Difference between revisions of "Part:BBa K2273034"
Line 1: Line 1:
 
__NOTOC__
 
__NOTOC__
 
{| style="color:black; margin: 20px 0px 20px 20px; float: right; text-align: justify;" cellpadding="6" cellspacing="1" border="2" align="right"
 
{| style="color:black; margin: 20px 0px 20px 20px; float: right; text-align: justify;" cellpadding="6" cellspacing="1" border="2" align="right"
−
! colspan="2" style="background:#66bbff;"| Codon-optimized mCherry for <i>Bacillus subtillus</i>
+
! colspan="2" style="background:#66bbff;"| Part Information
 
|-
 
|-
 
|'''BioBrick Nr.'''
 
|'''BioBrick Nr.'''
Line 14: Line 14:
 
|'''Original Biobrick Part'''
 
|'''Original Biobrick Part'''
 
|[https://parts.igem.org/Part:BBa_J06504 BBa_J06504: mCherry]
 
|[https://parts.igem.org/Part:BBa_J06504 BBa_J06504: mCherry]
−
|-
 
−
|'''Promoter'''
 
−
|[https://parts.igem.org/Part:BBa_K823003 BBa_K823003: Pveg]
 
 
|-
 
|-
 
|'''Submitted by'''
 
|'''Submitted by'''
Line 30: Line 27:
 
<body>
 
<body>
 
<title>BBa_K2273034</title>
 
<title>BBa_K2273034</title>
−
<h2 style="margin-left: 0cm; text-indent: 0cm; font-weight: bold; font-size: 20px;">Brief introduction
+
<h2 style="margin-left: 0cm; text-indent: 0cm; font-weight: bold; font-size: 22px;">Codon-optimized mCherry for <i>Bacillus subtilis</i></h2>
 +
<h2 style="margin-left: 0cm; text-indent: 0cm; font-weight: bold; font-size: 18px;">Brief introduction
 
in Fluorescent Proteins</h2>
 
in Fluorescent Proteins</h2>
 
<p class="MsoNormal"
 
<p class="MsoNormal"
Line 37: Line 35:
 
<p class="MsoNormal"><span lang="EN-US">Fluorescent proteins are small proteins with β-barrel-fold topology. They are useful for tracking global expression of target genes and localizations of these genes inside/outside cells. The unique chromophore in each fluorescent protein, originates from three intrinsic amino acids, at positions 65–67. The chromophore is tightly enclosed inside the protein and its formation does not require any cofactors or enzymes but only molecular oxygen. The rigidity of the β-barrel protects the chromophore from the environment and from radiationless decay. It also restricts chromophore flexibility as the correct folding of the protein is required for the chromophore formation. Proper orientation of the amino acids is necessary for chromophore maturation as it catalyzes chromophore synthesis.</p>
 
<p class="MsoNormal"><span lang="EN-US">Fluorescent proteins are small proteins with β-barrel-fold topology. They are useful for tracking global expression of target genes and localizations of these genes inside/outside cells. The unique chromophore in each fluorescent protein, originates from three intrinsic amino acids, at positions 65–67. The chromophore is tightly enclosed inside the protein and its formation does not require any cofactors or enzymes but only molecular oxygen. The rigidity of the β-barrel protects the chromophore from the environment and from radiationless decay. It also restricts chromophore flexibility as the correct folding of the protein is required for the chromophore formation. Proper orientation of the amino acids is necessary for chromophore maturation as it catalyzes chromophore synthesis.</p>
 
<br>
 
<br>
−
<h2 style="margin-left: 0cm; text-indent: 0cm; font-weight: bold; font-size: 20px; color: black;"><a
+
<h2 style="margin-left: 0cm; text-indent: 0cm; font-weight: bold; font-size: 18px;">Overview of mCherry</h2>
−
name="_Toc275817880"><span lang="EN-US" style="color: black;">Overview
+
−
of mCherry</span></a></h2>
+
−
  <p class="MsoNormal"
+
−
style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
+
−
align="center"><img style="width: 576px; height: 191px;"
+
−
id="Picture 4"
+
−
src="https://static.igem.org/mediawiki/2010/b/bc/Freiburg10_rep_synthetic_gene_fragment.png"
+
−
alt=""></p>
+
 
<p class="MsoNormal"
 
