Difference between revisions of "Part:BBa K404090"
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[[Image:Freiburg10_ReplicationBricks 1.png|thumb|center|480px]]<br>
 
[[Image:Freiburg10_ReplicationBricks 1.png|thumb|center|480px]]<br>
  
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<h3>Usage and Biology</h3>
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<h3>Rep40</h3>
 
{| style="margin: 0px 0px 300px 20px; color: black; float: right;" cellpadding="6" cellspacing="1" border="2"  
 
{| style="margin: 0px 0px 300px 20px; color: black; float: right;" cellpadding="6" cellspacing="1" border="2"  
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! colspan="2" style="background:#66bbff;"|[https://parts.igem.org/Part:BBa_K404107 <i>beta-globin</i> intron]
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! colspan="2" style="background:#66bbff;"|[https://parts.igem.org/Part:BBa_K40490 <i>beta-globin</i> intron]
 
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! colspan="2"|[[Image:Freiburg10 VectorplasmidBricks 4.png|200px]]
 
! colspan="2"|[[Image:Freiburg10 VectorplasmidBricks 4.png|200px]]
 
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|'''BioBrick Nr.'''
 
|'''BioBrick Nr.'''
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|[https://parts.igem.org/Part:BBa_K404107 BBa_K404107]
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|[https://parts.igem.org/Part:BBa_K40490 BBa_K40490]
 
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|'''RFC standard'''
 
|'''RFC standard'''
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Providing an element assumed to be an enhancer of transgene expression (Nott, Meislin, & Moore, 2003), the iGEM team Freiburg presents a beta-globin intron derived from the human beta globin gene which can be fused upstream of the desired gene of interest. <br/ >
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Introns are non-coding sequences that are spliced after gene transcription. Apart from the possibility of alternative splicing which leads to an increased variability of translated proteins from one single gene, additional functions of introns have been found. They regulate and enhance gene expression at multiple levels such as initiating transcription, gene editing and polyadenylation of pre-mRNA (Nott, Le Hir, & Moore, 2004).In Valencia, Dias, & Reed (2008) the authors described the influence of splicing transgenes containing one intron at the 5´ and 3´ position, respectively. They demonstrated that splicing promotes the nuclear export of mRNA and that the spliceosome is cross-coupled to the mRNA export machinery.  
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Rep40 in some minutes<br/>
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<br />
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• 40 kDa<br/>
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<img src="https://static.igem.org/mediawiki/parts/d/d7/Freiburg10_Nucleotide_sequence_beta-globin_intron.png" width="660"
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• ATPase and helicase activity <br/>
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height="auto"/>
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• Involved in genome encapsidation<br/>
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The smallest Rep protein (Rep40) possesses helicase and ATPase activity as well, but does not have strict requirements for DNA duplexes containing a 3´single-stranded end. Rep40 helicase activity requires bivalent ions such as Mg2+ or Mn2+ and is most active using ATP as substrate. Lacking the zinc finger domain, present in Rep52, Rep40 requires dimerization for functional helicase activity (Collaco, Kalman-Maltese, Smith, Dignam, & Trempe, 2003). Rep40/52 proteins are required for translocation of the single-stranded, viral genomes into the preformed capsids proceeding with the 3´end of the DNA (King, Dubielzig, Grimm, & Kleinschmidt, 2001).  
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<h3>References</h3>
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Revision as of 22:20, 26 October 2010

[AAV2]-Rep40ex

Freiburg10 ReplicationBricks 1.png

Rep40

beta-globin intron
Freiburg10 VectorplasmidBricks 4.png
BioBrick Nr. BBa_K40490
RFC standard RFC 10
Requirement pSB1C3
Source pAAV_MCS
Submitted by [http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010]

Rep40 in some minutes
• 40 kDa
• ATPase and helicase activity
• Involved in genome encapsidation
The smallest Rep protein (Rep40) possesses helicase and ATPase activity as well, but does not have strict requirements for DNA duplexes containing a 3´single-stranded end. Rep40 helicase activity requires bivalent ions such as Mg2+ or Mn2+ and is most active using ATP as substrate. Lacking the zinc finger domain, present in Rep52, Rep40 requires dimerization for functional helicase activity (Collaco, Kalman-Maltese, Smith, Dignam, & Trempe, 2003). Rep40/52 proteins are required for translocation of the single-stranded, viral genomes into the preformed capsids proceeding with the 3´end of the DNA (King, Dubielzig, Grimm, & Kleinschmidt, 2001).


Characterization

The BioBrick part beta-globin intron consists partially of a chimeric CMV promoter, followed by the intron II of the beta-globin gene. The 3´end of the intron is fused to the first 20 nucleotides of exon 3 of the beta globin gene. Our BioBrick part beta globin intron is assumed to enhance eukaryotic gene expression.
AAV-293 cells were transfected with all genes necessary for producing viral particles encapsidating two different rAAV genomes with and without beta-globin intron respectively. mVenus expression was determined by flow cytometry 24-hours post infection of HT1080 cells. The rAAV genomes missing the beta-globin intron showed a negligible difference in mVenus expression compared to viral genomes containing the beta-globin intron. Considering these results, we suggest using the beta-globin intron in dependence on the size of your transgene.

References

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 53
    Illegal XhoI site found at 920
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]