Team:Freiburg bioware/Project/invivo

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  href="https://2009.igem.org/Team:Freiburg_bioware/Project"><span
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class="l"></span><span class="r"></span><span
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class="r"></span><span class="t">The
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Project</span></a>
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  href="https://2009.igem.org/Team:Freiburg_bioware/Project#Summary">Summary</a>
  href="https://2009.igem.org/Team:Freiburg_bioware/Project#Summary">Summary</a>
       </li>
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href="https://2009.igem.org/Team:Freiburg_bioware/Project#Subprojects">Subprojects</a></li>
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  href="https://2009.igem.org/Team:Freiburg_bioware/Project#Highlights">Highlights</a></li>
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<div class="art-Post-inner">
<div class="art-Post-inner">
<div class="art-PostMetadataHeader">
<div class="art-PostMetadataHeader">
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<h2 class="art-PostHeaderIcon-wrapper"> &nbsp;<img
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<h2 class="art-PostHeaderIcon-wrapper"> <img
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style="width: 28px; height: 25px;" alt=""
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  src="https://static.igem.org/mediawiki/2009/2/2a/Freiburg09_Post_tanne_2.png" alt="PostHeaderIcon"
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  src="https://static.igem.org/mediawiki/2009/2/2a/Freiburg09_Post_tanne_2.png" />
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height="25" width="26" />&nbsp;<i>in vivo</i> assay</h2>
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In vivo assays<span class="art-PostHeader"></span> </h2>
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</div>
</div>
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<p class="MsoNormal" style="line-height: normal;"><b><span
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<div class="art-PostContent"> <br />
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style="font-size: 12pt; font-family: &quot;Lucida Sans&quot;,&quot;sans-serif&quot;;"
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<span style="font-weight: bold;"></span>
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  lang="EN-US"></span></b></p>
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<table style="text-align: left; width: 627px; height: 261px;"
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<table>
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  border="0" cellpadding="0" cellspacing="0">
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  <tbody bgcolor="#e2eff9">
     <tr align="center">
     <tr align="center">
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       <td><img style="width: 300px; height: 200px;"
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       <td><img style="width: 300px; height: 225px;"
  alt=""
  alt=""
  src="https://static.igem.org/mediawiki/2009/thumb/9/9a/Freiburg_09_2009-09-21_multidimensional_RV308_electro_mit_fluo_oligos-4_c1.JPG/800px-Freiburg_09_2009-09-21_multidimensional_RV308_electro_mit_fluo_oligos-4_c1.JPG" /></td>
  src="https://static.igem.org/mediawiki/2009/thumb/9/9a/Freiburg_09_2009-09-21_multidimensional_RV308_electro_mit_fluo_oligos-4_c1.JPG/800px-Freiburg_09_2009-09-21_multidimensional_RV308_electro_mit_fluo_oligos-4_c1.JPG" /></td>
     </tr>
     </tr>
     <tr align="center">
     <tr align="center">
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       <td><span
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       <td>Fluorescence microscope image of RV308 cells,
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style="font-size: 10pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"
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electroporated with fluorescein labeled oligonucleotides </td>
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lang="EN-US">Multidimensional RV308 electromicroscop, fluo oligos</span></td>