<p class="MsoNormal"
 
  style="margin-bottom: 0.0001pt; text-indent: 0cm;"><span
 
  style="margin-bottom: 0.0001pt; text-indent: 0cm;"><span
 
  style="font-size: 10pt; line-height: 200%;" lang="EN-US">&nbsp;</span></p>
 
  style="font-size: 10pt; line-height: 200%;" lang="EN-US">&nbsp;</span></p>
−
<p class="MsoNormal"><span lang="EN-US"> A mutant of the wild-type green fluorescent protein (GFP) from Aequorea victoria, super folder GFP (sfGFP). sfGFP is a novel and robust variant designed for in vivo high-throughput screening of protein expression levels. sfGFP shows increased thermal stability and is able to tolerate genetic fusion to poorly folding proteins while remaining fluorescent. It incorporates the red shift S65T mutation and the folding mutation F64L and six additional mutations which improve its folding: S30R, Y39N, N105T, Y145F, I171V and A206V. <span
+
<p class="MsoNormal"><span lang="EN-US"> mCherry is a red fluorescent protein that has an excitation peak 585 nm and a peak emission at 615 nm.   </span></p>
−
lang="EN-US">(Cotlet et. al 2006)</span></p>
+
<br>
 
<h2 style="margin-left: 0cm; text-indent: 0cm;"><a
 
<h2 style="margin-left: 0cm; text-indent: 0cm;"><a
 
  name="_Toc275817880"><span lang="EN-US"></span></a></h2>
 
  name="_Toc275817880"><span lang="EN-US"></span></a></h2>
−
 
 
<br>
 
<br>
 
<h3 style="margin-left: 0cm; text-indent: 0cm;"><a
 
<h3 style="margin-left: 0cm; text-indent: 0cm;"><a
 
  name="_Toc275885922"></a><a name="_Toc275817881"><span
 
  name="_Toc275885922"></a><a name="_Toc275817881"><span
−
  lang="EN-US">Modularization: Overview</span></a></h3>
+
  lang="EN-US">mCherry expression in <i>Bacillus subtilis</i></span></a></h3>
 
<p class="MsoNormal"
 
<p class="MsoNormal"
 
  style="text-indent: 0cm; line-height: 150%; page-break-after: avoid;"><span
 
  style="text-indent: 0cm; line-height: 150%; page-break-after: avoid;"><span
−
  lang="EN-US">In our terminology the term “RepVP123”
+
  lang="EN-US">  
−
encompasses the
+
 
−
whole AAV2 genome excluding the ITRs. The <i>rep</i> locus
+
 
−
comprises four
+
</span></p>
−
proteins related to genome replication while the <i>cap</i>
+
−
locus codes for the
+
−
proteins VP1, VP2, VP3 and the assembly-associated protein (AAP), which
+
−
are
+
−
required for viral capsid assembly. Source of the RepVP123 BioBrick
+
−
supplied
+
−
within iGEM team Freiburg_Bioware 2010 Virus Construction Kit is the
+
−
wild-type
+
−
AAV2 RepVP123, as provided e. g. in the pAAV vector from Stratagene. In
+
−
order
+
−
to introduce the iGEM standard and additionally enabling the
+
−
possibility to
+
−
modify
+
−
the viral capsid via integration of certain motives within the viral
+
−
loops 453
+
−
and 587, a total of twelve mutations within RepVP123 (see </span><span
+
−
lang="EN-US">Figure 1</span><span lang="EN-US">)
+
−
and additionally two mutations
+
−
within the pSB1C3 backbone were introduced by either Site-Directed
+
−
Mutagenesis
+
−
(SDM) or by ordering and cloning of specifically designed gene
+
−
sequences matching
+
−
the required demands. Modifying the pSB1C3 led to iGEM team
+
−
Freiburg_Bioware’s
+
−
variant of this backbone, pSB1C3_001.</span></p>
+
 
<br>
 
<br>
  

Revision as of 12:49, 22 October 2017

Part Information
BioBrick Nr. BBa_K2273034
RFC standard RFC 25
Requirement pSB1C3
Original Biobrick Part BBa_J06504: mCherry
Submitted by [http://2017.igem.org/Team:TU_Dresden TU Dresden]

BBa_K2273034

Codon-optimized mCherry for Bacillus subtilis

Brief introduction in Fluorescent Proteins

 

Fluorescent proteins are small proteins with β-barrel-fold topology. They are useful for tracking global expression of target genes and localizations of these genes inside/outside cells. The unique chromophore in each fluorescent protein, originates from three intrinsic amino acids, at positions 65–67. The chromophore is tightly enclosed inside the protein and its formation does not require any cofactors or enzymes but only molecular oxygen. The rigidity of the β-barrel protects the chromophore from the environment and from radiationless decay. It also restricts chromophore flexibility as the correct folding of the protein is required for the chromophore formation. Proper orientation of the amino acids is necessary for chromophore maturation as it catalyzes chromophore synthesis.


Overview of mCherry

 

mCherry is a red fluorescent protein that has an excitation peak 585 nm and a peak emission at 615 nm.



mCherry expression in Bacillus subtilis





Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]