Team:Amsterdam/extra/faq

From 2012.igem.org

(Difference between revisions)
 
Line 14: Line 14:
<h4>Won’t endogenous methylation cause interference?</h4>
<h4>Won’t endogenous methylation cause interference?</h4>
-
E. coli posses several endogenous methyltransferases which are used for epigenetic purposes of E. coli’s own genome. Our scan of these endogenous methyltransferases does not indicate that any of them posses the ability to bind to our M.ScaI-recognition site and are thus they are not able to methylate the detection site.
+
E. coli posses several endogenous methyltransferases which are used for epigenetic purposes of <i>E. coli</i>’s own genome. Our scan of these endogenous methyltransferases does not indicate that any of them posses the ability to bind to our M.ScaI-recognition site and are thus they are not able to methylate the detection site.
-
<h4>Will the fusion-protein fold correctly or be processed by E. coli’s native systems?</h4>
+
<h4>Will the fusion-protein fold correctly or be processed by <i>E. coli</i>’s native systems?</h4>
At the moment there is no indication that this will be the case. Separately the parts of the protein have been translated and expressed in E. coli without complications.  
At the moment there is no indication that this will be the case. Separately the parts of the protein have been translated and expressed in E. coli without complications.  
<h4>Will the introduced methyltranferase cause problems inside the microorganism?</h4>
<h4>Will the introduced methyltranferase cause problems inside the microorganism?</h4>
-
Right now all our experiments are conducted in the DH5alpha strain of E. coli which does not posses the M.ScaI methyltransferase we use. Actually the M.ScaI is a type 4 methyltransferase, that does not occur naturally in E. coli. After BLAST searching the E. coli genome we also did not find any potential binding site for our M.ScaI.
+
Right now all our experiments are conducted in the DH5alpha strain of <i>E. coli</i> which does not posses the M.ScaI methyltransferase we use. Actually the M.ScaI is a type 4 methyltransferase, that does not occur naturally in E. coli. After BLAST searching the E. coli genome we also did not find any potential binding site for our M.ScaI.
<h4>Won’t the memory plasmid be cut or processed by the endogenous restriction enzymes?</h4>
<h4>Won’t the memory plasmid be cut or processed by the endogenous restriction enzymes?</h4>
-
The chosen restriction sites in our plasmid are not recognized by E. coli’s endogenous restriction enzymes. The plasmid backbones and vectors that are used have all been previously used or expressed in E. coli without any complications.
+
The chosen restriction sites in our plasmid are not recognized by <i>E. coli</i>’s endogenous restriction enzymes. The plasmid backbones and vectors that are used have all been previously used or expressed in E. coli without any complications.
<h4>If there is leaky expression of the protein, would this cause interference?</h4>
<h4>If there is leaky expression of the protein, would this cause interference?</h4>
Line 29: Line 29:
<h4>How much specificity is created by the Zinc-finger?</h4>
<h4>How much specificity is created by the Zinc-finger?</h4>
-
The Zinc-finger uses an unique 18 bp sequence which is not found anywhere else on the plasmid or in the genome of E. coli. The Zinc-finger multiplies the overall probability of binding (PoB) to a much higher specificity when being attached to the M.ScaI which has its own PoB.  
+
The Zinc-finger uses an unique 18 bp sequence which is not found anywhere else on the plasmid or in the genome of <i>E. coli</i>. The Zinc-finger multiplies the overall probability of binding (PoB) to a much higher specificity when being attached to the M.ScaI which has its own PoB.  
<h4>Would constitutive active promoters cause random or unspecific methylation?</h4>
<h4>Would constitutive active promoters cause random or unspecific methylation?</h4>

Latest revision as of 03:57, 27 September 2012