.093	0.057	0.083	0.052	0.049	0.076	0.011	0.000
T2 (corrected)	0.130	0.073	0.110	0.076	0.057	0.085	0.048	0.043	0.076	0.008	0.000
T3 (corrected)	0.121	0.071	0.078	0.091	0.054	0.083	0.053	0.049	0.051	0.008	0.000
Average	0.125	0.072	0.099	0.087	0.056	0.084	0.051	0.047	0.068	0.009	0.000
Standard deviation	0.004	0.001	0.019	0.009	0.002	0.001	0.003	0.003	0.015	0.002	0.000

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 R² 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.

Figure 20
Standard curve ranging from 0 to 0.1 mM. The error bars represent the standard deviation between the technical replicates.

Leucine detection

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.

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.

Please note that we were only able to do one attempt on this experiment.

BioBricks construction

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.

BBa_J23101 was transformed in E. cloni to multiply the amount of vector DNA. After miniprepping, the BioBrick was cut with EagI together with a phosphatase to overcome self-ligation.
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).

Figure 21
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.

The correct colonies were selected, miniprepped and sent for sequencing.
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.
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).

Figure 22
Gel after purification. Lanes 2-5: insert (1400bp). Lane 6: linearized vector. Lanes 7-10 : insert. Lane 11: linearized vector

Figure 23
GFP is expressed in the cells. This confirms the correct construction of the BioBrick

Further characterization could be done by transforming the cheZ knockout Keio strain with this plasmid. These cells should then regain their possibility to swim.

Idea

A detailed description about the interaction between our two cells and the genetic circuit can be found here.

Methods

Here you can find the performed steps to create two different cell types and detailed quantification methods to determine interesting parameters.

Basic Parts

Our new, self-designed basic parts necessary to control cell-cell interactions and E. Coli motility.

Composite Parts

Our basic parts were combined with each other and with existing iGEM promotors, RBS and terminators.

Back

Go back to the Research page.

Idea

A detailed description about the interaction between our two cells and the genetic circuit can be found here.

Methods

Here you can find the performed steps to create two different cell types and detailed quantification methods to determine interesting parameters.

Basic Parts

Our new, self-designed basic parts necessary to control cell-cell interactions and E. Coli motility.

Composite Parts

Our basic parts were combined with each other and with existing iGEM promotors, RBS and terminators.

Back

Go back to the Research page.

Contact

Address: Celestijnenlaan 200G room 00.08 - 3001 Heverlee
Telephone: +32(0)16 32 73 19
Email: igem@chem.kuleuven.be

