Difference between revisions of "Part:BBa K3017011"

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<p>In our project, Combined CRISPRi and antisense RNA Toggle Switch, dCas9 plays a central role in CRISPR interference. dCas9 acts as an agent of steric hindrance to block mRNA polymerization to repress expression levels of target genes.</p>
 
<p>In our project, Combined CRISPRi and antisense RNA Toggle Switch, dCas9 plays a central role in CRISPR interference. dCas9 acts as an agent of steric hindrance to block mRNA polymerization to repress expression levels of target genes.</p>
  
<p>We sought help from Dr. Ho Yi Mak from our university, who is also working with dCas9. The plasmid Dr. Mak offered contains a <i>Streptococcus pyogenes</i> dCas9 gene optimized for mammalian cells but also functional in <i>E.coli</i>. We designed multiple PCR overhang primers to make the dCas9 gene compatible with the standard assembly so that we could clone it into RFC10 plasmids in further steps.</p>
 
  
<p>The dCas9 protein is tagged with 6XHis at the C-terminus (BBa_K3017011) for easy characterization. In BBa_K3017029, parts BBa_K608002 and BBa_B0015 are added before and after dCas9-6XHis respectively by PCR overhangs. As the dCas9 protein coding region contains intrinsic PstI cut sites, we could not add an iGEM suffix after the part. Instead, we added iGEM prefix and SpeI and SalI cut sites as a suffix for further digesting-ligation steps.</p>
+
<h2>Cloning</h2>
 +
<p>As one of the most important components of our circuit, dCas9 needed to be cloned and expressed in the cells. The dCas9 sequence used was kindly given by Dr. Hi Yi Mak, a professor of life science in HKUST who also have worked with dCas9. However, the sequence of that dCas9 was noticed to have 5 PstI cut sites within the coding sequence meaning that the standard assembly method using EcoRI, XbaI, SpeI and PstI would not be compatible. We thus have added a SalI cut site after PstI to replace PstI by PCR. The other basic parts (BBa_K608006 and BBa_B0015) that is used in subcloning were also added with the SalI cut site. The three parts were then constructed in a BBa_PSB1C3 vector to form a dCas9 expressing vector.</p>
  
  
 
<h2>Transformation and expression of dCas9 in DH5-α <i>E.coli</i></h2>
 
<h2>Transformation and expression of dCas9 in DH5-α <i>E.coli</i></h2>
  
<p>As one of the most important components of our circuit, dCas9 needed to be cloned and expressed in the cells. The dCas9 sequence used was kindly given by Dr. Hi Yi Mak, a professor of life science in HKUST who also have worked with dCas9. However, the sequence of that dCas9 was noticed to have 5 PstI cut sites within the coding sequence meaning that the standard assembly method using EcoRI, XbaI, SpeI and PstI would not be compatible. We thus have added a SalI cut site after PstI to replace PstI by PCR. The other basic parts (BBa_K608006 and BBa_B0015) that is used in subcloning were also added with the SalI cut site. The three parts were then constructed in a BBa_PSB1C3 vector to form a dCas9 expressing vector. The resulting plasmid was then transformed into DH5α E.coli using heat shock, and cells were selected by chloramphenicol followed by Miniprep plasmid extraction and PCR verification (Figure. 1).</p>
+
<p>The resulting plasmid was then transformed into DH5α <i>E. coli</i> using heat shock, and cells were selected by chloramphenicol followed by Miniprep plasmid extraction and PCR verification (Figure. 1).</p>
  
 
<p>Following transformation, we further went on checking the expression of dCas9. Cell lysis was prepared by sonication in NP-40 lysis buffer. The protein concentration of the lysis was checked by Bradford Assay against the BSA standard curve (Figure. 2). 10ul of cell lysis was used to run through the SDS-PAGE (5% stacking, 6% resolving). After the electrophoresis, the gel was stained with Coomassie Blue to visualize the protein (Figure. 3). As shown in the gel, there is an extra band much larger than 82kDa exist only in the dCas9 transformed cells but not appear in the cells transformed with an empty vector.</p>
 
<p>Following transformation, we further went on checking the expression of dCas9. Cell lysis was prepared by sonication in NP-40 lysis buffer. The protein concentration of the lysis was checked by Bradford Assay against the BSA standard curve (Figure. 2). 10ul of cell lysis was used to run through the SDS-PAGE (5% stacking, 6% resolving). After the electrophoresis, the gel was stained with Coomassie Blue to visualize the protein (Figure. 3). As shown in the gel, there is an extra band much larger than 82kDa exist only in the dCas9 transformed cells but not appear in the cells transformed with an empty vector.</p>
  
