Freiburg07/report fusion parts
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<h2>Proposal</h2> | <h2>Proposal</h2> | ||
| + | |||
| + | <p><b><font size="4">iGEM parts for mix-and-match construction of fusion | ||
| + | proteins </font></b></p> | ||
| + | <p><i>proposed by Katja M. Arndt, Kristian M. Müller, University of Freiburg </i> | ||
| + | </p> | ||
| + | <ul> | ||
| + | <li>Both parts are compatible with all standard iGEM parts as they have the | ||
| + | BioBricks prefix for coding sequences and the standard BioBrick suffix. </li> | ||
| + | <li>nnnnnn is a place holder for the coding sequence of the respective part. | ||
| + | </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 N-part is designed to be the start of a fusion protein or a | ||
| + | stand-alone protein part, to be cloned via XbaI/PstI to any iGEM RBS | ||
| + | expression part. </li> | ||
| + | <li>The FusionPart is designed to 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 fused to the N-part. </li> | ||
| + | <li>The FusionPart can also be a stand-alone part as it has a start codon | ||
| + | after the XbaI site (BioBrick prefix for coding sequence), however two | ||
| + | additional amino acids (A, G) are encoded before the start of the protein.<br> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <p><b style> | ||
| + | <span style="font-size: 10pt; font-family: "Courier New";"> </span></b> | ||
| + | <br> | ||
| + | <b style><span style="font-size: 14pt; font-family: Arial;">N-partt</span></b> | ||
| + | <br> | ||
| + | <b style><span style="font-size: 10pt; font-family: "Courier New";"> </span></b><br> | ||
| + | <b style><span style="font-size: 10pt; font-family: "Courier New";"> | ||
| + | <span style> </span>BsrFI<span style> | ||
| + | </span>SfcI<span style> </span></span></b><br> | ||
| + | <b style><span style="font-size: 10pt; font-family: "Courier New";"> | ||
| + | <span style> </span>ApoI<span style> </span>BsaWI<span style> | ||
| + | </span>MspA1I|<span style> </span></span></b><br> | ||
| + | <b style><span style="font-size: 10pt; font-family: "Courier New";"> | ||
| + | <span style> </span><span style="color: fuchsia;">EcoRI</span><span style> | ||
| + | </span><span style="color: rgb(51, 153, 102);">NotI</span><span style> </span><span style="color: red;">XbaI</span><span style> | ||
| + | </span><span style="color: blue;">AgeI</span><span style> </span> | ||
| + | <span style="color: red;">SpeI</span><span style> </span> | ||
| + | <span style="color: rgb(51, 153, 102);">NotI</span><span style> </span>||<span style="color: fuchsia;">PstI</span></span></b> | ||
| + | <br> | ||
| + | <b style><span style="font-size: 10pt; font-family: "Courier New";"> | ||
| + | <span style> </span>|<span style> </span>|<span style> </span>|<span style> | ||
| + | </span>|<span style> </span>|<span style> </span>|<span style> </span>||<span style> | ||
| + | </span>|</span></b> <br> | ||
| + | <b style><span style="font-size: 10pt; font-family: "Courier New";"> | ||
| + | <span style> </span><u><span style="color: fuchsia;">GAATTC</span><span style="color: rgb(51, 153, 102);">gcggccgc</span>t<span style="color: red;">TCTAG<i style>A</i></span><i style>tg</i></u>nnnnnn<span style="color: blue;">ACCGGT</span>taa<u>t<span style="color: red;">ACTAGT</span>a<span style="color: rgb(51, 153, 102);">gcggccg</span><span style="color: fuchsia;">CTGCAG</span></u></span></b> | ||
| + | <br> | ||
| + | <b style><span style="font-size: 10pt; font-family: "Courier New";"> | ||
| + | <span style> </span>1 | ||
| + | ---------+---------+---------+---------+---------+--------- 59</span></b> <br> | ||
| + | <b style><span style="font-size: 10pt; font-family: "Courier New";"> | ||
| + | <span style> </span> | ||
| + | CTTAAGcgccggcgaAGATCTacnnnnnnTGGCCAattaTGATCAtcgccggcGACGTC</span></b> <br> | ||
| + | <b style><span style="font-size: 10pt; font-family: Courier New"> </span></b> | ||
| + | <br> | ||
| + | <b style><span style="font-size: 10pt; font-family: "Courier New";">c<span style> | ||
| + | </span>I<span style> </span>R<span style> </span>G<span style> </span>R<span style> | ||
| + | </span>F<span style> </span>*<span style> </span>M<span style> </span>?<span style> | ||
| + | </span>?<span style> </span>T<span style> </span>G<span style> </span>*<span style> | ||
| + | </span>Y<span style> </span>*<span style> </span>*<span style> </span>R<span style> | ||
| + | </span>P<span style> </span>L<span style> </span>Q<span style> </span>-</span></b> | ||
| + | <br> | ||
| + | <b style><span style="font-size: 10pt; font-family: "Courier New";"> </span></b> | ||
| + | <br> | ||
| + | <b style><span style="font-size: 10pt; font-family: "Courier New";"> </span></b> | ||