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+<link rel="stylesheet" type="text/css"
+href="https://2015.igem.org/Template:KU_Leuven/Lightbox/CSS?action=raw&ctype=text/css" />
+</head>
 <body>
-<div class="example">
+<div class="summaryheader">
-<div class="one">
+  <div class="summaryimg">
-<h2>This is exaple one</h2>
+  <img src="https://static.igem.org/mediawiki/2015/5/5b/KU_Leuven_Banner_Geel.jpg" width="100%">
+  <div class="head">
+     <h2>Results</h2>
+  </div>
+ </div>
 </div>
-<div id="one">
-tiralalalala <br/>
-tiralalala <br/>
+<!------------------------------------------------------Begin Content--------------------------------------------------------->
-tiralalala<br/>
+<div class="summarytext1">
+<div class="center">
+<br/>
+<div class="togglebar">
+<div class="toggleone">
+<h2>Double Knockouts</h2>
 </div>
+<div id="toggleone">
+<p>
+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>
+<br/>
+<div class="center">
+<div id="image1">
+<a class="example-image-link" href="https://static.igem.org/mediawiki/2015/8/8f/KU_Leuven_knockoutTar.jpg" data-lightbox="example-set" data-title="knockout"><img class="example-image" src="https://static.igem.org/mediawiki/2015/8/8f/KU_Leuven_knockoutTar.jpg" alt="knockout" width="100%" height="100%"></a>
+<h4><div id=figure1>Figure 1</div> Excision of the kanamycin cassette of the knock-out of tar.
+As a positive control, the original knock-out of tar was used which should show a band at 1232 basepairs. If the kanamycin cassette is removed, a band at 438 bp will be visible. Besides, a 1 kb Plus DNA ladder of GeneRuler was used.</h4>
+</h4>
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-<div class="example">
-<div class="two">
-<h2>This is example two</h2>
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-<div id="two">
+<br/>
-pompompom<br/>
-pompompom<br/>
+<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>
-pompompom<br/>
+<br/>
+<div class="center">
+<div id="image2">
+<a class="example-image-link" href="https://static.igem.org/mediawiki/2015/8/88/KU_Leuven_Knockout_Tar_Kan_-P.jpg" data-lightbox="example-set" data-title="knockout"><img class="example-image" src="https://static.igem.org/mediawiki/2015/8/88/KU_Leuven_Knockout_Tar_Kan_-P.jpg" width="82.5%" alt="knockout" width="45%" height="45%"></a>
+<h4><div id=figure2>Figure 2</div>Test to prove that the ampicillin resistant PCP20 plasmid is removed. The left plate contains ampicillin, the right plate contains no antibiotic.</h4>
 </div>
 </div>
-<br>
+<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>
-<br>
+</br>
-<div>
-   <h1> Protocols </h1>
+<div class="center">
+<div id="image3">
+<a class="example-image-link" href="https://static.igem.org/mediawiki/2015/e/e4/KU_Leuven_dTsr_0.1P1.jpg" data-lightbox="example-set" data-title="dTsr"><img class="example-image" src="https://static.igem.org/mediawiki/2015/e/e4/KU_Leuven_dTsr_0.1P1.jpg" alt="knockout" width="49%" ></a>
+<a class="example-image-link" href="https://static.igem.org/mediawiki/2015/8/84/KU_Leuven_ECloni_plaques.jpg" data-lightbox="example-set" data-title="plaques"><img class="example-image" src="https://static.igem.org/mediawiki/2015/8/84/KU_Leuven_ECloni_plaques.jpg" alt="plaques" width="49%"></a>
+<h4><div id=figure3>Figure 3</div>Plaques after infection of our donor strains (Δ<i>cheZ</i> and <i>Δtsr</i>).</h4>
+</div>
 </div>
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-          <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordionprotocols" href="#collapse1" aria-expanded="false" aria-controls="collapse1">
- Competent cells
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-            <ol style="list-style: decimal inside; font-size:19px;">
-              <li>
-                <p class="lead">Plate Top10 cells and incubate at 37ºC overnight</p>
-              </li>
-              <li>
-                <p class="lead">Pick one colony, inoculate in LB media and incubate overnight while shaking at 37ºC</p>
-              </li>
-              <li>
+<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>
-                <p class="lead">Dilute the culture in fresh medium, and continue the incubation until the OD<sub>600</sub>= 0.4-0.6</p>
+<br/>
-              </li>
+<div class ="center">
+ <div id="image4">
+    <a class="example-image-link" href="https://static.igem.org/mediawiki/2015/4/48/KU_Leuven_-C_lysate_cheZ_and_Tsr.jpg"
+    data-lightbox="example-set" data-title="Tsr">
+    <img class="example-image" src="https://static.igem.org/mediawiki/2015/4/48/KU_Leuven_-C_lysate_cheZ_and_Tsr.jpg" width='92.5%'></a>
+<h4><div id=figure4>Figure 4</div>Control to check if the lysate of ΔcheZ and Δtsr is not contaminated with cells.</h4>
+</div>
+</div>
+<br/>
-              <li>
+<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>
-                <p class="lead">Centrifugation 5 min. at 4000 rpm.</p>
+<br/>
-              </li>
+<div class ="center">
+ <div id="image5">
+    <a class="example-image-link" href="https://static.igem.org/mediawiki/2015/a/a5/KU_Leuven_acceptor_strain_%2B_phages.jpg"
+    data-lightbox="example-set" data-title="acceptor_strain">
+    <img class="example-image" src="https://static.igem.org/mediawiki/2015/a/a5/KU_Leuven_acceptor_strain_%2B_phages.jpg" width='65%'></a>
+<h4>
+<div id=figure5>Figure 5</div>
+Infection of the acceptor strain (Δtar) by lysate originating from donor strains (ΔcheZ and Δtsr)</h4>
+</div>
+</div>
+<br/>
+<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.
+</p>
+<br/>
-              <li>
+<div class ="center">
-                <p class="lead">Pellet cells and resuspend in 100mM of CaCl<sub>2</sub> solution</p>
+ <div id="image6">
-              </li>
+    <a class="example-image-link" href="https://static.igem.org/mediawiki/2015/4/40/KU_Leuven_P1_-C_acceptor_cell.jpg"
+    data-lightbox="example-set" data-title="acceptor_cell">
+    <img class="example-image" src="https://static.igem.org/mediawiki/2015/4/40/KU_Leuven_P1_-C_acceptor_cell.jpg" width="50%"></a>
+<h4>
+<div id=figure6>Figure 6</div>
+Tar knock-out cells without the kanamycin cassette were plated out on kanamycin plates as control.</h4>
+</div>
+</div>
+<br/>
+<p>Different colonies were screened to confirm the knock-out in cheZ. If the cassette is not there, a band should show at 863 bp. If the cassette is still there, there should be a band at 581 bp. As a positive control we used a knockout Tar strain which does not contain the kanamycin cassette anymore.</p>
+<br/>
-              <li>
+<div class ="center">
-                <p class="lead">Incubate on ice for 20 min</p>
+ <div id="image7">
-              </li>
+    <a class="example-image-link" href="https://static.igem.org/mediawiki/2015/b/b2/KU_Leuven_dCheZ_Casette.jpg"
+    data-lightbox="example-set" data-title="dCheZ_Casette">
+    <img class="example-image" src="https://static.igem.org/mediawiki/2015/b/b2/KU_Leuven_dCheZ_Casette.jpg" width='50%'></a>
+<h4>
+<div id=figure7>Figure 7</div>
+PCR to check that cheZ is knocked out in Δtar. The positive control is a knock-out in tar which lost the plasmid. </h4>
+</div>
+</div>
-              <li>
+<br/>
-                <p class="lead">Centrifugation 5 min at 3000 rpm</p>
