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(Created page with "<html> <a name="gBlockDesign"></a> <li style="font-weight: bold;"> <h2>Design of IntI1 gBlock</h2> <ul style="font-weight: normal;"> The code for the Inti1 gene was sourced from...")
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<li style="font-weight: bold;"> <h2>Design of IntI1 gBlock</h2>
<li style="font-weight: bold;"> <h2>Design of IntI1 gBlock</h2>
<ul style="font-weight: normal;">
<ul style="font-weight: normal;">
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The code for the Inti1 gene was sourced from pUS2056 (theUSyd  Coleman- Holmes lab had this LINK). We tried to get the part from the Paris Bettencourt team (from 2010 part BBa_K329014 http://parts.igem.org/cgi/partsdb/puttext.cgi) but this was not possible so we had to make it. It was modified by Tom Geddes, Rokiah Alford and Nick Coleman to include:
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The code for the Inti1 gene was sourced from pUS2056 (theUSyd  Coleman- Holmes lab had this LINK). We tried to get the part from the Paris Bettencourt team (from 2010 part BBa_K329014 http://parts.igem.org/cgi/partsdb/puttext.cgi) but this was not possible so we had to make it. It was modified by Tom Geddes, Rokiah Alford and Nick Coleman to include: <ol>
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- a different ribosome binding site,  
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<li>- a different ribosome binding site, </li>
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- to remove an illegal spe1 site near the end,
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<li>- to remove an illegal spe1 site near the end, </li>
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- to include a Sac1 restriction enzyme site at the beginning before the inti1 gene so it could be excised independently of the promoter,  
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<li>- to include a Sac1 restriction enzyme site at the beginning before the inti1 gene so it could be excised independently of the promoter, </li>
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- to remove a promoter pC that was coded in the opposite direction to the promoter we wanted to use and would have clashed with the controllable promotion of this part, and
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<li>- to remove a promoter pC that was coded in the opposite direction to the promoter we wanted to use and would have clashed with the controllable promotion of this part, and </li>
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- to have Gibson ends that overlapped with the backbone we wanted to put it into which is described below.
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<li>- to have Gibson ends that overlapped with the backbone we wanted to put it into which is described below.<li>
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This backbone, made by honours student Sam Ross, is PSB1C3 with araC-pBAD (from BBa_K731201 a well characterized system) in between the suffix and prefix and will be called SamR’s plasmid throughout our documents. The code for the biobrick section of this plasmid was used to predict the sequence for junction PCR sequences and our biobrick’s sequence.  
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<br>
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<li>This backbone, made by honours student Sam Ross, is PSB1C3 with araC-pBAD (which was sourced from <a "href=http://parts.igem.org/Part:BBa_K731201">BBa_K731201</a> a well characterized part) in between the suffix and prefix and will be called SamR’s plasmid throughout our documents. The code for the biobrick section of this plasmid was used to predict the sequence for junction PCR sequences and our biobrick’s sequence. </li>
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<br>
Our first attempt to Gibson Assemble these two – the Inti1 Gblock and SamR’s plasmid – failed because the gibson end that overlapped the araC-pBAD section was incorrect. There was extra code after the code for pBAD which we had not anticipated. This triggered the redesign of the gblock.  
Our first attempt to Gibson Assemble these two – the Inti1 Gblock and SamR’s plasmid – failed because the gibson end that overlapped the araC-pBAD section was incorrect. There was extra code after the code for pBAD which we had not anticipated. This triggered the redesign of the gblock.  

Revision as of 00:59, 16 October 2014

  • Design of IntI1 gBlock

      The code for the Inti1 gene was sourced from pUS2056 (theUSyd Coleman- Holmes lab had this LINK). We tried to get the part from the Paris Bettencourt team (from 2010 part BBa_K329014 http://parts.igem.org/cgi/partsdb/puttext.cgi) but this was not possible so we had to make it. It was modified by Tom Geddes, Rokiah Alford and Nick Coleman to include:
      1. - a different ribosome binding site,
      2. - to remove an illegal spe1 site near the end,
      3. - to include a Sac1 restriction enzyme site at the beginning before the inti1 gene so it could be excised independently of the promoter,
      4. - to remove a promoter pC that was coded in the opposite direction to the promoter we wanted to use and would have clashed with the controllable promotion of this part, and
      5. - to have Gibson ends that overlapped with the backbone we wanted to put it into which is described below.

      7. This backbone, made by honours student Sam Ross, is PSB1C3 with araC-pBAD (which was sourced from BBa_K731201 a well characterized part) in between the suffix and prefix and will be called SamR’s plasmid throughout our documents. The code for the biobrick section of this plasmid was used to predict the sequence for junction PCR sequences and our biobrick’s sequence.

        8. Our first attempt to Gibson Assemble these two – the Inti1 Gblock and SamR’s plasmid – failed because the gibson end that overlapped the araC-pBAD section was incorrect. There was extra code after the code for pBAD which we had not anticipated. This triggered the redesign of the gblock. Back to top

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