Difference between revisions of "Part:BBa K157014:Design"

(→‎Design Notes)
(→‎Design Notes)
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Plasmid containing BioBrick 3.0 (Prefix and Suffix for protein fusion) restriction sites
 
Plasmid containing BioBrick 3.0 (Prefix and Suffix for protein fusion) restriction sites
 +
 +
<H1>Proposal for a BioBrick extension for fusion proteins</h1>
 +
iGEM team Freiburg
 +
 +
<h2>Introduction</h2>
 +
 +
<p>The generalized BioBrick prefix and suffix with its easy cloning strategy is an excellent and universal way to combine
 +
various parts, e.g. promoter region, gene of interest, terminator etc. However,
 +
also proteins consist of different parts and thus the &quot;mix-and-match&quot;
 +
combination of fusion proteins is an important task in Synthetic Biology.
 +
However, this modular assembly from BioBrick parts is currently not possible due to the generation of a
 +
stop codon at the SpeI/Xba scar.</p><br>
 +
<p>Suffix of the first part (<font color="#bababa">part in gray</font>,
 +
<font color="#cc00ff">PstI</font>, NotI,<font color="#ff5333"> SpeI</font>):</p>
 +
<p><b><span style="font-family: monospace; font-size: 125%; padding-left: 1em;">
 +
<font color="#bababa">...AC</font>t<font color="#ff5333">actagt</font>agcggccg<font color="#cc00ff">ctgcag</font></span></b>
 +
</p><br>
 +
<p>combined with Prefix of the second part (<font color="#cc00ff">EcoRI</font>, NotI,<font color="#ff5333">
 +
XbaI</font>,<font color="#bababa">part in gray</font>):</p>
 +
<p><b><span style="font-family: monospace; font-size: 125%; padding-left: 1em;">
 +
<font color="#cc00ff">gaattcc</font>gcggccgct<font color="#ff5333">tctaga</font>g<font color="#bababa">CA...</font></span></b><br>
 +
</p>
 +
<p>results after an SpeI/Xba combination in:</p>
 +
<p><b><span style="font-family: monospace; font-size: 125%; padding-left: 1em;">
 +
<font color="#bababa">...AC</font>t<font color="#ff5333">act</font></span><span style="font-family: monospace; font-size: 125%; "><font color="#ff5333">aga</font>g<font color="#bababa">CA...</font></span></b></p>
 +
<p>encoding the amino acids Tyr (codon: tac), STOP (codon: tag) and Ser or Arg (codon
 +
agn).</p>
 +
<p>Consequently, fusion proteins can so far only be designed as one part but not
 +
in a modular fashion. Therefore, we developed a new generation of BioBricks
 +
completely compatible with the first BioBrick version but with two additional compatible
 +
restriction sites to allow for the modular construction of protein fusion parts.
 +
Appropriate enzymes were chosen carefully to include ensure coding for amino
 +
acids, which are compatible with flexible linkers as well as with the N-end rule
 +
for protein stability. We call this new version BioBricks 3.0.</p>
 +
<p>
 +
<h2>Proposal</h2>
 +
We propose to extend the standard BioBrick suffix and prefix with two
 +
additional compatible restriction sites. The frame of the standard suffix and
 +
prefix remains the same resulting in complete compatibility with any previously
 +
designed parts. We chose the restriction sites NgoMVI and AgeI as they code for
 +
the amino acids Ala-Gly or Thr-Gly, respectively, which are compatible with
 +
flexible linkers commonly used in fusion proteins and also compatible with the
 +
N-end rule for protein stabiliy. Consequently, we name these parts <b>
 +
FusionParts</b>. A combination of two such FusionParts creates an
 +
AgeI/NgoMIV scar coding for Thr-Gly, which can easily be integrated in any
 +
linker sequence. Furthermore, the NgoMIV site (coding for Ala-Gly) after the
 +
start Methionin of the standard BioBrick suffix adheres completely to the N-end
 +
rule. For proteins, which are sensitive to amino acid addition at the
 +
N-terminus, we also devised an <b>N-Part</b> with a suffix that lacks the NgoMIV site.</p><br>
 +
 +
<p>The following list summarizes the most important factors of the BioBrick 3.0