+<p>To confirm that the knock-out in tsr was successful, a PCR was performed. A knock-out in tsr should give a band at 1304 bp while the original gene should give a band at 1964 bp. As positive control, a knock-out in tar which lost the kanamycin cassette was used. </p>
-              </li>
+<br/>
-              <li>
+<div class ="center">
-                <p class="lead">Pellet cells and suspend again in 100mM CaCl<sub>2</sub> solution</p>
+ <div id="image8">
-              </li>
+    <a class="example-image-link" href="https://static.igem.org/mediawiki/2015/9/9f/KU_Leuven_dTsr_Casette.jpg"
+    data-lightbox="example-set" data-title="dTsr_Casette">
+    <img src="https://static.igem.org/mediawiki/2015/9/9f/KU_Leuven_dTsr_Casette.jpg" width='50%'></a>
+<h4>
+<div id=figure8>Figure 8</div>
+PCR to check that tsr is knocked out in Δtar. The positive control is a knock-out in tar which lost the plasmid. </h4>
+</div>
+</div>
-              <li>
+<br/>
-                <p class="lead">Incubate on ice for 60 min</p>
+<p>Especially in the new constructed ΔtarΔcheZ, the chance exists that the knock-out in tar is undone due the sloppy packaging mechanism of the phage P1. Therefore, the knock-out in tar is checked again by PCR (see figure 9). The first positive control (c1) is a tar knock-out who has lost the kanamycin cassette. As a second control (c2), a cheZ knock-out strain was used. The gel of figure 8 shows that all our ΔtarΔtsr strains are ok, while only two of the ΔtarΔcheZ are still ok. </p>
-              </li>
+<br/>
+<div class ="center">
+ <div id="image9">
+     <a class="example-image-link" href="https://static.igem.org/mediawiki/2015/0/06/KU_Leuven_dTar_Check.jpg"
+    data-lightbox="example-set" data-title="dTar_Check">
+    <img src="https://static.igem.org/mediawiki/2015/0/06/KU_Leuven_dTar_Check.jpg" width='50%'></a>
+<h4>
+<div id=figure9>Figure 9</div>
+PCR to check the knock-out in tar after the P1 transduction</h4>
+</div>
+</div>
+<br/>
+<p>Finally, the all the genes of the operon containing cheZ were checked by PCR (see figure 10). As positive control the tar knock-out which does not contain the plasmid anymore was used, but each time with the  primers corresponding to the checked gene. The negative control contains the mastermix but without template.</p>
+<br/>
-              <li>
+<div class ="center">
-                <p class="lead">Centrifugation 5 min at 3000 rpm</p>
+ <div id="image10">
-              </li>
+    <a class="example-image-link" href="https://static.igem.org/mediawiki/2015/0/0c/KU_Leuven_operoncheck_dTardCheZ.jpeg"
+    data-lightbox="example-set" data-title="operoncheck_dTardCheZ">
+    <img src="https://static.igem.org/mediawiki/2015/0/0c/KU_Leuven_operoncheck_dTardCheZ.jpeg" width='50%'></a>
+<h4>
+<div id=figure10>Figure 10</div>
+PCR to check the operon of ΔtarΔcheZ</h4>
+</div>
+</div>
+<div class="whiterow"></div>
+</div>
+</div>
+<br/>
+<div class="togglebar">
+<div class="toggletwo">
+<h2>Motility Test</h2>
+</div>
+<div id="toggletwo">
+<p>
+The cheZ knock-outs are not able to swim anymore. Therefor we performed a phenotypical motility test. The result is visible in figure 11. </p>
+<br/>
+<div class ="center">
+ <div id="image11">
+    <a class="example-image-link" href="https://static.igem.org/mediawiki/2015/3/3c/KU_Leuven_Motility_Test_CheZ.jpg"
+    data-lightbox="example-set" data-title="Test_CheZ">
+    <img src="https://static.igem.org/mediawiki/2015/3/3c/KU_Leuven_Motility_Test_CheZ.jpg" width='50%'></a>
+<h4>
+<div id=figure11>Figure 11</div>
+Motility test of our cheZ knock-out</h4>
+</div>
+</div>
-              <li>
+<div class="whiterow"></div>
-                <p class="lead">Add a solution of 100mM CaCl<sub>2</sub> + 40% glycerol</p>
+</div>
-              </li>
+</div>
+<br/>
+<div class="togglebar">
+<div class="togglethree">
+<h2>Gibson Assembly Method</h2>
+</div>
+<div id="togglethree">
-              <li>
+<p><b>Method 1</b><br/>
-                <p class="lead">Store immediately at -80ºC</p>
+The first Gibson assembly was performed using only gBlocks without a vector. Since the T5 exonuclease cleaves nucleotides from 5’ to 3’, an extra fill-in step was added after the 1 hour incubation at 50°C. Appropriate primers were added together with Phusion DNA polymerase and T4 DNA ligase to recreate the blunt insert fragment. The ligated fragments were checked by PCR and both reactions showed a band at the expected height (Figure 12). The 1-2-3 reaction however showed a lot of aspecific bands. The 5-6 fragment was stored untill further use and the 1-2-3 fragment was then digested to be directionally cloned in a pSB1C3 backbone and transformed in electrocompetent <i>E. cloni</i> cells.
-              </li>
+</p>
-            </ol>
-          </div>
-        </div>
-      </div>
-      <div class="panel panel-default">
+<div class ="center">
-        <div class="panel-heading panel-title" role="tab" id="heading2">
+ <div id="image12">
-          <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordionprotocols" href="#collapse2" aria-expanded="false" aria-controls="collapse2">
+    <a class="example-image-link" href="https://static.igem.org/mediawiki/2015/d/da/KU_Leuven_Method1.jpg"
-           Transpormation
+    data-lightbox="example-set" data-title="Method1Test">
-          </a>
+    <img src="https://static.igem.org/mediawiki/2015/d/da/KU_Leuven_Method1.jpg" width='50%'></a>
-        </div>
+<h4>
-        <div id="collapse2" class="panel-collapse collapse" role="tabpanel" aria-labelledby="heading2">
+<div id=figure12>Figure 12</div>PCR check of assembled gBlocks.</h4>
-          <div class="panel-body">
+<div class="whiterow"></div>
-            <ol style="list-style: decimal inside; font-size:10px;">
+</div>
-              <li>
+</div>
-                <p class="lead">Take the competent cells from the storage at -80ºC and leave them on ice for 10-15 min</p>
-              </li>
-              <li>
+<p>Positive colonies of 1-2-3 were further checked by colony PCR. Of this check, only one colony seemed to have the right insert (Figure 13). Restriction digestion was however negative (Figure not shown). </p>
-                <p class="lead">Add 1-2 µL of plasmid solution to the 50 µL cell tube</p>
-              </li>
-              <li>
+<div class ="center">
-                <p class="lead">Incubate on ice for 30 min</p>
+ <div id="image13">
-              </li>
+    <a class="example-image-link" href="https://static.igem.org/mediawiki/2015/8/85/KU_Leuven_123CPCR_M1.jpg"
+    data-lightbox="example-set" data-title="Colony PCR 123-Method1">
+    <img src="https://static.igem.org/mediawiki/2015/8/85/KU_Leuven_123CPCR_M1.jpg" width='50%'></a>
+<h4>
+<div id=figure13>Figure 13</div>Colony PCR of 1-2-3 assembled pSB1C3 plasmids</h4>
+<div class="whiterow"></div>
+</div>
+</div>
-              <li>
+<p><b>Method 2</b><br/>
-                <p class="lead">Heat-shock the cells at 42ºC for 45s</p>
+Because our method was aberrant compared to the IDT protocol, a vector with correct overhangs was PCR amplified. This vector was used in a one-reaction Gibson assembly. After transformation, liquid cultures were grown from which the plasmid DNA was isolated. Restriction mapping was performed (Figure 14) and showed presence of only pUC19. </p>
