Difference between revisions of "Team:DTU-Denmark/Description"

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                         <h1>Title<p class="lead">leader under the title, short introduction. Ubique moderatius efficiantur eum et, dico oporteat recusabo ius cu, pro id modus sadipscing. Maluisset patrioque eum ad, mel eius doctus accommodare eu, minimum deleniti repudiandae mel ea. Noster nostrud diceret sea no. Eos an nullam molestiae signiferumque, vel ne laudem ignota oblique. Duo te luptatum percipitur signiferumque, at dicunt iriure dolorem his.</p></h1>
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                         <h1>Title<p class="lead">leader under the title, short introduction.  
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        <h2 class="h2">Section 3</h2>
 
  
 
            
 
            
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                 <div class="col-md-9 col-sm-9 col-xs-12">
                     <p>Description of biobrick</p>
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                     <p>Constitutive TEF1 promoter</p>
 
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                     <p>Description of biobrick</p>
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                     <p>Human proinsulin codon-optimised for <i>Y. lipolytica</i></p>
 
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                 <div class="col-md-9 col-sm-9 col-xs-12">
                     <p>Description of biobrick</p>
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                     <p>Composite part consisting of the TEF1 promoter and the human proinsulin gene</p>
 
                 </div>
 
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                 <div class="col-md-9 col-sm-9 col-xs-12">
                     <p>Description of biobrick</p>
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                     <p>hrGFP codon-optimized for <i>Y. lipolytica</i></p>
 
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                 <div class="col-md-9 col-sm-9 col-xs-12">
                     <p>Description of biobrick</p>
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                     <p>hrGFP expressed by the constitutive TEF1 promoter </p>
 
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                     <p>Description of biobrick</p>
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                     <p>SCR1'-tRNA promoter expressing gRNA</p>
 
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                     <p>Description of biobrick</p>
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                     <p>Semi-synthetic shuttle vector pSB1A8YL</p>
 
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                     <p>Description of biobrick</p>
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                     <p>Composite part of TEF1 and enhanced YFP </p>
 
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                     <p>Description of biobrick</p>
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                     <p>Composite part of TEF1 and eCFP</p>
 
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                     <p>Description of biobrick</p>
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                     <p>Improved CrtE</p>
 
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                     <p>Description of biobrick</p>
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                     <p>Improved CrtI</p>
 
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         <ul class="nav" id="sidebar">
 
         <ul class="nav" id="sidebar">
 
             <li><a href="#section-1">Improvement and Charactization</a></li>
 
             <li><a href="#section-1">Improvement and Charactization</a></li>
             <li><a href="#section-2">Section 2</a></li>
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             <li><a href="#section-2">Parts list</a></li>
 
             <li><a href="#section-3">Section 3</a></li>
 
             <li><a href="#section-3">Section 3</a></li>
 
             <li><a href="#section-4">Section 4</a></li>
 
             <li><a href="#section-4">Section 4</a></li>

Revision as of 18:50, 18 October 2016

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Title

leader under the title, short introduction.


Improvement and Characterization of Two Existing Parts

Quote Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer posuere erat a ante.

Someone famous in Source Title

Improvement and Characterization of Two Existing Parts We have improved two existing BioBricks by successful removal of two illegal restriction sites and further improved the characterization.

In our laboratory work, we wanted to work with the two BioBricks; BBa_K530001 (CrtE gene) and BBa_K530002 (CrtI gene) created by the John Hopkins iGEM team 2011. The genes encode the two enzymes Geranylgeranyl Diphosphate Synthase and Phytoene Desaturase, respectively, both from the wildtype strain of xanthophyllomyces dendrorhous used in the biosynthesis of B-Carotene.

When looking into the specifications of these BioBricks, we realized that both genes contained illegal restriction sites. BBa_K530001 contained an AgeI restriction site making the part incompatible with the RFC25 Freiburg Standard. The RFC25 standard allows for in-frame assembly of protein domains. BBa_K530002 contained the illegal restriction site BglII making the part incompatible with the RFC21 Berkely Standard, which enables in-frame assembly of proteins.

