Team:Alberta/Project/Recombineering

From 2009.igem.org

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               <li>There must be a system for targeting the construct to a specific site for insertion </p>
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               <li>There must be a system for targeting the construct to a specific site of insertion </p>
<li>Activation of the recombination system must be under experimenter control </p>
<li>Activation of the recombination system must be under experimenter control </p>
<li>It must be possible to select for and verify colonies in which the insertion was successful </p>
<li>It must be possible to select for and verify colonies in which the insertion was successful </p>
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<P>
<P>
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The BioBytes team has chosen to use a recombination system from bacteriophage lambda. Lambda Red recombinase specifically recombines on the ends of a linear fragment of DNA. If the ends of this fragment are homologous to two separate sites on the <i>E. coli</i> chomosome, the genetic material between these two homologous regions will be exchanged. This will be the basis for our targeting system.</p>
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We have chosen to employ the Red Recombination System from bacteriophage lambda. Lambda Red recombinase causes specific crossover events between the ends of a linear and homologous chromosomal DNA. If the ends of a fragment contain at least 50 bp of homology to two separate sites on the <i>E. coli</i> chromosome, the genetic material between these two homologous regions will be exchanged. This provides the basis for our chromosome assembly system.</p>
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<p><b>Figure 1.</b></p>
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<img src="https://static.igem.org/mediawiki/2009/b/b9/UofA_iGEM09_RecombFig1.png" width="720">
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<img src="https://static.igem.org/mediawiki/2009/2/21/UofA_iGEM09_RecombFig2.png">
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<P>
<P>
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The homologous regions must be a minimum of 50 base pairs in length for recombination to occur at a significant frequency. This can be achieved in different ways:</p>
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The homologous regions must be a minimum of 50 base pairs in length for recombination to occur at a significant frequency. These homologous regions can be produced in different ways:</p>
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<P>
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<ul>
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First 5' extensions corresponding to the homologous sequence can be added to any gene using PCR amplification. This would allow a PCR product to be targeted to a specific site for insertion. Because our constructs will be recircularized and grown (see <i>DNA assembly</i>), this would require us to PCR each plasmid construct seperately in order to add the homologous regions to the ends.</p>
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<P>An alternative is to use the genes on either end of our construct as the homologous regions. As an example, we could first locate a region of genes which were deemed inessential through literature review and our Matlab modelling. This region would necessarily be flanked by an essential gene at either end. We would then assemble a plasmid containing these two essential genes. If the insertion is successful, we would be left with a chromosome without this region of inessential genes.</p>
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<li>Regions corresponding to homologous genomic sequences can be added to any gene through PCR amplification with 5' extended primers. However, our synthetic constructs are rather large and long-extension PCR proves rather finicky. PCR amplification across an entire linear construct in order to add regions of homology regions is not a viable option.</li>
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<li>An alternative is to use the flanking essential genes on either end of our linear construct as the homologous regions. This provides much greater sequence homology and should provide increased recombination efficiency.</li>
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The Lambda Red Recombinase system consists primarily of three proteins: lambda exonuclease, which progressively digests the 5'-ended strand of a dsDNA end; beta protein, which binds to ssDNA and promotes strand annealing; and gamma protein, which binds to the bacterial RecBCD enzyme and inhibits its activities.  These three genes are contained on the plasmid pKD46 under an arabinose promoter.  For further regulation, this plasmid contains the RepA temperature sensitive origin.  Therefore, one can specifically induce recombination of a desired linear piece of DNA, then cure the cell of the pKD46 plasmid at 42C.  This system leaves a host cell with a specific chromosomal mutation, but is free of the Lambda Red system in order to prevent random, unwanted recombination within the cell.</p>   
+
The lambda Red recombinase system consists primarily of three proteins: lambda exonuclease, which progressively removes 5' nucleotides from linear dsDNA; beta protein, which binds to ssDNA and promotes strand annealing; and gamma protein, which binds to the bacterial RecBCD enzyme to inhibit its activity.  These three genes are contained on the plasmid pKD46 downstream from an arabinose promoter.  For further regulation, this plasmid contains the RepA temperature sensitive origin.  Therefore, one can induce chromosomal integration of a transformed linear DNA fragment with arabinose induction, then cure the cell of the pKD46 plasmid with growth at 42&deg;C.  This system leaves a host cell with a specific chromosomal mutation, and free of lambda Red recombinase in order to prevent future random, unwanted recombination within the cell.</p>   