-              </li>
-              <li>
+<div class ="center">
-                <p class="lead">Incubate on ice for 2 min</p>
+ <div id="image14">
-              </li>
+    <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">
+    <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"
+    data-lightbox="example-set" data-title="Method1Test">
+    <img src="https://static.igem.org/mediawiki/2015/a/a0/KU_Leuven_5-6_RD.jpg" height='200px'></a>
+<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 class="whiterow"></div>
+</div>
+</div>
-              <li>
+<p><b>Method 3</b><br/>
-                <p class="lead">Add 500 µL of LB media and incubate 60 min at 37ºC</p>
+To get rid of the template pUC, the PCR amplified backbone was digested with DpnI. This restriction enzyme only cuts methylated DNA and is thus inactive on PCR amplified pUC and synthesised gBlocks.<br/>
-              </li>
+<br/>
+Even after digestion with DpnI, pUC19 kept appearing (Figure 15). The correct plasmid was gel purified and again transformed in <i>E. cloni</i>. After miniprep, gel analysis was negative for the assembled plasmid and positive for pUC19 only. </p>
+<div class ="center">
+ <div id="image15">
+    <a class="example-image-link" href="https://static.igem.org/mediawiki/2015/3/3d/KU_Leuven_GelSeparation.jpg"
+    data-lightbox="example-set" data-title="Method1Test">
+    <img src="https://static.igem.org/mediawiki/2015/3/3d/KU_Leuven_GelSeparation.jpg" width='50%'></a>
+<h4>
+<div id=figure15>Figure 15</div>Miniprepped positive pSB1C3-1-2-3 (lane a-b), pUC-1-2-3 (lane c-k) and pUC-5-6 (lane l-r) clones</h4></div></div>
+<br/>
+<p><b>Method 4</b><br/>
+To optimize the vector PCR, pUC19 was first linearized using the unique restriction enzyme XbaI. Again, the PCR product was digested with DpnI. This plasmid was also transformed as a negative control and did not show any positive colonies. The plates that should contain the insert did show positive colonies. Gel analysis of the positive colonies again showed only a positive band for pUC19. Even though it appeared that the assembly worked (shown by PCR and gel separation) and that the template was completely degraded, the pUC19 vector reappears almost after every purification. In the future, plasmid insert load should be reduced to make the plasmid more stable over time. </p>
-              <li>
-                <p class="lead">Plate the cultures on agar plate</p>
-              </li>
-            </ol>
-          </div>
-        </div>
-      </div>
-      <div class="panel panel-default">
-        <div class="panel-heading panel-title" role="tab" id="heading3">
-          <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordionprotocols" href="#collapse3" aria-expanded="false" aria-controls="collapse3">
-            Cell Cultures
-          </a>
-        </div>
-        <div id="collapse3" class="panel-collapse collapse" role="tabpanel" aria-labelledby="heading3">
-          <div class="panel-body">
-            <ol style="list-style: decimal inside; font-size:19px;">
-              <li>
+<div class="whiterow"></div>
-                <p class="lead">Pick a single colony from a plate or cryostock</p>
+</div>
-              </li>
+</div>
+<br/>
+<div class="togglebar">
+<div class="togglefour">
+<h2>OHHL detection</h2>
+</div>
+<div id="togglefour">
+<p>
+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>
-              <li>
+<div class ="center">
-                <p class="lead">Put the colony in a 50 mL sterile tube and add 5-10 mL of LB fresh medium</p>
+ <div id="imageAHL1">
-              </li>
+    <a class="example-image-link" href="https://static.igem.org/mediawiki/2015/d/d2/KU_Leuven_ResultOHHL1.jpeg"
+    data-lightbox="example-set" data-title="acceptor_cell">
+    <img class="example-image" src="https://static.igem.org/mediawiki/2015/d/d2/KU_Leuven_ResultOHHL1.jpeg" width="50%"></a>
+<h4>
+<div id=figure16>Figure 16</div>
+Culture tubes of inoculated <i>Chromobacterium violacein</i> CV026 with different amounts of OHHL. click to enlarge</h4>
+</div>
+</div>
-              <li>
+<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).
-                <p class="lead">Put the tube to incubate for at least 16 h, at 37ºC and 200 rpm</p>
+</p>
-              </li>
-            </ol>
-          </div>
-        </div>
-      </div>
-      <div class="panel panel-default">
+<div class ="center">
-        <div class="panel-heading panel-title" role="tab" id="heading4">
+ <div id="imageAHL2">
-          <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordionprotocols" href="#collapse4" aria-expanded="false" aria-controls="collapse4">
+    <a class="example-image-link" href="https://static.igem.org/mediawiki/2015/8/84/KU_Leuven_ResultOHHL2.jpeg"
-            DNA Electrophoresis
+    data-lightbox="example-set" data-title="acceptor_cell">
-          </a>
+    <img class="example-image" src="https://static.igem.org/mediawiki/2015/8/84/KU_Leuven_ResultOHHL2.jpeg" width="50%"></a>
-        </div>
+<h4>
-        <div id="collapse4" class="panel-collapse collapse" role="tabpanel" aria-labelledby="heading4">
+<div id=figure17>Figure 17</div>
-          <div class="panel-body">
+Violacein was removed from the samples by centrifugation and resuspension in DMSO. Click to enlarge</h4>
-            <h3>Gel making</h3>
+</div>
-            <ol style="list-style: decimal inside; font-size:10px;">
+</div>
-              <li>
+<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).
-                <p class="lead">Prepare 200 mL of TAE buffer</p>
+</p>
-              </li>
-              <li>
-                <p class="lead">Mix the TAE solution with 2g of agarose (for 1%)</p>
-              </li>
-              <li>
+<div class ="center">
-                <p class="lead">Heat the solution to boiling, and then cool it to 50ºC aprox.</p>
+ <div id="imageAHL3">
-              </li>
+    <a class="example-image-link"https://static.igem.org/mediawiki/2015/5/5f/KU_Leuven_ResultOHHL.jpg"
+    data-lightbox="example-set" data-title="acceptor_cell">
+    <img class="example-image" src="https://static.igem.org/mediawiki/2015/5/5f/KU_Leuven_ResultOHHL.jpg" width="50%"></a>
+<h4>
+<div id=figure18>Figure 18</div>
+-well falcon microtiter plate containing the three technical replicates of the dilution series. Click to enlarge</h4>
+</div>
+</div>
-              <li>
+<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.
-                <p class="lead">Add 5 µL of Ethidium bromide to the solution</p>
+</p>
-              </li>
+<div class ="center">
+ <div id="imageAHL4">
+    <a class="example-image-link" href="https://static.igem.org/mediawiki/2015/2/24/KU_Leuven_ResultOHHL4.jpeg"
+    data-lightbox="example-set" data-title="acceptor_cell">
+    <img class="example-image" src="https://static.igem.org/mediawiki/2015/2/24/KU_Leuven_ResultOHHL4.jpeg" width="50%"></a>
+<h4>
+<div id=figure19>Figure 19</div>
+First estimation of the OHHL standard curve. click to enlarge</h4>
+</div>
+</div>
-              <li>
