Difference between revisions of "Team:KU Leuven/Research/Methods"
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− | <p>On this page you can find all of the methods and protocols used in the lab to obtain our results. For some techniques | + | <p>On this page, you can find all of the methods and protocols used in the lab to obtain our results. For some techniques we included some basic theory since it is a prerequisite to get acquainted with the details behind these techniques before using them. To learn more about them click the titles below.</p> |
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<p><b>Theory</b><br/> | <p><b>Theory</b><br/> | ||
− | To be able to create patterns, two different cell types called A and B will interact with each other. In order to achieve the desired behavior, the cells used in the experiments were derived from K12 <i>Escherichia coli</i> strains with introduction of specific knockouts. Cell type A has a deletion of <i>tar</i> and <i>tsr</i>, whereas in cell type B both <i>tar</i> and <i>cheZ</i> are knocked out. The Keio collection is composed of a set of precisely defined single-gene | + | To be able to create patterns, two different cell types called A and B will have to interact with each other. In order to achieve the desired behavior, the cells used in the experiments were derived from K12 <i>Escherichia coli</i> strains with introduction of specific knockouts. Cell type A has a deletion of <i>tar</i> and <i>tsr</i> genes, whereas in cell type B both <i>tar</i> and <i>cheZ</i> genes are knocked out. The Keio collection is composed of a set of precisely defined single-gene deletion mutants of all non-essential genes in <i>E. coli</i> K-12. The targeted genes were replaced by a kanamycin resistance cassette. The kanamycin cassette is enclosed between two FRT sites making excision possible using FLP recombinase (reference 1). FLP recombinase triggers an intramolecular recombination between FRT repeats in the chromosome. Since both the antibiotic resistance gene and the plasmid replication region are surrounded by two FRT sites, both are to be eliminated (Figure 1, step 1). <br/> |
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− | A genetic procedure for moving selectable mutations of interest called the P1 transduction was used. Since the packaging of the bacteriophage P1 is rather inaccurate, it will on occasion package the DNA of its bacterial host instead of its own phage chromosome. This implies that the lysate | + | A genetic procedure for moving selectable mutations of interest, called the P1 transduction, was used. Since the packaging of the bacteriophage P1 is rather inaccurate, it will on occasion package the DNA of its bacterial host instead of its own phage chromosome. This implies that the lysate will contain either packaged phage or bacterial DNA. After infection of a second host with this lysate, a transfer of parts of the chromosome from the donor strain into the receiver strain will take place. Those DNA pieces can then recombine using the FRT sites and hereby be incorporated permanently into the chromosome of the new strain. Here, the recombination was triggered by selection on kanamycin. (reference 2) <br/> |
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− | In general, we used three steps to obtain our double knock-outs (Figure 1). In the first step, the kanamycin cassette of our <i>tar</i> knock-out strain was removed by flippase, coded on plasmid PCP20. Afterwards, the temperature sensitive plasmid was removed by growing the cells overnight at 42°C. In a third step, the <i>tar</i> knock-out strain | + | In general, we used three steps to obtain our double knock-outs (Figure 1). In the first step, the kanamycin cassette of our <i>tar</i> knock-out strain was removed by flippase, coded on plasmid PCP20. Afterwards, the temperature sensitive plasmid was removed by growing the cells overnight at 42°C. In a third step, the <i>tar</i> knock-out strain was infected by lysate originating from the <i>tsr</i> and <i>cheZ</i> knock-out strains. After selection on kanamycin plates, we obtained the double knock-outs. These knock-outs were confirmed by PCR. For more information, please check our result page. <br/> |
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<p><b>Theory</b><br/> | <p><b>Theory</b><br/> | ||
− | The Gibson assembly as described by Gibson et al., is a rapid DNA assembly method that assures directional cloning of fragments in one single reaction. To perform the Gibson assembly, three essential enzymes are needed: a mesophylic nuclease, a thermophylic ligase and a high fidelity polymerase. Therefore, the NEBuilder<sup>Ⓡ</sup> HiFi DNA Assembly Master Mix (New England Biolabs) was used. In the first step of this reaction, the exonuclease rapidly cleaves off the 5’ DNA ends. The exonuclease is unstable at 50°C and gets degraded early in the process. In the second step, the designed sequence overlaps anneal and the polymerase starts filling in the gaps. The ligase then covalently joins both ends finalizing the plasmid assembly for transformation. This text was based on <a href="https://www.idtdna.com/pages/docs/default-source/user-guides-and-protocols/gibson-assembly.pdf?sfvrsn=16">the IDT | + | The Gibson assembly, as described by Gibson et al., is a rapid DNA assembly method that assures directional cloning of fragments in one single reaction. To perform the Gibson assembly, three essential enzymes are needed: a mesophylic nuclease, a thermophylic ligase and a high fidelity polymerase. Therefore, the NEBuilder<sup>Ⓡ</sup> HiFi DNA Assembly Master Mix (New England Biolabs) was used. In the first step of this reaction, the exonuclease rapidly cleaves off the 5’ DNA ends. The exonuclease is unstable at 50°C and gets degraded early in the process. In the second step, the designed sequence overlaps anneal and the polymerase starts filling in the gaps. The ligase then covalently joins both ends finalizing the plasmid assembly for transformation. This text was based on <a href="https://www.idtdna.com/pages/docs/default-source/user-guides-and-protocols/gibson-assembly.pdf?sfvrsn=16">the IDT description as seen on 13/09/2015</a>. In the following paragraph, our optimized protocol is given.</p> |