 +
design</p><br>
 +
 +
<p><b><font size="4">Strategy for iGEM BioBrick 3.0 parts for mix-and-match
 +
construction of fusion proteins  </font></b></p>
 +
<p><i>developed by the iGEM Team Freiburg </i>
 +
</p>
 +
<ul>
 +
<li>Both parts, the FusionPart and the N-part are fully compatible with all standard iGEM parts as they have the
 +
BioBricks prefix for coding sequences and the standard BioBrick suffix. </li>
 +
<li>Both parts have two additional enzymes, NgoMIV and AgeI, which have
 +
compatible cohesive ends and enable in-frame fusion of protein parts with
 +
the linker sequence TG (no stop codons). </li>
 +
<li>The only difference of the N-part and FusionPart is the additional
 +
NgoMIV site in the FusionPart. </li>
 +
<li>The FusionPart is the universal part for fusion proteins, and it can be a stand-alone
 +
protein part as it has a start codon
 +
after the XbaI site (BioBrick prefix for coding sequence), with two
 +
additional amino acids (A, G) encoded before the start of the protein.</li>
 +
<li>The N-part is designed to be the start of a fusion protein or a
 +
stand-alone protein part,, in which the N-terminus is sensitive to any amino
 +
acid addition,&nbsp; to be cloned via XbaI/PstI to any iGEM RBS
 +
expression part. </li>
 +
<li>The FusionPart can be fused to the N-part by digesting the
 +
N-part with AgeI/SpeI and the FusionPart with NgoMIV/SpeI. </li>
 +
<li>Any number of FusionParts can be combined and optionally fused to the N-part. </li>
 +
<li>nnnnnn is a place holder for the coding sequence of the respective part.
 +
</li>
 +
</ul>
 +
 +
 +
 +
<p><b>Prefix</b></p>
 +
<div style="border-style: solid; border-width: 1px; padding-left: 4px; padding-right: 4px; padding-top: 1px; padding-bottom: 1px">
 +
BioBrick 3.0 FusionPrefix (<font color="#cc00ff">EcoRI</font>, NotI,<font color="#ff5333">
 +
XbaI</font>,<font color="#bababa"> </font><font color="#009999">NgoMIV</font>,<font color="#bababa"> part in gray</font>;
 +
original BioBrick prefix for coding sequences underlined):<p><b><span style="font-family: monospace; font-size: 125%; padding-left: 1em;">
 +
<u>
 +
<font color="#cc00ff">gaattcc</font>gcggccgct</u><font color="#ff5333"><u>tctag</u>a</font>tg<font color="#009999">gccggc</font><font color="#bababa">CA...</font></span></b>
 +
</p><p>BioBrick 3.0 N-partPrefix is identical to the BioBrick prefix for coding
 +
sequences (<font color="#cc00ff">EcoRI</font>, NotI,<font color="#ff5333">
 +
XbaI</font>,<font color="#bababa"> part in gray)</font>:</p>
 +
<p><b><span style="font-family: monospace; font-size: 125%; padding-left: 1em;">
 +
<font color="#cc00ff">gaattcc</font>gcggccgct<font color="#ff5333">tctag</font><font color="#bababa">ATG...</font></span></b>
 +
</p></div>
 +
<p><b>Suffix</b></p>
 +
<div style="border-style: solid; border-width: 1px; padding-left: 4px; padding-right: 4px; padding-top: 1px; padding-bottom: 1px">
 +
BioBrick 3.0 FusionSuffix (<font color="#bababa">part in gray</font>,
 +
<font color="#0000FF"> AgeI</font>,<font color="#ff5333"> SpeI</font>, NotI,
 +
<font color="#cc00ff">PstI</font>; original BioBrick suffix underlined):<p><b><span style="font-family: monospace; font-size: 125%; padding-left: 1em;">
 +
<font color="#bababa">...AC</font><font color="#0000FF">accggt</font>taa<u>t<font color="#ff5333">actagt</font>agcggccg<font color="#cc00ff">ctgcag</font></u></span></b>
 +
</p> </div>
 +
<p>&nbsp;</p>
 +
<p>Combining the respective prefix and suffix generates the following FusionPart
 +
and N-part:</p>
 +
<p><br>
 +
<b><font size="3">FusionPart</font></b><br>
 +
</p>
 +
<div style="border-style: solid; border-width: 1px; padding-left: 4px; padding-right: 4px; padding-top: 1px; padding-bottom: 1px">
 +
<b><font face="Courier New"><br>
 +
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
NaeI&nbsp;&nbsp;&nbsp;&nbsp; BsrFI&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