+<p>In the second experiment, a range between 0.01 and 0.10 mM was investigated. The difference with the previous experiment is that the OD was first measured in 1 cm cuvettes and that the violacein was afterwards isolated. In this way, the plate reader does not contain cells. This optimisation is done, because we noticed in the previous experiment that violacein was produced more quickly in a culture tube than in a microtiterplate, probably due to the amount of supplied oxygen. Another reason for this working method is because estimating the amount of cells is more standardised by using cuvettes of 1 cm than using a microtiterplate.
-                <p class="lead">Pour the solution in the electrophoresis vessel. Apply the combs.</p>
+</p>
-              </li>
+<p> Table 1 contains the processed values of the absorbance measurements. The processing is similar to the previous experiment. First the absorbance of DMSO was subtracted from the absorbance of the standards. Thereafter, these values were divided by the OD measured in 1 cm cuvettes. And finally, these values were corrected by putting the standard with a concentration of 0 mM OHHL in the origin.
+</p>
+<h4>Table 1: Absorbance at 585 nm divided by the OD </h4>
+<div class="center">
+<div class="datatable">
+<table>
+  <tr>
+    <th colspan="12" class="emerged">Concentration (mM) </th>
+  </tr>
+  <tr>
+    <th></th>
+    <th>0.10</th>
+    <th>0.09</th>
+    <th>0.08</th>
+    <th>0.07</th>
+    <th>0.06</th>
+    <th>0.05</th>
+    <th>0.04</th>
+    <th>0.03</th>
+    <th>0.02</th>
+    <th>0.01</th>
+    <th>0.00</th>
+  </tr>
+  <tr class="lightrow">
+    <th>T1 </br> (corrected)</th>
+    <td>0.126</td>
+    <td>0.072</td>
+    <td>0.110</td>
+    <td>0.093</td>
+    <td>0.057</td>
+    <td>0.083</td>
+    <td>0.052</td>
+    <td>0.049</td>
+    <td>0.076</td>
+    <td>0.011</td>
+    <td>0.000</td>
+  </tr>
+  <tr>
+    <th>T2 </br> (corrected)</th>
+    <td>0.130</td>
+    <td>0.073</td>
+    <td>0.110</td>
+    <td>0.076</td>
+    <td>0.057</td>
+    <td>0.085</td>
+    <td>0.048</td>
+    <td>0.043</td>
+    <td>0.076</td>
+    <td>0.008</td>
+    <td>0.000</td>
+  </tr>
+    <tr class="lightrow">
+    <th>T3 </br> (corrected)</th>
+    <td>0.121</td>
+    <td>0.071</td>
+    <td>0.078</td>
+    <td>0.091</td>
+    <td>0.054</td>
+    <td>0.083</td>
+    <td>0.053</td>
+    <td>0.049</td>
+    <td>0.051</td>
+    <td>0.008</td>
+    <td>0.000</td>
+  </tr>
+  <tr>
+    <th>Average</th>
+    <td>0.125</td>
+    <td>0.072</td>
+    <td>0.099</td>
+    <td>0.087</td>
+    <td>0.056</td>
+    <td>0.084</td>
+    <td>0.051</td>
+    <td>0.047</td>
+    <td>0.068</td>
+    <td>0.009</td>
+    <td>0.000</td>
+  </tr>
+    <tr class="lightrow">
+    <th>Standard deviation</th>
+    <td>0.004</td>
+    <td>0.001</td>
+    <td>0.019</td>
+    <td>0.009</td>
+    <td>0.002</td>
+    <td>0.001</td>
+    <td>0.003</td>
+    <td>0.003</td>
+    <td>0.015</td>
+    <td>0.002</td>
+    <td>0.000</td>
+  </tr>
+</table>
+</div></div>
-              <li>
+<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.
-                <p class="lead">Let it polymerize, and then cover it with TAE</p>
+</p>
-              </li>
-            </ol>
-            <h3>Gel running</h3>
-            <ol style="list-style: decimal inside; font-size:10px;">
-              <li>
-                <p class="lead">Add 1/6 of total volume of Loading buffer to every DNA sample.
-                  <li>
-                    <p class="lead">Remove the combs from the gel, and pipette DNA samples and DNA ladder
-                      <li>
-                        <p class="lead">Run at 100-130V for 30-60 min (depends on the fragments)</p>
-                      </li>
-            </ol>
-          </div>
-        </div>
-      </div>
-      <div class="panel panel-default">
-        <div class="panel-heading panel-title" role="tab" id="heading5">
-          <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordionprotocols" href="#collapse5" aria-expanded="false" aria-controls="collapse5">
-Ligation
-          </a>
-        </div>
-        <div id="collapse5" class="panel-collapse collapse" role="tabpanel" aria-labelledby="heading5">
-          <div class="panel-body">
-            <ol style="list-style: decimal inside; font-size:10px;">
-              <li>
-                <p class="lead">Add 20-100 ng of vector DNA (can be calculated from the DNA concentration in the sample)</p>
-              </li>
-              <li>
+<div class ="center">
-                <p class="lead">Add X ng of insert DNA. X is calculated using the length of both vector and insert and the molar ratio desired.</p>
+ <div id="imageAHL5">
-              </li>
+    <a class="example-image-link" href="https://static.igem.org/mediawiki/2015/8/89/KU_Leuven_ResultOHHL5.png"
+    data-lightbox="example-set" data-title="acceptor_cell">
+    <img class="example-image" src="https://static.igem.org/mediawiki/2015/8/89/KU_Leuven_ResultOHHL5.png" width="50%"></a>
+<h4>
+<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>
+</div>
+</div>
-              <li>
+<div class="whiterow"></div>
-                <p class="lead">Add 2µL of ligation buffer</p>
+</div>
-              </li>
+</div>
+<br/>
+<div class="togglebar">
+<div class="togglefive">
+<h2>Leucine detection</h2>
+</div>
+<div id="togglefive">
+<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>
+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>
-              <li>
+<br/>
-                <p class="lead">Add MQ water to set the final volume to 15-20</p>
+<br/>
-              </li>
-              <li>
+<div class="whiterow"></div>
-                <p class="lead"> Add 1 µL of T4 ligase (always at the end to keep the enzyme in optimal conditions)</p>
+</div>
-              </li>
+</div>
+<br/>
+<div class="togglebar">
+<div class="togglesix">
+<h2>BioBricks construction</h2>
+</div>
+<div id="togglesix">
+<p>
+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/>
+<br/>
+BBa_J23101 was transformed in <i>E. cloni</i> to multiply the amount of vector DNA.
+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/>
-              <li>
+<p>The correct colonies were selected, miniprepped and sent for sequencing. <br/>
-                <p class="lead">Incubate for at least 3 hours at 16ºC</p>
+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/>
-              </li>
+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>
-            </ol>
-          </div>
-        </div>
-      </div>
-      <div class="panel panel-default">
+<div class ="center">
-        <div class="panel-heading panel-title" role="tab" id="heading6">
+ <div id="image22">
-          <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordionprotocols" href="#collapse6" aria-expanded="false" aria-controls="collapse6">
+    <a class="example-image-link" href="https://2015.igem.org/File:KU_Leuven_GelPurification.jpeg"
-           Cell Cryostock
+    data-lightbox="example-set" data-title="Method1Test">
-          </a>
+    <img src="https://static.igem.org/mediawiki/2015/2/22/KU_Leuven_GelPurification.jpeg" width='50%'></a>
-        </div>
+<h4>
-        <div id="collapse6" class="panel-collapse collapse" role="tabpanel" aria-labelledby="heading6">
+<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>
-          <div class="panel-body">
+<br/>
-            <ol style="list-style: decimal inside; font-size:10px;">
-              <li>