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     </tr>
     </tr>
   </tbody>
   </tbody>
</table>
</table>
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<p class="MsoNormal" style="line-height: normal;"><b><span
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<span style="font-weight: bold;"></span><br />
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style="font-size: 12pt; font-family: &quot;Lucida Sans&quot;,&quot;sans-serif&quot;;"
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<h3>Introduction</h3>
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lang="EN-US"></span></b></p>
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<p class="MsoNormal" style="line-height: normal;"><b><span
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-
style="font-size: 12pt; font-family: &quot;Lucida Sans&quot;,&quot;sans-serif&quot;;"
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lang="EN-US">Introduction</span></b><span
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style="font-size: 12pt; font-family: &quot;Lucida Sans&quot;,&quot;sans-serif&quot;;"
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lang="EN-US"><br />
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In vivo use of programmable restriction enzymes can provide the
In vivo use of programmable restriction enzymes can provide the
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opportunity of
+
opportunity of genome engineering. Here we develop and test strategies
-
genome engineering. Here we develop and test strategies for the
+
for the application of our programmable restriction endonuclease.<br />
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application of
+
<br />
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our programmable restriction endonuclease. <o:p></o:p></span></p>
+
In our strategy we cotransformed Fok_i and Fok_a constructs fused to
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<p class="MsoNormal" style="line-height: normal;"><span
+
binding domains into a certain bacteria strain leading to the basal
-
style="font-size: 12pt; font-family: &quot;Lucida Sans&quot;,&quot;sans-serif&quot;;"
+
-
lang="EN-US">In our strategy we cotransformed Fok_i and
+
-
Fok_a constructs
+
-
fused to binding domains into a certain bacteria strain leading to the
+
-
basal
+
expression of the Fok heterodimers. Transformation of single-stranded
expression of the Fok heterodimers. Transformation of single-stranded
bacteriophage M13 DNA hybridized with modified oligonucleotides enables
bacteriophage M13 DNA hybridized with modified oligonucleotides enables
-
the Fok
+
the Fok heterodimers to bind the DNA. Inside the cells the phage DNA
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heterodimers to bind the DNA. Inside the cells the phage DNA should be
+
should be cut by the dimerized and thereby activated Fok_a should
-
cut by
+
result in a decreased progeny of phages within the bacterial culture in
-
the dimerized and thereby activated Fok_a should result in a decreased
+
comparison to control assays. Two ways of detection were envisioned. A
-
progeny
+
qPCR should reveal successful cutting of the phage DNA by the lack of
-
of phages within the bacterial culture in comparison to control assays.
+
amplified phage DNA. In addition an adjacent phage titer determination
-
Two
+
via a blue white screening ressembling phage assay by plating the cells
-
ways of detection were envisioned. A qPCR should reveal successful
+
to IPTG/Xgal plates should lead to a decreased number of plaques in
-
cutting of
+
comparison to the control assays. <br />
-
the phage DNA by the lack of amplified phage DNA. In addition an
+
<br />
-
adjacent phage
+
<table style="text-align: left; width: 322px; height: 436px;"
-
titer determination via blue white screening <span style="">&nbsp;</span>by
+
  border="0" cellpadding="0" cellspacing="0">
-
plating the cells to IPTG/Xgal plates for
+
   <tbody bgcolor="#e2eff9">
-
an adjacent phage titer determination via blue white screening should
+
     <tr align="center">
-
lead to a
+
       <td><img style="width: 300px; height: 400px;"
-