SfcI <br>
 +
&nbsp;ApoI&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
BsrFI |&nbsp;&nbsp;&nbsp;&nbsp; BsaWI&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
MspA1I| <br>
 +
<font color="#FF00FF">EcoRI</font>&nbsp;&nbsp; <font color="#009900">NotI</font>&nbsp;&nbsp;&nbsp;
 +
<font color="#FF0000">XbaI</font>&nbsp; <font color="#009999">NgoMIV</font> |&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
<font color="#0000FF">AgeI</font>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
<font color="#FF0000">SpeI</font>&nbsp;&nbsp;&nbsp; <font color="#009900">NotI</font>&nbsp;&nbsp;&nbsp;
 +
||<font color="#FF00FF">PstI</font><br>
 +
&nbsp;&nbsp;&nbsp; |&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; |&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
|&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; | |&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
|&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; |&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
|&nbsp;&nbsp;&nbsp; ||&nbsp;&nbsp; |<br>
 +
&nbsp;&nbsp;&nbsp; <u><font color="#FF00FF">GAATTC</font><font color="#009900">gcggccgc</font>t<font color="#FF0000">TCTAG</font></u><i><font color="#FF0000">A</font>tg</i><font color="#009999">GCCGGC</font>nnnnnn<font color="#0000FF">ACCGGT</font>taa<u>t<font color="#FF0000">ACTAGT</font>a<font color="#009900">gcggccg</font><font color="#FF00FF">CTGCAG</font></u><br>
 +
&nbsp; 1 ---------+---------+---------+---------+---------+---------+----- 65<br>
 +
&nbsp;&nbsp;&nbsp;
 +
CTTAAGcgccggcgaAGATCTacCGGCCGnnnnnnTGGCCAattaTGATCAtcgccggcGACGTC<br>
 +
c&nbsp;&nbsp;&nbsp;&nbsp; I&nbsp; R&nbsp; G&nbsp; R&nbsp; F&nbsp; *&nbsp; M&nbsp;
 +
A&nbsp; G&nbsp; ?&nbsp; ?&nbsp; T&nbsp; G&nbsp; *&nbsp; Y&nbsp; *&nbsp; *&nbsp;
 +
R&nbsp; P&nbsp; L&nbsp; Q&nbsp;&nbsp; -<br>
 +
</font></b></div>
 +
 +
 +
 +
<p><b><font size="3">N-part</font></b><br>
 +
</p>
 +
<div style="border-style: solid; border-width: 1px; padding-left: 4px; padding-right: 4px; padding-top: 1px; padding-bottom: 1px">
 +
<b><font face="Courier New"><br>
 +
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
BsrFI&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
SfcI <br>
 +
&nbsp;ApoI&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
BsaWI&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
MspA1I| <br>
 +
<font color="#FF00FF">EcoRI</font>&nbsp;&nbsp; <font color="#009900">NotI</font>&nbsp;&nbsp;&nbsp;
 +
<font color="#FF0000">XbaI</font>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
<font color="#0000FF">AgeI</font>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
<font color="#FF0000">SpeI</font>&nbsp;&nbsp;&nbsp; <font color="#009900">NotI</font>&nbsp;&nbsp;&nbsp;
 +
||<font color="#FF00FF">PstI</font><br>
 +
&nbsp;&nbsp;&nbsp; |&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; |&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
|&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; |&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
|&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; |&nbsp;&nbsp;&nbsp; ||&nbsp;&nbsp; |<br>
 +
&nbsp;&nbsp;&nbsp; <u><font color="#FF00FF">GAATTC</font><font color="#009900">gcggccgc</font>t<font color="#FF0000">TCTAG</font></u><font color="#FF0000"><i>A</i></font><i>tg</i>nnnnnn<font color="#0000FF">ACCGGT</font>taa<u>t<font color="#FF0000">ACTAGT</font>a<font color="#009900">gcggccg</font><font color="#FF00FF">CTGCAG</font></u><br>
 +
&nbsp; 1 ---------+---------+---------+---------+---------+--------- 59<br>
 +
&nbsp;&nbsp;&nbsp; CTTAAGcgccggcgaAGATCTacnnnnnnTGGCCAattaTGATCAtcgccggcGACGTC<br>
 +
c&nbsp;&nbsp;&nbsp;&nbsp; I&nbsp; R&nbsp; G&nbsp; R&nbsp; F&nbsp; *&nbsp; M&nbsp;
 +
?&nbsp; ?&nbsp; <font color="#0000FF">T&nbsp; G&nbsp; </font>*&nbsp; Y&nbsp; *&nbsp; *&nbsp;
 +
R&nbsp; P&nbsp; L&nbsp; Q&nbsp;&nbsp; -<br>
 +
</font></b></div>
 +
<p><br></p>
  