-                <p class="lead">Take 1.5 mL from a freshly grown culture and put it in a 1.5 mL tube</p>
-              </li>
-              <li>
+<div class ="center">
-                <p class="lead">Spin the tube for 10 min at 2000 rpm</p>
+ <div id="image23">
-              </li>
+    <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/>
-              <li>
+<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>
-                <p class="lead"> Decant the supernatant without disturbing the pellet</p>
+<br/>
-              </li>
+<br/>
-              <li>
+<div class="whiterow"></div>
-                <p class="lead">Add 0.5 mL of LB media and 0.5 mL of glycerol 80% solution</p>
+</div>
-              </li>
+</div>
-              <li>
+</div>
-                <p class="lead">Mix by vortexing</p>
+</div>
-              </li>
+<br/>
-              <li>
+<div class="whiterow">
-                <p class="lead">Save in the -80ºC freezer</p>
+</div>
-              </li>
-            </ol>
-          </div>
-        </div>
-      </div>
-      <div class="panel panel-default">
-        <div class="panel-heading panel-title" role="tab" id="heading7">
-          <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordionprotocols" href="#collapse7" aria-expanded="false" aria-controls="collapse7">
-            Restriction (NEB enzymes)
-          </a>
-        </div>
-        <div id="collapse7" class="panel-collapse collapse" role="tabpanel" aria-labelledby="heading7">
-          <div class="panel-body">
-            <ol style="list-style: decimal inside; font-size:10px;">
-              <li>
-                <p class="lead">Add 1 µg of DNA (can be calculated from concentration in the sample)</p>
-              </li>
-              <li>
+<!--------------------------------------------------Foot don't touch!------------------------------------------------------>
-                <p class="lead">Add 5 µL of NEB buffer</p>
-              </li>
-              <li>
+<div class="subsections">
-                <p class="lead">Add 1 µL of restriction enzyme 1</p>
+<div class="subsectionwrapper">
-              </li>
+<div class="subimgrow">
-              <li>
+<div class="whitespaceside"></div>
-                <p class="lead">Add 1 µL of restriction enzyme 2</p>
+<div class="subimg">
-              </li>
+<a href="https://2015.igem.org/Team:KU_Leuven/Research/Idea">
+ <img src="https://static.igem.org/mediawiki/2015/5/5e/KU_Leuven_Wiki_Button_-_Idea2.png" width="100%" >
+</a>
+</div>
-              <li>
+<div class="whitespace">
-                <p class="lead">Add MQ water to set the final volume at 50 µL </p>
+</div>
-              </li>
-              <li>
+<div class="subimg">
-                <p class="lead">Mix the solution by flicking the tube</p>
+<a href="https://2015.igem.org/Team:KU_Leuven/Research/Methods">
-              </li>
+ <img src="https://static.igem.org/mediawiki/2015/1/1a/KU_Leuven_Wiki_Button_-_Methods2.png" width="100%">
+</a>
+</div>
+<div class="whitespaceside"></div>
+</div>
-              <li>
+<div class="subtextrow">
-                <p class="lead">Spin-down in a microcentrifuge for 15 s</p>
+<div class="whitespaceside"></div>
-              </li>
+<div class="subtext">
+<a href="https://2015.igem.org/Team:KU_Leuven/Research/Idea">
+ <h2>Idea</h2>
+ <p> A detailed description about the interaction between our two cells and the genetic circuit can be found here.
+<br/>
+</p>
+</a>
+</div>
-              <li>
+<div class="whitespace">
-                <p class="lead">Incubate at 37ºC for 1-2 hours</p>
+</div>
-              </li>
-            </ol>
-          </div>
-        </div>
-      </div>
-      <div class="panel panel-default">
+<div class="subtext">
-        <div class="panel-heading panel-title" role="tab" id="heading8">
+<a href="https://2015.igem.org/Team:KU_Leuven/Research/Methods">
-          <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordionprotocols" href="#collapse8" aria-expanded="false" aria-controls="collapse8">
+ <h2>Methods</h2>
-            Plasmid isolation (from Promega PureYield ® Kit)
+ <p> Here you can find the performed steps to create two different cell types and detailed quantification methods to determine interesting parameters.
-          </a>
+</p>
-        </div>
+</a>
-        <div id="collapse8" class="panel-collapse collapse" role="tabpanel" aria-labelledby="heading8">
+</div>
-          <div class="panel-body">
+<div class="whitespaceside"></div>
-            <ol style="list-style: decimal inside; font-size:10px;">
+</div>
-              <li>
-                <p class="lead">Add 1.5 mL of bacterial culture in LB medium to a 1.5 mL micro-centrifuge tube. Centrifuge that tube at max speed for 3 min</p>
-              </li>
-              <li>
+<div class="subimgreadmore">
-                <p class="lead">Remove the supernatant, and add 600 µL of MQ water to the pellet</p>
+<div class="whitespaceside"></div>
-              </li>
+<div class="subimgrm">
+<a href=" https://2015.igem.org/Team:KU_Leuven/Research/Idea">
+<div id="more">
+<img src="https://static.igem.org/mediawiki/2015/7/73/KUL_Wiki_Button_-_Read_more.png" height="40%" width="85%" alt="Read more">
+</div>
+</a>
+</div>
-              <li>
+<div class="whitespace">
-                <p class="lead">Add 100 µL of Cell Lysis Buffer, and mix by inverting 6 times. The color change to blue indicates complete lysis</p>
+</div>
-              </li>
-              <li>
+<div class="subimgrm">
-                <p class="lead">Add 350 µL of cold (4-8ºC) Neutralization Buffer, and mix by inverting the tube. The color change to yellow indicates total neutralization</p>
+<a href="https://2015.igem.org/Team:KU_Leuven/Research/Methods">
-              </li>
+<div id="more">
+<img src="https://static.igem.org/mediawiki/2015/7/73/KUL_Wiki_Button_-_Read_more.png" height="40%" width="85%" alt="Read more">
+</div>
+</a>
+</div>
+<div class="whitespaceside"></div>
+</div>
+</div>
+<div class="whiterow"></div>
+<div class="subsectionwrapper">
+<div class="subimgrow">
+<div class="whitespace1"></div>
+<div class="subimg">
+<a href="https://2015.igem.org/Team:KU_Leuven/Research/Basic_Part">
+ <img src="https://static.igem.org/mediawiki/2015/e/e6/KU_Leuven_Wiki_Button_-_Parts2.png" width="100%">
+</a>
+</div>
-              <li>
+<div class="whitespace"></div>
-                <p class="lead">Centrifugate at maximum speed for 3 minutes, and transfer the supernatant to a PureYield Minicolumn
-                </p>
+<div class="subimg">
-              </li>
+<a href="https://2015.igem.org/Team:KU_Leuven/Research/Composite_Part">
+ <img src="https://static.igem.org/mediawiki/2015/0/08/KU_Leuven_Wiki_Button_-_Composite_parts2.png" width="100%">
+</a>
+</div>
-              <li>
+<div class="whitespace"></div>
-                <p class="lead">Place the minicolumn into a PureYield Collection Tube and centrifuge at maximum speed for 15 seconds
-                </p>
+<div class="subimg">
-              </li>
+<a href="https://2015.igem.org/Team:KU_Leuven/Research">
+ <img src="https://static.igem.org/mediawiki/2015/c/cb/KUL_Wiki_Button_-_Back.png" width="100%" ></a>
+</div>
+<div class="whitespace1"></div>
+</div>
-              <li>
+<div class="subtextrow">