<p>To further verify if that extra band is really the dCas9, we repeated the experiment again with all the condition remain the same expect that instead of visualizing by Coomassie Blue, we changed to use western blot targeting the 6XHis tag at the C-terminal of dCas9. The western blotting was done using a His-probe Antibody (1:2000, Rabbit) and a secondary HRP-goat anti-rabbit antibody (1:2000). 500ul of ECL detection reagents (Amersham) was then added and the band can subsequently be visualized using light-sensitive X-ray films. As shown in the figure, a clear band only appeared in the dCas9 transformed cells verifying the extra band shown in figure 2 is indeed the dCas9.</p>
+
<p>To further verify if that extra band is really the dCas9, we repeated the experiment again with all the condition remain the same except that instead of visualizing by Coomassie Blue, we changed to use western blot targeting the 6XHis tag at the C-terminal of dCas9. The western blotting was done using a His-probe Antibody (1:2000, Rabbit) and a secondary HRP-goat anti-rabbit antibody (1:2000). 500ul of ECL detection reagents (Amersham) was then added and the band can subsequently be visualized using light-sensitive X-ray films. As shown in the figure, a clear band only appeared in the dCas9 transformed cells verifying the extra band shown in figure 2 is indeed the dCas9.</p>
  
  

Revision as of 07:30, 17 October 2019


dCas9-6XHis

The CRISPR associated protein, in our case, dCas9, is a non-specific deactivated endonuclease. dCas9 is catalytically dead with mutations D10A and H840A. It binds to the target protein, when coupled with single-guide RNA, but does not induce any DNA breakage as in Cas9 enzyme.

In our project, Combined CRISPRi and antisense RNA Toggle Switch, dCas9 plays a central role in CRISPR interference. dCas9 acts as an agent of steric hindrance to block mRNA polymerization to repress expression levels of target genes.


Cloning

As one of the most important components of our circuit, dCas9 needed to be cloned and expressed in the cells. The dCas9 sequence used was kindly given by Dr. Hi Yi Mak, a professor of life science in HKUST who also have worked with dCas9. However, the sequence of that dCas9 was noticed to have 5 PstI cut sites within the coding sequence meaning that the standard assembly method using EcoRI, XbaI, SpeI and PstI would not be compatible. We thus have added a SalI cut site after PstI to replace PstI by PCR. The other basic parts (BBa_K608006 and BBa_B0015) that is used in subcloning were also added with the SalI cut site. The three parts were then constructed in a BBa_PSB1C3 vector to form a dCas9 expressing vector.


Transformation and expression of dCas9 in DH5-α E.coli

The resulting plasmid was then transformed into DH5α E. coli using heat shock, and cells were selected by chloramphenicol followed by Miniprep plasmid extraction and PCR verification (Figure. 1).

Following transformation, we further went on checking the expression of dCas9. Cell lysis was prepared by sonication in NP-40 lysis buffer. The protein concentration of the lysis was checked by Bradford Assay against the BSA standard curve (Figure. 2). 10ul of cell lysis was used to run through the SDS-PAGE (5% stacking, 6% resolving). After the electrophoresis, the gel was stained with Coomassie Blue to visualize the protein (Figure. 3). As shown in the gel, there is an extra band much larger than 82kDa exist only in the dCas9 transformed cells but not appear in the cells transformed with an empty vector.

To further verify if that extra band is really the dCas9, we repeated the experiment again with all the condition remain the same except that instead of visualizing by Coomassie Blue, we changed to use western blot targeting the 6XHis tag at the C-terminal of dCas9. The western blotting was done using a His-probe Antibody (1:2000, Rabbit) and a secondary HRP-goat anti-rabbit antibody (1:2000). 500ul of ECL detection reagents (Amersham) was then added and the band can subsequently be visualized using light-sensitive X-ray films. As shown in the figure, a clear band only appeared in the dCas9 transformed cells verifying the extra band shown in figure 2 is indeed the dCas9.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 790
    Illegal PstI site found at 2212
    Illegal PstI site found at 2416
    Illegal PstI site found at 2446
    Illegal PstI site found at 3658
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 790
    Illegal PstI site found at 2212
    Illegal PstI site found at 2416
    Illegal PstI site found at 2446
    Illegal PstI site found at 3658
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 251
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 790
    Illegal PstI site found at 2212
    Illegal PstI site found at 2416
    Illegal PstI site found at 2446
    Illegal PstI site found at 3658
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 790
    Illegal PstI site found at 2212
    Illegal PstI site found at 2416
    Illegal PstI site found at 2446
    Illegal PstI site found at 3658
    Illegal NgoMIV site found at 1078
    Illegal NgoMIV site found at 2182
    Illegal NgoMIV site found at 2255
    Illegal NgoMIV site found at 2740
    Illegal NgoMIV site found at 3649
  • 1000
    COMPATIBLE WITH RFC[1000]