| + | <br> | ||
| + | <b style><span style="font-size: 14pt; font-family: Arial;">FusionPart</span></b> | ||
| + | <br> | ||
| + | <b style><span style="font-size: 10pt; font-family: "Courier New";"> </span></b> | ||
| + | <br> | ||
| + | <b style><span style="font-size: 10pt; font-family: "Courier New";"> | ||
| + | <span style> </span>NaeI<span style> </span> | ||
| + | BsrFI<span style> </span>SfcI<span style> </span></span> | ||
| + | </b><br> | ||
| + | <b style><span style="font-size: 10pt; font-family: "Courier New";"> | ||
| + | <span style> </span>ApoI<span style> </span>BsrFI |<span style> | ||
| + | </span>BsaWI<span style> </span>MspA1I|<span style> </span> | ||
| + | </span></b><br> | ||
| + | <b style><span style="font-size: 10pt; font-family: "Courier New";"> | ||
| + | <span style> </span><span style="color: fuchsia;">EcoRI</span><span style> | ||
| + | </span><span style="color: rgb(51, 153, 102);">NotI</span><span style> </span><span style="color: red;">XbaI</span><span style> | ||
| + | </span><span style="color: rgb(51, 204, 204);">NgoMIV</span> |<span style> </span><span style="color: blue;">AgeI</span><span style> | ||
| + | </span><span style="color: red;">SpeI</span><span style> </span> | ||
| + | <span style="color: rgb(51, 153, 102);">NotI</span><span style> </span>||<span style="color: fuchsia;">PstI</span></span></b> | ||
| + | <br> | ||
| + | <b style><span style="font-size: 10pt; font-family: "Courier New";"> | ||
| + | <span style> </span>|<span style> </span>|<span style> </span>|<span style> | ||
| + | </span>| |<span style> </span>|<span style> </span>|<span style> | ||
| + | </span>|<span style> </span>||<span style> </span>|</span></b> <br> | ||
| + | <b style><span style="font-size: 10pt; font-family: "Courier New";"> | ||
| + | <span style> </span><u><span style="color: fuchsia;">GAATTC</span><span style="color: rgb(51, 153, 102);">gcggccgc</span>t<span style="color: red;">TCTAG<i style>A</i></span><i style>tg</i></u><span style="color: rgb(51, 204, 204);">GCCGGC</span>nnnnnn<span style="color: blue;">ACCGGT</span>taa<u>t<span style="color: red;">ACTAGT</span>a<span style="color: rgb(51, 153, 102);">gcggccg</span><span style="color: fuchsia;">CTGCAG</span></u></span></b> | ||
| + | <br> | ||
| + | <b style><span style="font-size: 10pt; font-family: "Courier New";"> | ||
| + | <span style> </span>1 | ||
| + | ---------+---------+---------+---------+---------+---------+----- 65</span></b> | ||
| + | <br> | ||
| + | <b style><span style="font-size: 10pt; font-family: "Courier New";"> | ||
| + | <span style> </span> | ||
| + | CTTAAGcgccggcgaAGATCTacCGGCCGnnnnnnTGGCCAattaTGATCAtcgccggcGACGTC</span></b> <br> | ||
| + | <b style><span style="font-size: 10pt; font-family: Courier New"> </span></b> | ||
| + | <br> | ||
| + | <b style><span style="font-size: 10pt; font-family: "Courier New";">c<span style> | ||
| + | </span>I<span style> </span>R<span style> </span>G<span style> </span>R<span style> | ||
| + | </span>F<span style> </span>*<span style> </span>M<span style> </span>A<span style> | ||
| + | </span>G<span style> </span>?<span style> </span>?<span style> </span>T<span style> | ||
| + | </span>G<span style> </span>*<span style> </span>Y<span style> </span>*<span style> | ||
| + | </span>*<span style> </span>R<span style> </span>P<span style> </span>L<span style> | ||
| + | </span>Q<span style> </span>-</span></b> </p> | ||
| + | <pre> </pre> | ||
| + | |||
<h2>Discussion</h2> | <h2>Discussion</h2> | ||
<h2>References</h2> | <h2>References</h2> | ||
Revision as of 21:48, 20 October 2007
Contents |
Proposal for a BioBrick extension for fusion proteins
iGEM team Freiburg
Introduction
Proposal
iGEM parts for mix-and-match construction of fusion proteins
proposed by Katja M. Arndt, Kristian M. Müller, University of Freiburg
- Both parts are compatible with all standard iGEM parts as they have the BioBricks prefix for coding sequences and the standard BioBrick suffix.
- nnnnnn is a place holder for the coding sequence of the respective part.
- 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 N-part is designed to be the start of a fusion protein or a stand-alone protein part, to be cloned via XbaI/PstI to any iGEM RBS expression part.
- The FusionPart is designed to 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 fused to the N-part.
- The FusionPart can also be a stand-alone part as it has a start codon
after the XbaI site (BioBrick prefix for coding sequence), however two
additional amino acids (A, G) are encoded before the start of the protein.
N-partt
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 -
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