-                <p class="lead">Discard the flowthrough and place the minicolumn again into the same PureYield Collection tube</p>
+<div class="whitespace1"></div>
-              </li>
+<div class="subtext">
+<a href="https://2015.igem.org/Team:KU_Leuven/Research/Basic_Part">
+ <h2>Basic Parts</h2>
+ <p>Our new, self-designed basic parts necessary to control cell-cell interactions and <i>E. Coli</i> motility.
+</p>
+</a>
+</div>
-              <li>
-                <p class="lead">Add 200 µL of Endotoxin Removal Wash to the minicolumn. Centrifuge at maximum speed for 15 seconds. Do not empty the Collection Tube now</p>
-              </li>
-              <li>
+<div class="whitespace">
-                <p class="lead"> Add 400 µL of Column Wash Solution to the minicolumn, and centrifuge at maximum speed for 30 seconds
+</div>
-                </p>
+<div class="subtext">
-              </li>
+<a href="https://2015.igem.org/Team:KU_Leuven/Research/Composite_Part">
+ <h2>Composite Parts</h2>
+ <p> Our basic parts were combined with each other and with existing iGEM promotors, RBS and terminators.
+</p>
+</a>
+</div>
-              <li>
+<div class="whitespace">
-                <p class="lead">Transfer the minicolumn to a clean 1.5 mL tube, and 30 µL of hot (50ºC, pre-warmed) MQ water directly to the minicolumn matrix. Let stand for 5 minutes at room temperature</p>
+</div>
-              </li>
-              <li>
+<div class="subtext">
-                <p class="lead">Centrifuge at maximum speed in a microcentrifuge for 15 seconds to elute plasmidic DNA. Cap the tube, and store the DNA solution at -20 ºC (or use it directly for cloning experiments)</p>
+<a href="https://2015.igem.org/Team:KU_Leuven/Research">
-              </li>
+ <h2>Back</h2>
-            </ol>
+ <p> Go back to the Research page.
-          </div>
+</p>
-        </div>
+</a>
-      </div>
+</div>
-      <div class="panel panel-default">
+<div class="whitespace1"></div>
-        <div class="panel-heading panel-title" role="tab" id="heading9">
+</div>
-          <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordionprotocols" href="#collapse9" aria-expanded="false" aria-controls="collapse9">
-          Gel isolation (from Promega Wizard ® Kit)
-          </a>
-        </div>
-        <div id="collapse9" class="panel-collapse collapse" role="tabpanel" aria-labelledby="heading9">
-          <div class="panel-body">
-            <ol style="list-style: decimal inside; font-size:10px;">
-              <li>
-                <p class="lead">Weigh a 1.5 mL microcentrifuge tube for each DNA fragment to be isolated, and record the weight</p>
-              </li>
-              <li>
+<div class="subimgreadmore">
-                <p class="lead">Visualize the DNA in the agarose gel using a long-wavelength UV lamp and an intercalating dye (Ethidium bromide). Irradiate the gel the minimum possible time to reduce nicking</p>
+<div class="whitespace1"></div>
-              </li>
+<div class="subimgrm">
+<a href="https://2015.igem.org/Team:KU_Leuven/Research/Basic_Part">
+<div id="more">
+<img src="https://static.igem.org/mediawiki/2015/7/73/KUL_Wiki_Button_-_Read_more.png" height="40%" width="85%" alt="Read more">
+</div>
+</a>
+</div>
-              <li>
+<div class="whitespace">
-                <p class="lead">Excise the DNA fragment of interest in a minimal volume of agarose using a clean scalpel or razor blade. Transfer the gel slice to a weighted 1.5 mL tube and record the weight, again. Subtract the previously measured tube weight to obtain
+</div>
-                  the weight of the gel slice containing the DNA fragment</p>
-              </li>
-              <li>
+<div class="subimgrm">
-                <p class="lead">Add Membrane Binding Solution at a ratio of 10 µL of solution per 10 mg of agarose gel slice</p>
+<a href="https://2015.igem.org/Team:KU_Leuven/Research/Composite_Part">
-              </li>
+<div id="more">
+<img src="https://static.igem.org/mediawiki/2015/7/73/KUL_Wiki_Button_-_Read_more.png" height="40%" width="85%" alt="Read more">
+</div>
+</a>
+</div>
-              <li>
+<div class="whitespace">
-                <p class="lead">Vortex the mixture and incubate at 50-65ºC for 10 minutes, or until the gel slice is completely dissolve in the liquid. You can vortex the tube every few minutes to increase the rate of agarose melting
+</div>
-                </p>
+<div class="subimgrm">
-              </li>
+<a href="https://2015.igem.org/Team:KU_Leuven/Research">
+<div id="back">
+<img src="https://static.igem.org/mediawiki/2015/7/73/KUL_Wiki_Button_-_Read_more.png" height="40%" width="85%" alt="Read more">
+</div>
+</a>
+</div>
+<div class="whitespace1"></div>
+</div>
-              <li>
+<div class="subimgrowm">
-                <p class="lead">Centrifuge the tube briefly at room temperature to ensure the contents are at the bottom of the tube. Once the agarose gel is melted, the gel will not re-solidify at room temperature</p>
+<div class="subimgm">
-              </li>
+ <a href="https://2015.igem.org/Team:KU_Leuven/Research/Idea" >
+     <b>Idea</b>
+ <img src="https://static.igem.org/mediawiki/2015/5/5e/KU_Leuven_Wiki_Button_-_Idea2.png" width="100%" ></a>
+</div>
-              <li>
+<div class="whitespace">
-                <p class="lead">Place one SV Minicolumn in a Collection Tube for each dissolved gel slice</p>
+</div>
-              </li>
-              <li>
-                <p class="lead">Transfer the dissolved gel mixture to the SV minicolumn assembly and incubate for 1 minute at room temperature</p>
-              </li>
-              <li>
-                <p class="lead">Centrifuge the SV Minicolumn assembly in a microcentrifuge at max speed for 1 minute. Remove the SV Minicolumn from the Spin Column assembly and discard the liquid in the Collection Tube. Return the SV Minicolumn to the Collection Tube
-                  afterwards</p>
-              </li>
-              <li>
-                <p class="lead">Wash the column by adding 700 µL of Membrane Wash Solution, previously diluted with 95% ethanol to the SV Minicolumn. Centrifuge the SV Minicolumn assembly for 1 minute at maximum speed
-                </p>
+  <div class="subtextm">
-              </li>
+      <a href="https://2015.igem.org/Team:KU_Leuven/Research/Idea" >
-              <li>
+         <p>
-                <p class="lead">Empty the Collection Tube as before, and place the SV Minicolumn back in the Collection Tube. Repeat the wash with 500 µL of Membrane Wash Solution, and centrifuge the SV Minicolumn assembly for 5 minutes at maximum speed</p>
+A detailed description about the interaction between our two cells and the genetic circuit can be found here.
-              </li>
-              <li>
-                <p class="lead"> Remove the SV Minicolumn assembly from the centrifuge (not wetting the bottom of the column with the supernatant). Empty the Collection Tube and centrifuge the assembly for 1 minute with the microcentrifuge lid open (or off) to allow ethanol
-                  evaporation</p>
-              </li>
-              <li>
-                <p class="lead">Carefully transfer the SV Minicolumn to a clean 1.5 mL tube. Apply 50 µL of Nuclease-Free Water (at 50ºC) directly to the center of the column, without touching the membrane with the pipette. Incubate at room temperature for 5 minutes</p>
