Team:The University of Alberta/16 May 2008

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(New page: ==To Do== Plant Seminar @ 4:00pm)
 
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Plant Seminar @ 4:00pm
Plant Seminar @ 4:00pm
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==In the Lab==
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<u>'''Winnie and Saima'''</u><br>
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carried out transformation using DNA from the part registry from the 2007 butanol project (DNA: I725021, I725022,I725023,I725025,I725099)
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we followed the procedure outlined in the part registry book except we used 100µl of competent cells instead of 50µl and diluted the DNA with additional 5µl of MilliQ
 +
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Winnie found a page on interaction of PIFs and PhyB, it is posted under project in  our wiki
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<u>'''Chris'''</u><br>
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<ul>
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<li> I finished sequencing the thiolase gene again; the samples need to be run up to the MBSU<br>
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<li>Jason and I designed the BioBrick for PIF3; I spent some time trying to do codon optimization for E.Coli
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I downloaded a program onto one of the computers in the back of the lab for optimizing the BioBricks but it doesnt seem to work very well; use [http://genomes.urv.es/OPTIMIZER this] website instead if you want to work on codon optimization.<br>
 +
<li>The PIF3 BioBrick doesn't have a His-tag added to it. Can't forget to do that before we get them synthesized.
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</ul><br><br>
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If anyone wants to work on building BioBricks, these sites are a good help:<ul>
 +
<li> [http://www.arabidopsis.org - '''The Arabidposis Information Resource (TAIR)''']: This site will help you find the sequences for the genes and proteins that we want from Arabidopsis. It's best if you use cDNA sequences because these already have the introns spliced from them!
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<li>[http://www.ebi.ac.uk/Tools/clustalw2/index.html - '''ClustalW''']: This site is great for aligning two DNA or protein sequences. This makes it super easy to check to make sure your edited/optimized sequences still code from the same thing as the reference sequences from TAIR.
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<li>[http://www.expasy.ch/ - '''ExPASy Proteomics Server''']: This site has tons of tools that we'll probably never use, but there are still a few useful things:<ul>
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<li> [http://www.expasy.ch/tools/dna.html Translate] This will let you translate your cDNA sequence into an amino acid sequence at the click of a button!
 +
<li> [http://www.expasy.ch/prosite/ ProSite and ScanProSite] These let you search your amino acid sequence to check for domains, modification sites, etc. Probably not useful but it does have a tool that allows you to make some neat schematic diagrams of genes/proteins like the one below:<br>
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[[Image:examplediagram.png]]</ul></ul>
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<br>
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<u>Keep in mind</u>: simply attaching the BioBrick prefix and suffix to the cDNA won't necessairly work. The cDNAs have 3' and 5' Untranslated Regions (UTRs). These need to be edited out. Probably the easist way to do this is to translate the cDNA and compare it to the reference sequence on TAIR. You'll see a string of amino acids at the beginning and end of the translated sequence that are not in the reference sequence; these correspond to the untranslated regions. Delete the nucleotide sequences from the cDNA that correspond to these extra amino acids.
 +
 +
<u>Also remember</u>: we dont want to have any internal restriction sites (particularly EcoR1 and Pst1) in the BioBricks, lest they be chopped up when we're doing our digestions. This would be bad. The optimization tool I listed above has an option that lets you see if there are any sites in the BioBrick and lets you change the sequence accordingly.
 +
 +
<u>'''Jason'''</u><br>
 +
<ul>
 +
<li> I also started working on the Phytochromobilin operon. This involved finding the sequences and removing all useless DNA (UTRs, introns etc.)This operon does not have a his tag added yet either.
 +
<li> Me and James where thinking of using I0500 as the vector for our constructs as it has an arabinose induced promoter that would allow us to control transcription if these genes turn out to be toxic. If it turns out these genes are not toxic we might then start thinking of moving them into a plasmid with a constituative promoter.</ul>
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<br>'''Lab Tip of the Day'''
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Tweezers can be sharp and can sometimes break the skin if fiddled with. One should use the utmost caution when handling and always read the manual and wear proper PPE when handling.

Latest revision as of 20:56, 22 May 2008

To Do

Plant Seminar @ 4:00pm

In the Lab

Winnie and Saima

carried out transformation using DNA from the part registry from the 2007 butanol project (DNA: I725021, I725022,I725023,I725025,I725099) we followed the procedure outlined in the part registry book except we used 100µl of competent cells instead of 50µl and diluted the DNA with additional 5µl of MilliQ

Winnie found a page on interaction of PIFs and PhyB, it is posted under project in our wiki


Chris

  • I finished sequencing the thiolase gene again; the samples need to be run up to the MBSU
  • Jason and I designed the BioBrick for PIF3; I spent some time trying to do codon optimization for E.Coli I downloaded a program onto one of the computers in the back of the lab for optimizing the BioBricks but it doesnt seem to work very well; use [http://genomes.urv.es/OPTIMIZER this] website instead if you want to work on codon optimization.
  • The PIF3 BioBrick doesn't have a His-tag added to it. Can't forget to do that before we get them synthesized.


If anyone wants to work on building BioBricks, these sites are a good help:
  • [http://www.arabidopsis.org - The Arabidposis Information Resource (TAIR)]: This site will help you find the sequences for the genes and proteins that we want from Arabidopsis. It's best if you use cDNA sequences because these already have the introns spliced from them!
  • [http://www.ebi.ac.uk/Tools/clustalw2/index.html - ClustalW]: This site is great for aligning two DNA or protein sequences. This makes it super easy to check to make sure your edited/optimized sequences still code from the same thing as the reference sequences from TAIR.
  • [http://www.expasy.ch/ - ExPASy Proteomics Server]: This site has tons of tools that we'll probably never use, but there are still a few useful things:
    • [http://www.expasy.ch/tools/dna.html Translate] This will let you translate your cDNA sequence into an amino acid sequence at the click of a button!
    • [http://www.expasy.ch/prosite/ ProSite and ScanProSite] These let you search your amino acid sequence to check for domains, modification sites, etc. Probably not useful but it does have a tool that allows you to make some neat schematic diagrams of genes/proteins like the one below:
      Examplediagram.png


Keep in mind: simply attaching the BioBrick prefix and suffix to the cDNA won't necessairly work. The cDNAs have 3' and 5' Untranslated Regions (UTRs). These need to be edited out. Probably the easist way to do this is to translate the cDNA and compare it to the reference sequence on TAIR. You'll see a string of amino acids at the beginning and end of the translated sequence that are not in the reference sequence; these correspond to the untranslated regions. Delete the nucleotide sequences from the cDNA that correspond to these extra amino acids.

Also remember: we dont want to have any internal restriction sites (particularly EcoR1 and Pst1) in the BioBricks, lest they be chopped up when we're doing our digestions. This would be bad. The optimization tool I listed above has an option that lets you see if there are any sites in the BioBrick and lets you change the sequence accordingly.

Jason

  • I also started working on the Phytochromobilin operon. This involved finding the sequences and removing all useless DNA (UTRs, introns etc.)This operon does not have a his tag added yet either.
  • Me and James where thinking of using I0500 as the vector for our constructs as it has an arabinose induced promoter that would allow us to control transcription if these genes turn out to be toxic. If it turns out these genes are not toxic we might then start thinking of moving them into a plasmid with a constituative promoter.


Lab Tip of the Day

Tweezers can be sharp and can sometimes break the skin if fiddled with. One should use the utmost caution when handling and always read the manual and wear proper PPE when handling.

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