We wanted to overcome these obstacle by removing the illegal restriction sites from the above described Biobricks.

How did we do it?

The illegal restriction sites were removed using site directed mutagenesis with primers containing nucleotide substitutions in the two restriction sites (see figure. Protocol https://static.igem.org/mediawiki/2014/b/b5/Wageningen_UR_protocols_Site_Directed_Mutagenesis.pdf).

Dog

Text for figure:Primers overlapping the restriction sites are designed with a single nucleotide change to disrupt the restriction site. The primers anneal to the template plasmid and replicate while introducing the point mutation. The elongated plasmids are digested with the enzyme DpnI, which cleaves at the methylated sites breaking down the circular template, resulting in a higher transformation efficiency of the linear PCR product.

Insert table

The new plasmid, BBa_K2117012, with the removed AgeI restriction site was double digested with the enzyme and SpeI to test if we had successfully removed the AgeI restriction site.

Dog

Figure text: AgeI + SpeI digestion. Electrophoresis on a 1% agarose gel showing the digestion of BBa_K2117012 (lane 1-3). BBa_530001 digested with AgeI + SpeI and undigested BBa_530001 were used as controls (lane 4+5).

The digestion showed the removal of the AgeI restriction site, shown on the gel picture by only one band on the BBa_K2117012 compared to two bands on the control BBa_K530001.

BBa_K2117012 and BBa_K2117013 were sent for sequencing with the verification primers VR and VF2 primers to further verify the removal of the restriction sites.

insert plasmids

The sequencing results show a substitution in the restriction sites corresponding to the nucleotide substitution designed in the primers (table ??).

By deleting the two restriction sites in the BBa_K2117012 and BBa_K2117013 we have made the two BioBricks compatible with RFC25 and RFC21 standards, respectively.

Improvement of Characterization

While working with BBa_K530001 and BBa_K530002 we were struggling with assembly of the genes. Therefore, we sequenced the plasmids received in the distribution kit.

Sequencing results of BBa_K530002 showed nucleotide substitutions in the prefix and a large deletion in the suffix.

Dog Dog

figure text 1: Figure text: Alignment of BBa_K530002 sequence received from the parts page (top) and sequencing results of BBa_K530002 from the distribution kit. Alignment shows many nucleotide substitutions in the prefix seuqence

Figure text 2: Figure text: Alignment of BBa_K530002 sequence received from the parts page (top) and sequencing results of BBa_K530002 from the distribution kit. Alignment shows a deletion in the suffix sequence.

The alterations in prefix and suffix make the BBa_K530002 incompatible with the BioBrick standard

Parts list

Constitutive TEF1 promoter

Human proinsulin codon-optimised for Y. lipolytica

Composite part consisting of the TEF1 promoter and the human proinsulin gene

hrGFP codon-optimized for Y. lipolytica

hrGFP expressed by the constitutive TEF1 promoter

SCR1'-tRNA promoter expressing gRNA

Semi-synthetic shuttle vector pSB1A8YL

Composite part of TEF1 and enhanced YFP

Composite part of TEF1 and eCFP

Improved CrtE

Improved CrtI

Has ut facer debitis, quo eu agam purto. In eum justo aeterno. Sea ut atqui efficiantur, mandamus deseruisse at est, erat natum cum eu. Quot numquam in vel. Salutatus euripidis moderatius qui ex, eu tempor volumus vituperatoribus has, ius ea ullum facer corrumpit.

Section 4

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Section 5

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Section 6

Has ut facer debitis, quo eu agam purto. In eum justo aeterno. Sea ut atqui efficiantur, mandamus deseruisse at est, erat natum cum eu. Quot numquam in vel. Salutatus euripidis moderatius qui ex, eu tempor volumus vituperatoribus has, ius ea ullum facer corrumpit.

Section 7

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