decreased number of plaques in comparison to the control assays. <o:p></o:p></span></p>
+
  alt=""
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<br>
+
  src="https://static.igem.org/mediawiki/2009/b/b4/Freiburg09_In.vivo.assay.scheme.png" /></td>
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<table style="text-align: left; width: 412px; height: 303px;"
+
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  border="0" cellpadding="2" cellspacing="2">
+
-
   <tbody>
+
-
     <tr>
+
-
       <td><a
+
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href="http://www.molbiotech.uni-freiburg.de/iGEM/wiki2009/index.php/Image:Freiburg09_021009-HisFluASplitFoki_%2B_%C3%9Flac-his_highres%2Bbearbeitet.jpg"
+
-
class="image"
+
-
title="Western Blot: His-Flu_a-Split_Fok_i in pEx; lanes: NEB prestained marker broad range, pool of elution fractions 2-5, empty lane, 3 positive controls"><img
+
-
  alt="Western Blot: His-Flu_a-Split_Fok_i in pEx; lanes: NEB prestained marker broad range, pool of elution fractions 2-5, empty lane, 3 positive controls"
+
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  src="https://static.igem.org/mediawiki/2009/2/2f/Freiburg09_Invivoscheme.png"
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class="thumbimage" border="0" height="350"
+
-
width="400" /></a></td>
+
     </tr>
     </tr>
-
     <tr>
+
     <tr align="center">
       <td>in vivo scheme</td>
       <td>in vivo scheme</td>
     </tr>
     </tr>
   </tbody>
   </tbody>
</table>
</table>
-
<p class="MsoNormal" style="line-height: normal;"><b
+
<br />
-
style=""><span
+
<br />
-
style="font-size: 12pt; font-family: &quot;Lucida Sans&quot;,&quot;sans-serif&quot;;"
+
<h3>Methods</h3>
-
lang="EN-US">Methods<o:p></o:p></span></b></p>
+
In detail two heterodimeric Fok constructs His-Dig-Split-Fok_a
-
<h1><span
+
(BBa_K243036) and His-FluA-Split-Fok_i (BBa_K243010) are encoded by two
-
style="font-size: 12pt; line-height: 115%; font-family: &quot;Lucida Sans&quot;,&quot;sans-serif&quot;; color: windowtext; font-weight: normal;"
+
different plasmids, plasmid pJS419 with a chloramphenicol and pEx with
-
lang="EN-US">In detail two heterodimeric Fok constructs
+
an ampicillin resistance. This enables a positive selection of the
-
His-Dig-Split-Fok_a (</span><span
+
bacteria strain XL1 blue cotransformed with both plasmids. Subsequent
-
style="font-size: 12pt; line-height: 115%; font-family: &quot;Lucida Sans&quot;,&quot;sans-serif&quot;; color: windowtext; font-weight: normal;"
+
to the preparation of electrocompetent cotransformed XL1 blue, the M13
-
lang="EN-US">BBa_K243036</span><span
+
ssDNA hybridized with the modified oligonucleotides were inserted into
-
style="font-size: 12pt; line-height: 115%; font-family: &quot;Lucida Sans&quot;,&quot;sans-serif&quot;; color: windowtext; font-weight: normal;"
+
these bacteria by electroporation at 1.7 kV. The infected cells were
-
lang="EN-US">) and
+
incubated at 37&deg;C for 0.5 h. At different dilutions the phages
-
His-FluA-Split-Fok_i (</span><span
+
were mixed with 180&mu;l ER2738 cells with an OD600 of about 0.5.
-
style="font-size: 12pt; line-height: 115%; font-family: &quot;Lucida Sans&quot;,&quot;sans-serif&quot;; color: windowtext; font-weight: normal;"
+
Then they were mixed with 3 ml of 42&deg;C warm top agar and plated
-
lang="EN-US">BBa_K243010)</span><span
+
on IPTG/X-Gal plates.<br />
-
style="font-size: 12pt; line-height: 115%; font-family: &quot;Lucida Sans&quot;,&quot;sans-serif&quot;; color: windowtext; font-weight: normal;"
+
<br />
-
lang="EN-US"> are encoded by two different
+
XL1 Blue as well as ER2738 harbour a F-plasmid coding for the
-
plasmids, plasmid pJS419 with a chloramphenicol and pEx with an
+
omega-fragment of beta-galactosidase. The M13 phages in turn encodes
-
ampicillin
+
-
resistance. This enables a positive selection of the bacteria strain
+
-
XL1 blue
+
-
cotransformed with both plasmids. Subsequent to the preparation of
+
-
electrocompetent
+
-
cotransformed XL1 blue, the M13 ssDNA hybridized with the modified
+
-
oligonucleotides were inserted into these bacteria by electroporation
+
-
at 1.7
+
-
kV. The infected cells were incubated at 37&deg;C for 1.5 h. At