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Our first attempt at recombineering entailed the insertion of an ampicillin resistance cassette into a gene-less region of the host chromosome.  Amp cassette primers were engineered with 50 bp extenstion that were homologous to flanking portions of the region of the chromosome to be excised.  The linear construct produced by these primers was then electroporated into pre-Arabinose pKD46 containing competent cells.  Cells were left to incubate at 30C for four hours, then plated on ampicillin containing agarose plates.</p>  
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Our first attempt at recombineering entailed the integration of an ampicillin resistance cassette into a non-coding, non-regulatory region of the host chromosome.  Amp<sup>R</sup> cassette primers were engineered with 50 bp extensions that were homologous to flanking portions of the region of the chromosome to be excised.  The linear construct produced through PCR amplification with these primers was then electroporated into cells containing pKD46 that were pre-induced with arabinose.  Cells were left to incubate at 30&deg;C for four hours, then plated under selection for ampicillin resistance.</p>  
<P>
<P>
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Colonies with proper recombination were selected for by PCR verification.  PCRing across the excised region was expected to show markedly different fragment sizes between wildtype and ampicillin resistant cells, as the ampicillin cassette was a substantially different size than the region excised.</p>
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Colonies with the intended recombination event were screened for using PCR.  PCR amplification across the expected integration region was expected to produce markedly different fragment sizes between wildtype and ampicillin-resistant cells if integration of the Amp<sup>R</sup> cassette occurred.</p>
<P>
<P>
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PCR verification showed no difference in fragment size between wildtype (not ampicillin resistant) and ampicillin resistant cells.  This has lead us to believe that non-specific recombination occurred and that the 50 bp of homology used was not great enough for site-specific recombination using our methods.</p>
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PCR verification showed no difference in fragment size between wildtype and ampicillin-resistant cells.  This led us to believe that non-specific recombination occurred and that the 50 bp of homology used was not great enough for site-specific recombination using this method.</p>
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In order to decrease the degree of non-specific recombination, we attempted to insert a linear construct with whole-gene homology to the chromosome.  To do this, we flanked a chloramphenicol resistance cassette with an essential gene on either side.  The region selected for excision contained no known essential genes.</p>
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In order to decrease the degree of non-specific recombination, we attempted to integrate a linear construct with whole-gene homology into the chromosome.  To do this, we flanked a chloramphenicol resistance cassette with an essential gene on either side.  The region that would be excised during integration contained no known essential genes. The construct was built from five bytes using the BioBytes assembly method </p>
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Our 5 byte construct was built using our Biobyte system and looked like:
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<p><b>Figure 2.</p></b>
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</p>
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            <center><img src="https://static.igem.org/mediawiki/2009/6/6b/UofA_iGEM09_RecombConstruct.png"></center>
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    <li>               orn - 75% promoter - amp - 75% promoter - yjeE </p>
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The gel purified construct was once again electroporated into pre-Arabinose pKD46 containing competent cells.  Cells were left to incubate at 30C for four hours, then plated on chloramphenicol containing agarose plates. This time no colonies grew. </p>   
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The gel-purified construct was electroporated into cells containing pKD46 that were pre-induced with arabinose.  Cells were left to incubate at 30&deg;C for four hours, then plated under selection for chloramphenicol resistance. However, no colonies grew. </p>   
<P>
<P>
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This shows that either recombination did not occur, or the cassette was non-functional.  We did not have time for further trouble-shooting. </p>
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This indicated either that recombination did not occur, or that the cassette was non-functional.  We did not have time for further troubleshooting.</p>
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Latest revision as of 02:54, 22 October 2009

University of Alberta - BioBytes










































































































What is Recombineering?