 
===Source===
 
===Source===

Revision as of 12:03, 27 October 2008

Tm-Fluo-linker-cYFP


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 4
    Illegal AgeI site found at 385
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Plasmid containing BioBrick 3.0 (Prefix and Suffix for protein fusion) restriction sites

Proposal for a BioBrick extension for fusion proteins

iGEM team Freiburg

Introduction

The generalized BioBrick prefix and suffix with its easy cloning strategy is an excellent and universal way to combine various parts, e.g. promoter region, gene of interest, terminator etc. However, also proteins consist of different parts and thus the "mix-and-match" combination of fusion proteins is an important task in Synthetic Biology. However, this modular assembly from BioBrick parts is currently not possible due to the generation of a stop codon at the SpeI/Xba scar.


Suffix of the first part (part in gray, PstI, NotI, SpeI):

...ACtactagtagcggccgctgcag


combined with Prefix of the second part (EcoRI, NotI, XbaI,part in gray):

gaattccgcggccgcttctagagCA...

results after an SpeI/Xba combination in:

...ACtactagagCA...

encoding the amino acids Tyr (codon: tac), STOP (codon: tag) and Ser or Arg (codon agn).

Consequently, fusion proteins can so far only be designed as one part but not in a modular fashion. Therefore, we developed a new generation of BioBricks completely compatible with the first BioBrick version but with two additional compatible restriction sites to allow for the modular construction of protein fusion parts. Appropriate enzymes were chosen carefully to include ensure coding for amino acids, which are compatible with flexible linkers as well as with the N-end rule for protein stability. We call this new version BioBricks 3.0.

Proposal

We propose to extend the standard BioBrick suffix and prefix with two additional compatible restriction sites. The frame of the standard suffix and prefix remains the same resulting in complete compatibility with any previously designed parts. We chose the restriction sites NgoMVI and AgeI as they code for the amino acids Ala-Gly or Thr-Gly, respectively, which are compatible with flexible linkers commonly used in fusion proteins and also compatible with the N-end rule for protein stabiliy. Consequently, we name these parts FusionParts. A combination of two such FusionParts creates an AgeI/NgoMIV scar coding for Thr-Gly, which can easily be integrated in any linker sequence. Furthermore, the NgoMIV site (coding for Ala-Gly) after the start Methionin of the standard BioBrick suffix adheres completely to the N-end rule. For proteins, which are sensitive to amino acid addition at the

N-terminus, we also devised an N-Part with a suffix that lacks the NgoMIV site.