+
-
different dilutions
+
-
the phages were mixed with 180</span><span
+
-
style="font-size: 12pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;; color: windowtext; font-weight: normal;">&mu;</span><span
+
-
style="font-size: 12pt; line-height: 115%; font-family: &quot;Lucida Sans&quot;,&quot;sans-serif&quot;; color: windowtext; font-weight: normal;"
+
-
lang="EN-US">l ER2738
+
-
cells with an OD600 of about 0.5. After an incubation time of 10
+
-
minutes they
+
-
were plated in 3 ml top agar on IPTG/X-Gal plates. </span><span
+
-
style="font-size: 12pt; line-height: 115%; font-family: &quot;Lucida Sans&quot;,&quot;sans-serif&quot;; color: windowtext; font-weight: normal;"
+
-
lang="EN-US"><o:p></o:p></span></h1>
+
-
<p class="MsoNormal" style="line-height: normal;"><span
+
-
style="font-size: 12pt; font-family: &quot;Lucida Sans&quot;,&quot;sans-serif&quot;;"
+
-
lang="EN-US">XL1 Blue as well as ER2738 harbour a F-plasmid
+
-
coding
+
-
for the omega-fragment of beta-galactosidase. The M13 phages in turn
+
-
encodes
+
the lacking alpha-fragment. Consequently, alpha-complementation can be
the lacking alpha-fragment. Consequently, alpha-complementation can be
-
detected
+
detected via a screening for blue plaques. After incubation overnight
-
by a blue/white screening. After incubation overnight at 37&deg;C
+
at 37&deg;C the infected cells were inspected for blue plaques
-
the infected
+
formation because of the enzymatic cleavage of X-Gal and production of
-
cells were inspected for blue plaques fomration because of the
+
the blue dye 5,5'-dibromine-4,4'-dichloride-indigo and plaques can be
-
enzymatic
+
seen where the phages lysed the XL1 blue cells. In comparison with
-
cleavage of X-Gal and production of the blue dye
+
plated bacteria only transformed with M13 ssDNA, M13 ssDNA with other
-
5,5'-dibromine-4,4'-dichloride-indigo and plaques can be seen where the
+
oligos not modified or infected with entire phages directly the number
-
phages
+
of pfu (plaque forming units) should be decreased.<br />
-
lysed the ER2738 cells. In comparison with plated bacteria only
+
<br />
-
transformed
+
Another possible approach is a qPCR after the insertion and incubation
-
with M13 ssDNA, M13 ssDNA with other oligos not modified or infected
+
of the phage DNA hybridized with a modified oligo or pure ssDNA into
-
with
+
the XL1 blue. This quantitative real time polymerase chain reaction
-
entire phages directly the number of pfu (plaque forming units) should
+
allows to amplify and simultaneously quantify a certain sequence of the
-
be
+
phage DNA. The additional feature of the qPCR is the quantification of
-
decreased.<o:p></o:p></span></p>
+
the accumulating DNA happens in real time after each amplification
-
<p class="MsoNormal" style="line-height: normal;"><span
+
step. The phage DNA would be extracted by boiling preparation, a method
-
style="font-size: 12pt; font-family: &quot;Lucida Sans&quot;,&quot;sans-serif&quot;;"
+
faster than conventional alkaline lysis minipreps leaving the DNA
-
lang="EN-US">Another possible approach is a qPCR after the
+
somewhat dirtier, but adequate for qPCR. After the qPCR with
-
insertion and incubation of the phage DNA hybridized with a modified
+
appropriate primers, the DNA can be quantified with a fluorescent dye,
-
oligo or
+
intercalating in the double-stranded DNA. The expected result would be
-
pure ssDNA into the XL1 blue. This quantitative real time polymerase
+
a lower amount of double-stranded phage DNA for our XL1 blue
-
chain
+
transformed with DNA and oligo in comparison to the control assay.<br />
-
reaction allows to amplify and simultaneously quantify a certain
+
<br />
-
sequence of
+
<br />
-
the phage DNA. The additional feature of the qPCR is the quantification
+
<h3>Results and Discussion</h3>
-
of the
+
Our attempt to plate cotransformated XL1 Blue on IPTG/X-Gal plates
-
accumulating DNA happens in real time after each amplification step.
+
showed several blue plaques for the M13 DNA without hybridized
-
The phage
+
oligonucleotides. In comparison the M13 DNA hybridized with the
-
DNA would be extracted by boiling preparation, a method faster than
+
