B11111

From 2008.igem.org

(Difference between revisions)

Revision as of 08:40, 29 October 2008

无标题文档

Background

Site-specific recombination
Site-specific recombination differs from general recombination in that short specific sequences which are required for the recombination, are the only sites at which recombination occurs. These reactions invariably require specialized proteins to recognize these sites and to catalyze the recombination reaction at these sites.

Inverted repeats
If the two sites at which recombination will take place are oriented oppositely to one another in the same DNA module then the following illustrates the sequence of events that will take place:

Direct repeats
While if oriented in the same direction, then the following illustrates the sequence of events:

The net result is that the segment of DNA between the two recombinogenic sites has inverted with respect to the rest of the DNA module.
In other words, recombination at inverted repeats causes an inversion

The net result is that the segment of DNA between the two recombinogenic sites has been deleted from the rest of the DNA module and appears as a circular module.
In other words, recombination at direct repeats causes a deletion.

Note that the reverse reaction -- the recombination of a circular module with another DNA module (either circular or linear), brings about a fusion of both modules or the integration of one module into the other. The integrated segment will be flanked by directly repeating sequences which can, of course, be used to excise the integrated segment again.

Integration of bacteriophage lambda

In order for the lambda prophage to exist in a host E. coli cell, it must integrate into the host chromosome which it does by means of a site-specific recombination reaction.

The E. coli chromosome contains one attachment site which is designated attB. The site is only 30 bp in size and contains a conserved central 15 bp region where the recombination reaction will take place. The structure of the recombination site is usually represented as BOB'.

The bacteriophage recombination site - attP - contains the identical central 15 bp region as attB. The overall structure can be represented as POP'.

Integration of bacteriophage lambda requires one phage-encoded protein - INT, which is the integrase - and one bacterial protein - IHF, which is Integration Host Factor. Both of these proteins bind to sites on the P and P' arms of attP to form a complex in which the central conserved 15 bp elements of attP and attB are properly aligned.

The result of recombination is that the integrated prophage is flanked by two attachment sites but now they are slightly different: attL has the structure BOP' and attR has the structure POB'.

Cre-Lox recombination is a special type of site-specific recombination, which is often applied as a gene knockout tool.
Cre is a site-specific DNA recombinase, which can catalyse the recombination of DNA between specific sites, e.g. loxP in a DNA module. When cells that have loxP sites in their genome express Cre, a reciprocal recombination event will occur between the loxP sites. The double stranded DNA is cut at both loxP sites by the Cre protein. The strands are then rejoined with DNA ligase. The efficiency of recombination depends on the orientation of the loxP sites. For two lox sites on the same chromosome arm, inverted loxP sites will cause an inversion, while a direct repeat of loxP sites will cause a deletion event.

Lox P site
Lox P (locus of X-over P1) is a site on the Bacteriophage P1 consisting of 34 bp. There exists an asymmetric 8 bp sequence in between with two sets of palindromic, 13 bp sequences flanking it. The detailed structure is given below.

aaaaaa

@@ Line 91: / Line 91: @@
          <td height="23" colspan="2" bgcolor="#03438A" class="STYLE24"><p class="STYLE4"><span class="STYLE11 STYLE4 STYLE23 STYLE5">The result of  recombination is that the integrated prophage is flanked by two attachment  sites but now they are slightly different: <em>attL</em> has the structure BOP' and <em>attR</em> has the structure POB'. </span></p>
              <p class="STYLE4">Cre-Lox  recombination is a special  type of site-specific recombination, which is often applied as a gene knockout  tool. <br />
-               Cre is a site-specific  DNA recombinase, which can catalyse the recombination of DNA between specific  sites, e.g. loxP in a DNA module. When cells that have loxP sites in  their genome express Cre, a reciprocal recombination event will occur between  the loxP sites. The double stranded DNA is cut at both loxP sites  by the Cre protein. The strands are then rejoined with <a href="http://en.wikipedia.org/wiki/DNA_ligase" title="DNA ligase">DNA ligase</a>. The efficiency of recombination  depends on the orientation of the loxP sites. For two lox sites on the  same chromosome arm, inverted loxP sites will cause an inversion, while  a direct repeat of loxP sites will cause a deletion event.</p>
+               Cre is a site-specific  DNA recombinase, which can catalyse the recombination of DNA between specific  sites, e.g. loxP in a DNA module. When cells that have loxP sites in  their genome express Cre, a reciprocal recombination event will occur between  the loxP sites. The double stranded DNA is cut at both loxP sites  by the Cre protein. The strands are then rejoined with DNA ligase. The efficiency of recombination  depends on the orientation of the loxP sites. For two lox sites on the  same chromosome arm, inverted loxP sites will cause an inversion, while  a direct repeat of loxP sites will cause a deletion event.</p>
            <p class="STYLE4"> Lox P site<br />
              Lox P (locus of X-over P1) is  a site on the Bacteriophage P1 consisting of 34 bp. There exists an asymmetric  8 bp sequence in between with two sets of palindromic, 13 bp sequences flanking  it. The detailed structure is given below.</p></td>