The following list summarizes the most important factors of the BioBrick 3.0 design


Strategy for iGEM BioBrick 3.0 parts for mix-and-match construction of fusion proteins

developed by the iGEM Team Freiburg

  • Both parts, the FusionPart and the N-part are fully compatible with all standard iGEM parts as they have the BioBricks prefix for coding sequences and the standard BioBrick suffix.
  • Both parts have two additional enzymes, NgoMIV and AgeI, which have compatible cohesive ends and enable in-frame fusion of protein parts with the linker sequence TG (no stop codons).
  • The only difference of the N-part and FusionPart is the additional NgoMIV site in the FusionPart.
  • The FusionPart is the universal part for fusion proteins, and it can be a stand-alone protein part as it has a start codon after the XbaI site (BioBrick prefix for coding sequence), with two additional amino acids (A, G) encoded before the start of the protein.
  • The N-part is designed to be the start of a fusion protein or a stand-alone protein part,, in which the N-terminus is sensitive to any amino acid addition,  to be cloned via XbaI/PstI to any iGEM RBS expression part.
  • The FusionPart can be fused to the N-part by digesting the N-part with AgeI/SpeI and the FusionPart with NgoMIV/SpeI.
  • Any number of FusionParts can be combined and optionally fused to the N-part.
  • nnnnnn is a place holder for the coding sequence of the respective part.


Prefix

BioBrick 3.0 FusionPrefix (EcoRI, NotI, XbaI, NgoMIV, part in gray;

original BioBrick prefix for coding sequences underlined):

gaattccgcggccgcttctagatggccggcCA...

BioBrick 3.0 N-partPrefix is identical to the BioBrick prefix for coding

sequences (EcoRI, NotI,

XbaI, part in gray):

gaattccgcggccgcttctagATG...

Suffix

BioBrick 3.0 FusionSuffix (part in gray, AgeI, SpeI, NotI,

PstI; original BioBrick suffix underlined):

...ACaccggttaatactagtagcggccgctgcag

 

Combining the respective prefix and suffix generates the following FusionPart and N-part:


FusionPart


                          NaeI     BsrFI                    SfcI
 ApoI                  BsrFI |     BsaWI                 MspA1I|
EcoRI   NotI    XbaI  NgoMIV |      AgeI      SpeI    NotI    ||PstI
    |      |       |       | |         |         |       |    ||   |
    GAATTCgcggccgctTCTAGAtgGCCGGCnnnnnnACCGGTtaatACTAGTagcggccgCTGCAG
  1 ---------+---------+---------+---------+---------+---------+----- 65
    CTTAAGcgccggcgaAGATCTacCGGCCGnnnnnnTGGCCAattaTGATCAtcgccggcGACGTC
c     I  R  G  R  F  *  M  A  G  ?  ?  T  G  *  Y  *  *  R  P  L  Q   -


N-part


                             BsrFI                    SfcI
 ApoI                        BsaWI                 MspA1I|
EcoRI   NotI    XbaI          AgeI      SpeI    NotI    ||PstI
    |      |       |             |         |       |    ||   |
    GAATTCgcggccgctTCTAGAtgnnnnnnACCGGTtaatACTAGTagcggccgCTGCAG
  1 ---------+---------+---------+---------+---------+--------- 59
    CTTAAGcgccggcgaAGATCTacnnnnnnTGGCCAattaTGATCAtcgccggcGACGTC
c     I  R  G  R  F  *  M  ?  ?  T  G  *  Y  *  *  R  P  L  Q   -


Source

Derived by cloning of the parts BBa_K157002, BBa_K157009 and BBa_K157007. For further information see the description of the parts mentioned above.

References