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<p><b>Theory</b></br> | <p><b>Theory</b></br> | ||
N-acyl homoserine lactones (AHL) are small diffusible molecules used in cell-to-cell signaling by Gram-negative bacteria. <i>Chromobacterium violaceum</i> is a Gram-negative bacterium that produces the violet pigment violacein as a result of sensing AHL. AHL is produced by the autoinducer synthase CviI and released in the environment. When a quorum has been reached, AHL diffuses back into the bacteria and binds the transcriptional regulator CviR. This activates the expression of specific genes which leads to the production of violacein. </p> | N-acyl homoserine lactones (AHL) are small diffusible molecules used in cell-to-cell signaling by Gram-negative bacteria. <i>Chromobacterium violaceum</i> is a Gram-negative bacterium that produces the violet pigment violacein as a result of sensing AHL. AHL is produced by the autoinducer synthase CviI and released in the environment. When a quorum has been reached, AHL diffuses back into the bacteria and binds the transcriptional regulator CviR. This activates the expression of specific genes which leads to the production of violacein. </p> | ||
− | <p>In our project, the mutant <i>C. violaceum</i> CV026 was used to quantify the amount of N-(3-Oxohexanoyl)-L-homoserine lactone (OHHL), a specific type of AHL, produced by the LuxI enzyme our <i>E. coli</i> strains. The CV026 strain is <i>cviI</i> gene deficient and therefore requires exogenous addition of AHL to produce violacein. The idea is to plot a standard curve in which CV026 is induced with different concentrations of AHL. In the standard curve, results were normalized to the OD.</p> | + | <p>In our project, the mutant <i>C. violaceum</i> CV026 was used to quantify the amount of N-(3-Oxohexanoyl)-L-homoserine lactone (OHHL), a specific type of AHL, produced by the LuxI enzyme in our <i>E. coli</i> strains. The CV026 strain is <i>cviI</i> gene deficient and therefore requires exogenous addition of AHL to produce violacein. The idea is to plot a standard curve in which CV026 is induced with different concentrations of AHL. In the standard curve, results were normalized to the OD.</p> |
− | <p>To quantify | + | <p>To quantify the amount of AHL produced by <i>E. coli</i>, these were grown and pelleted after which CV026 was added. After incubating for several hours, CV026 and violacein were spun down and the supernatant was removed. The pellet was resuspended in dimethyl sulfoxide and again centrifuged to separate the violacein dissolved in dimethyl sulfoxide from the cells. Absorbance of the supernatans was measured at 585 nm.</p> |
<p><b>Protocol</b></br> | <p><b>Protocol</b></br> | ||
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<p><b>Theory</b></br> | <p><b>Theory</b></br> | ||
− | The chemiluminescence detection assay of Kugimiya and Fukada (2015) was used as a reference to quantify the amount of leucine produced by IlvE. In this technique, leucine-tRNA synthetase in combination with a luminol chemiluminescence reaction was used to detect leucine. In the article, a selective quantification from 1 to 20 µM leucine was mentioned and | + | The chemiluminescence detection assay of Kugimiya and Fukada (2015) was used as a reference to quantify the amount of leucine produced by IlvE gene containing cells. In this technique, leucine-tRNA synthetase in combination with a luminol chemiluminescence reaction was used to detect leucine. In the article, a selective quantification from 1 to 20 µM leucine was mentioned and was further optimized in our protocol.<br> |
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− | Below, the reaction equations can be seen. In the first step, leucine-tRNA synthetase (LeuRS) is activated by ATP to form leucyl-AMP with formation of a side product | + | Below, the reaction equations can be seen. In the first step, leucine-tRNA synthetase (LeuRS) is activated by ATP to form leucyl-AMP with formation of a side product - pyrophosphate. The formation of pyrophosphate is further used to detect the amount of leucine. After the addition of inorganic pyrophosphatase, this enzyme hydrolyses pyrophosphate to phosphate. When pyruvate oxidase and pyruvate is added to phosphate, it results in the formation of acetyl phosphate and hydrogen peroxide. In the fourth reaction, the hydrogen peroxide in combination with luminol and horseradish peroxidase leads to the emition of light. This luminescence is detected with a luminometer.<br></p> |
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− | The idea is to grow the bacteria on minimal medium without leucine. These bacteria | + | The idea is to grow the bacteria on minimal medium without leucine. These bacteria are spun down and the supernatant is further investigated in the presence of leucine. The luminescence originating from the bacterial samples is compared with the standard curve. Firstly, a big range is used for the standard curve (0 to 100 μM) to then further zoom in to the biologically relevant concentrations.<br> |
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Revision as of 01:18, 19 September 2015
Methods
On this page, you can find all of the methods and protocols used in the lab to obtain our results. For some techniques we included some basic theory since it is a prerequisite to get acquainted with the details behind these techniques before using them. To learn more about them click the titles below.
Contact
Address: Celestijnenlaan 200G room 00.08 - 3001 Heverlee
Telephone: +32(0)16 32 73 19
Email: igem@chem.kuleuven.be