Recombineering refers to the strategic use of recombination in vivo in order to reach a defined goal. In the case of BioBytes, a method is required to target the final construct for insertion at a specific place on the E. coli chromosome.

To do this successfully, three components must be taken into account:

  • There must be a system for targeting the construct to a specific site of insertion

  • Activation of the recombination system must be under experimenter control

  • It must be possible to select for and verify colonies in which the insertion was successful

Targeting

We have chosen to employ the Red Recombination System from bacteriophage lambda. Lambda Red recombinase causes specific crossover events between the ends of a linear and homologous chromosomal DNA. If the ends of a fragment contain at least 50 bp of homology to two separate sites on the E. coli chromosome, the genetic material between these two homologous regions will be exchanged. This provides the basis for our chromosome assembly system.

Figure 1.

The homologous regions must be a minimum of 50 base pairs in length for recombination to occur at a significant frequency. These homologous regions can be produced in different ways:

  • Regions corresponding to homologous genomic sequences can be added to any gene through PCR amplification with 5' extended primers. However, our synthetic constructs are rather large and long-extension PCR proves rather finicky. PCR amplification across an entire linear construct in order to add regions of homology regions is not a viable option.
  • An alternative is to use the flanking essential genes on either end of our linear construct as the homologous regions. This provides much greater sequence homology and should provide increased recombination efficiency.

Inducible Recombination System

The lambda Red recombinase system consists primarily of three proteins: lambda exonuclease, which progressively removes 5' nucleotides from linear dsDNA; beta protein, which binds to ssDNA and promotes strand annealing; and gamma protein, which binds to the bacterial RecBCD enzyme to inhibit its activity. These three genes are contained on the plasmid pKD46 downstream from an arabinose promoter. For further regulation, this plasmid contains the RepA temperature sensitive origin. Therefore, one can induce chromosomal integration of a transformed linear DNA fragment with arabinose induction, then cure the cell of the pKD46 plasmid with growth at 42°C. This system leaves a host cell with a specific chromosomal mutation, and free of lambda Red recombinase in order to prevent future random, unwanted recombination within the cell.

Our Efforts at Recombineering

Attempt I:

Our first attempt at recombineering entailed the integration of an ampicillin resistance cassette into a non-coding, non-regulatory region of the host chromosome. AmpR cassette primers were engineered with 50 bp extensions that were homologous to flanking portions of the region of the chromosome to be excised. The linear construct produced through PCR amplification with these primers was then electroporated into cells containing pKD46 that were pre-induced with arabinose. Cells were left to incubate at 30°C for four hours, then plated under selection for ampicillin resistance.

Colonies with the intended recombination event were screened for using PCR. PCR amplification across the expected integration region was expected to produce markedly different fragment sizes between wildtype and ampicillin-resistant cells if integration of the AmpR cassette occurred.

PCR verification showed no difference in fragment size between wildtype and ampicillin-resistant cells. This led us to believe that non-specific recombination occurred and that the 50 bp of homology used was not great enough for site-specific recombination using this method.

Attempt II:

In order to decrease the degree of non-specific recombination, we attempted to integrate a linear construct with whole-gene homology into the chromosome. To do this, we flanked a chloramphenicol resistance cassette with an essential gene on either side. The region that would be excised during integration contained no known essential genes. The construct was built from five bytes using the BioBytes assembly method

Figure 2.

The gel-purified construct was electroporated into cells containing pKD46 that were pre-induced with arabinose. Cells were left to incubate at 30°C for four hours, then plated under selection for chloramphenicol resistance. However, no colonies grew.

This indicated either that recombination did not occur, or that the cassette was non-functional. We did not have time for further troubleshooting.

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