modified oligonucleotide showed no blue plaques, demonstrating that the
-
conventional alkaline lysis minipreps leaving the DNA somewhat dirtier,
+
binding of the basally expressed heterodimeric Fok constructs triggered
-
but
+
by the labeled guide oligonucleotides and the following cleavage of the
-
adequate for qPCR. After the qPCR with appropriate primers, the DNA can
+
phage DNA was successful and the progeny of the phages and thus
-
be quantified
+
alpha/complementation decreased.<br />
-
with a fluorescent dye, intercalating in the double-stranded DNA. The
+
<br />
-
expected
+
<table style="text-align: left; width: 464px; height: 272px;"
-
result would be a lower amount of double-stranded phage DNA for our XL1
+
  border="0" cellpadding="0" cellspacing="0">
-
blue
+
  <tbody bgcolor="#e2eff9">
-
transformed with DNA and oligo in comparison to the control assay.<o:p></o:p></span></p>
+
    <tr>
-
<p class="MsoNormal" style="line-height: normal;"><b
+
      <td><img style="width: 450px; height: 220px;"
-
style=""><span
+
  alt=""
-
style="font-size: 12pt; font-family: &quot;Lucida Sans&quot;,&quot;sans-serif&quot;;"
+
  src="https://static.igem.org/mediawiki/2009/e/ee/Freiburg09_plaques_plates_P1000313.jpg" /></td>
-
  lang="EN-US">Results and Discussion<o:p></o:p></span></b></p>
+
    </tr>
-
<p class="MsoNormal" style="line-height: normal;"><span
+
    <tr>
-
  style="font-size: 12pt; font-family: &quot;Lucida Sans&quot;,&quot;sans-serif&quot;;"
+
      <td>M13 DNA without (left) and with (right) hybridized
-
  lang="EN-US">After the first test attempt to plate
+
modified oligonucleotide, no plaques formation in presence of modified
-
cotransformated
+
oligonucleotide</td>
-
XL1 Blue on IPTG/X-Gal plates no blue plaques could be seen, indicating
+
    </tr>
-
that we
+
  </tbody>
-
plated the wrong dilutions, the M13 ssDNA wasn&rsquo;t good, our
+
</table>
-
cells aren&rsquo;t
+
<br />
-
electrocompetent or the basal activity of Fok_a and Fok_i is already
+
The qPCR assay is still in progress. Expected results have still to be
-
sufficient
+
verified.
-
to destroy the whole phage DNA without an adapter-oligo. A
+
<p><span class="art-button-wrapper"></span></p>
-
transformation of a
+
<p><span class="art-button-wrapper"><br />
-
test plasmid in the electrocompetent XL1 blue and another
+
</span></p>
-
electrocompetent
+
<br />
-
strain, RV 308, showed in comparison a weaker but existing
+
</div>
-
transformation rate
+
-
in XL1 blue. Another test assay was to test our infection method by
+
-
infecton of
+
-
ER2738 and XL1 blue with phages from the ph.d 7 phage display peptide
+
-
library,
+
-
i.e. incubation of cells with OD600 of about 0.5 at different phage
+
-
dilutions
+
-
and plating them in prewarmed top agar on IPTG/X-Gal plates. Only the
+
-
ER2738
+
-
but not XL1 blue showed entire blue coloration of the plates.
+
-
(Bedeutung f&uuml;r
+
-
Xblue?) At another test assay we transformed the XL1 blue with new M13
+
-
ssDNA
+
-
and incubated them for 1.5h on a 37&deg;C shaker at 750rpm. Then we
+
-
mixed them at
+
-
different concentrations with ER2738 of an OD600 of 0.42 and plated
+
-
them after
+
-
10 min incubation time in top agar on IPTG/X-Gal plates. As result all
+
-
the
+
-
plates were totally blue. Also with a shorter incubation time of XL1
+
-
blue of
+
-
0.5h on the 37&deg;C shaker we obtained the same result indicating
+
-
that our ER2738
+
-
are already contaminated with phages. Now the next is to try the
+
-
boiling prep
+
-
and the qPCR or redo the same assay with new ER2738.<o:p></o:p></span></p>
+
-
<p class="MsoNormal" style="line-height: normal;"><span
+
-
style="font-size: 12pt; font-family: &quot;Lucida Sans&quot;,&quot;sans-serif&quot;;"
+
-
lang="EN-US"><small></small><o:p></o:p></span></p>
+
<div class="cleared"></div>
<div class="cleared"></div>
</div>
</div>
</div>
</div>
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<b><br />
+
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</b></div>
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<p> <span class="art-button-wrapper"> &nbsp;</span></p>
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Latest revision as of 16:00, 24 November 2009