-              </li>
-              <li>
-                <p class="lead">Centrifuge for 1 minute at 14000 rpm. Discard the SV Minicolumn, and store the tube containing the eluted DNA at 4ºC or -20ºC</p>
-              </li>
-            </ol>
-          </div>
-        </div>
-      </div>
-      <div class="panel panel-default">
-         <div class="panel-heading panel-title" role="tab" id="heading10">
-          <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordionprotocols" href="#collapse10" aria-expanded="false" aria-controls="collapse10">
-            Restriction product purification (from Promega Wizard ® Kit)
-          </a>
-        </div>
-        <div id="collapse10" class="panel-collapse collapse" role="tabpanel" aria-labelledby="heading10">
-          <div class="panel-body">
-            <ol style="list-style: decimal inside; font-size:10px;">
-              <li>
-                <p class="lead">Add an equal volume of Membrane Binding Solution to the restriction product tube</p>
-              </li>
-              <li>
+        </p>
-                <p class="lead">Place one SV Minicolumn in a Collection Tube for each restriction product solution</p>
+     </a>
-              </li>
+  </div>
+</div>
-              <li>
+<div class="subimgrowm">
-                <p class="lead">Transfer the mixture to the SV Minicolumn assembly and incubate for 1 minute at room temperature
+<div class="whiterow">
+</div>
+</div>
-                </p>
+<div class="subimgrowm">
-              </li>
+<div class="subimgm">
+ <a href="https://2015.igem.org/Team:KU_Leuven/Research/Methods" >
+     <b>Methods</b>
+ <img src="https://static.igem.org/mediawiki/2015/1/1a/KU_Leuven_Wiki_Button_-_Methods2.png" width="100%" ></a>
+</div>
-              <li>
+<div class="whitespace">
-                <p class="lead">Centrifuge the SV Minicolumn assembly in a microcentrifuge at max speed for 1 minute. Remove the SV Minicolumn from the Spin Column assembly and discard the liquid in the Collection Tube. Return the SV Minicolumn to the Collection Tube
+</div>
-                  afterwards</p>
-              </li>
-              <li>
+  <div class="subtextm">
-                <p class="lead"> Wash the column by adding 700 µL of Membrane Wash Solution, previously diluted with 95% ethanol to the SV Minicolumn. Centrifuge the SV Minicolumn assembly for 1 minute at maximum speed
+      <a href="https://2015.igem.org/Team:KU_Leuven/Research/Methods" >
+        <p>
+Here you can find the performed steps to create two different cell types and detailed quantification methods to determine interesting parameters.
+        </p>
+     </a>
+  </div>
+</div>
-                </p>
+<div class="subimgrowm">
-              </li>
+<div class="whiterow">
+</div>
+</div>
-              <li>
+<div class="subimgrowm">
-                <p class="lead">Empty the Collection Tube as before, and place the SV Minicolumn back in the Collection Tube. Repeat the wash with 500 µL of Membrane Wash Solution, and centrifuge the SV Minicolumn assembly for 5 minutes at maximum speed</p>
+<div class="subimgm">
-              </li>
+ <a href="https://2015.igem.org/Team:KU_Leuven/Research/Basic_Part" >
+     <b>Basic Parts</b>
+ <img src="https://static.igem.org/mediawiki/2015/e/e6/KU_Leuven_Wiki_Button_-_Parts2.png" width="100%" ></a>
+</div>
-              <li>
+<div class="whitespace">
-                <p class="lead">Remove the SV Minicolumn assembly from the centrifuge (not wetting the bottom of the column with the supernatant). Empty the Collection Tube and centrifuge the assembly for 1 minute with the microcentrifuge lid open (or off) to allow ethanol
+</div>
-                  evaporation</p>
-              </li>
-              <li>
+  <div class="subtextm">
-                <p class="lead">Carefully transfer the SV Minicolumn to a clean 1.5 mL tube. Apply 50 µL of Nuclease-Free Water (at 50ºC) directly to the center of the column, without touching the membrane with the pipette. Incubate at room temperature for 5 minutes</p>
+      <a href="https://2015.igem.org/Team:KU_Leuven/Research/Basic_Part" >
-              </li>
+        <p>
+          Our new, self-designed basic parts necessary to control cell-cell interactions and <i>E. Coli</i> motility.
+        </p>
+     </a>
+  </div>
+</div>
-              <li>
-                <p class="lead">Centrifuge for 1 minute at 14000 rpm. Discard the SV Minicolumn, and store the tube containing the eluted DNA at 4ºC or -20ºC</p>
-              </li>
-            </ol>
-          </div>
-        </div>
-      </div>
+<div class="subimgrowm">
+<div class="whiterow">
+</div>
+</div>
-    </div>
+<div class="subimgrowm">
+<div class="subimgm">
+ <a href="https://2015.igem.org/Team:KU_Leuven/Research/Composite_Part" >
+     <b>Composite Parts</b>
+ <img src="https://static.igem.org/mediawiki/2015/0/08/KU_Leuven_Wiki_Button_-_Composite_parts2.png" width="100%" ></a>
+</div>
+<div class="whitespace">
+</div>
+  <div class="subtextm">
+      <a href="https://2015.igem.org/Team:KU_Leuven/Research/Composite_Part" >
+        <p>
+          Our basic parts were combined with each other and with existing iGEM promotors, RBS and terminators.
+        </p>
+     </a>
    </div>
-</body>
+</div>
+<div class="subimgrowm">
+<div class="whiterow">
+</div>
+</div>
+<div class="subimgrowm">
+<div class="subimgm">
+ <a href="https://2015.igem.org/Team:KU_Leuven/Research" >
+     <b>Back</b>
+ <img src="https://static.igem.org/mediawiki/2015/c/cb/KUL_Wiki_Button_-_Back.png" width="100%" ></a>
+</div>
+<div class="whitespace">
+</div>
+  <div class="subtextm">
+      <a href="https://2015.igem.org/Team:KU_Leuven/Research" >
+        <p>
+         Go back to the Research page.
+        </p>
+     </a>
+  </div>
+</div>
+</div>
+</div>
+<div id="summarywrapper">
+<div class="summary">
+<h3> Contact </h3>
+<p style="font-size:1.3em; text-align: center">
+Address: Celestijnenlaan 200G room 00.08 - 3001 Heverlee<br>
+Telephone: +32(0)16 32 73 19<br>
+Email: igem@chem.kuleuven.be<br>
+</p>
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Difference between revisions of "Team:KU Leuven/Research/Results"

Latest revision as of 09:35, 20 October 2015

Results

Double Knockouts

Figure 2Test to prove that the ampicillin resistant PCP20 plasmid is removed. The left plate contains ampicillin, the right plate contains no antibiotic.

Figure 3Plaques after infection of our donor strains (ΔcheZ and Δtsr).

Figure 4Control to check if the lysate of ΔcheZ and Δtsr is not contaminated with cells.

Figure 5 Infection of the acceptor strain (Δtar) by lysate originating from donor strains (ΔcheZ and Δtsr)

Figure 6 Tar knock-out cells without the kanamycin cassette were plated out on kanamycin plates as control.

Figure 7 PCR to check that cheZ is knocked out in Δtar. The positive control is a knock-out in tar which lost the plasmid.

Figure 8 PCR to check that tsr is knocked out in Δtar. The positive control is a knock-out in tar which lost the plasmid.

Figure 9 PCR to check the knock-out in tar after the P1 transduction

Figure 10 PCR to check the operon of ΔtarΔcheZ