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Fluorescence microscope image of RV308 cells, electroporated with fluorescein labeled oligonucleotides

Introduction

In vivo use of programmable restriction enzymes can provide the opportunity of genome engineering. Here we develop and test strategies for the application of our programmable restriction endonuclease.

In our strategy we cotransformed Fok_i and Fok_a constructs fused to binding domains into a certain bacteria strain leading to the basal expression of the Fok heterodimers. Transformation of single-stranded bacteriophage M13 DNA hybridized with modified oligonucleotides enables the Fok heterodimers to bind the DNA. Inside the cells the phage DNA should be cut by the dimerized and thereby activated Fok_a should result in a decreased progeny of phages within the bacterial culture in comparison to control assays. Two ways of detection were envisioned. A qPCR should reveal successful cutting of the phage DNA by the lack of amplified phage DNA. In addition an adjacent phage titer determination via a blue white screening ressembling phage assay by plating the cells to IPTG/Xgal plates should lead to a decreased number of plaques in comparison to the control assays.

in vivo scheme


Methods

In detail two heterodimeric Fok constructs His-Dig-Split-Fok_a (BBa_K243036) and His-FluA-Split-Fok_i (BBa_K243010) are encoded by two different plasmids, plasmid pJS419 with a chloramphenicol and pEx with an ampicillin resistance. This enables a positive selection of the bacteria strain XL1 blue cotransformed with both plasmids. Subsequent to the preparation of electrocompetent cotransformed XL1 blue, the M13 ssDNA hybridized with the modified oligonucleotides were inserted into these bacteria by electroporation at 1.7 kV. The infected cells were incubated at 37°C for 0.5 h. At different dilutions the phages were mixed with 180μl ER2738 cells with an OD600 of about 0.5. Then they were mixed with 3 ml of 42°C warm top agar and plated on IPTG/X-Gal plates.

XL1 Blue as well as ER2738 harbour a F-plasmid coding for the omega-fragment of beta-galactosidase. The M13 phages in turn encodes the lacking alpha-fragment. Consequently, alpha-complementation can be detected via a screening for blue plaques. After incubation overnight at 37°C the infected cells were inspected for blue plaques formation because of the enzymatic cleavage of X-Gal and production of the blue dye 5,5'-dibromine-4,4'-dichloride-indigo and plaques can be seen where the phages lysed the XL1 blue cells. In comparison with plated bacteria only transformed with M13 ssDNA, M13 ssDNA with other oligos not modified or infected with entire phages directly the number of pfu (plaque forming units) should be decreased.

Another possible approach is a qPCR after the insertion and incubation of the phage DNA hybridized with a modified oligo or pure ssDNA into the XL1 blue. This quantitative real time polymerase chain reaction allows to amplify and simultaneously quantify a certain sequence of the phage DNA. The additional feature of the qPCR is the quantification of the accumulating DNA happens in real time after each amplification step. The phage DNA would be extracted by boiling preparation, a method faster than conventional alkaline lysis minipreps leaving the DNA somewhat dirtier, but adequate for qPCR. After the qPCR with appropriate primers, the DNA can be quantified with a fluorescent dye, intercalating in the double-stranded DNA. The expected result would be a lower amount of double-stranded phage DNA for our XL1 blue transformed with DNA and oligo in comparison to the control assay.


Results and Discussion

Our attempt to plate cotransformated XL1 Blue on IPTG/X-Gal plates showed several blue plaques for the M13 DNA without hybridized oligonucleotides. In comparison the M13 DNA hybridized with the modified oligonucleotide showed no blue plaques, demonstrating that the binding of the basally expressed heterodimeric Fok constructs triggered by the labeled guide oligonucleotides and the following cleavage of the phage DNA was successful and the progeny of the phages and thus alpha/complementation decreased.

M13 DNA without (left) and with (right) hybridized modified oligonucleotide, no plaques formation in presence of modified oligonucleotide

The qPCR assay is still in progress. Expected results have still to be verified.