Difference between revisions of "Part:BBa K808032"
Line 5: Line 5:
 
It contains the following units:
 
It contains the following units:
 
AraC-AraO2-AraO1-AraPromo(PBAD-RBS-PhoA-His6tag-pNBEst13-Myctag-EstA
 
AraC-AraO2-AraO1-AraPromo(PBAD-RBS-PhoA-His6tag-pNBEst13-Myctag-EstA
−
which is similiar to BBa_K808000 & BBa_K808030
+
which is similiar to [https://parts.igem.org/wiki/index.php?title=Part:BBa_K808000 BBa_K808000] & [https://parts.igem.org/wiki/index.php?title=Part:BBa_K808030 BBa_K808030]
 
It is an operon, being induced by adding 0.02% to 0.5% of L-Arabinose. It is made for regulating the cell surface expression of our PET cleaving enzyme pNB-Est13, which is anchored C-terminal to EstA.
 
It is an operon, being induced by adding 0.02% to 0.5% of L-Arabinose. It is made for regulating the cell surface expression of our PET cleaving enzyme pNB-Est13, which is anchored C-terminal to EstA.
  
  
−
<!-- Add more about the biology of this part here
+
 
 
===Usage and Biology===
 
===Usage and Biology===
 +
This part regulates the expression of our chimeric protein consisting of the following parts:
 +
* PhoA signal sequence ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K808028 BBa_K808028]), which leads to periplasmatic expression.
 +
* pNB-Est13 ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K808026 BBa_K808026]) is an enzyme that is able to hydrolyse PET
 +
* EstA ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K808027 BBa_K808027]) is used as a C-terminal membrane anchor, being able to express even large enzymes on the bacterial surface
 +
 +
In order to get a more information about our composite part [https://parts.igem.org/wiki/index.php?title=Part:BBa_K808030 BBa_K808030] please take a look at its own registry page.
 +
 +
 +
This site is about its expression rate and its activity in three different ''E.coli'' strains:
 +
* [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10]
 +
* [http://ecoliwiki.net/colipedia/index.php/DH5_alpha DH5alpha]
 +
* [http://ecoliwiki.net/colipedia/index.php/MG1655 Mg1655]
 +
 +
====Functionality====
 +
 +
The functionality of BBa_K808032 is shown by the following methods:
 +
* SDS PAGE
 +
* Western blot
 +
* flow cytometry (after antibody staining)
 +
* screening for hydrolysis by bacterial colonies using Tributyrin agar plates
 +
* pNP-Assay with living cells using para-Nitrophenylbutyrate
 +
 +
=====SDS PAGE=====
 +
 +
Of course the first step for showing any kind of functionality is to perform a [http://2012.igem.org/Team:TU_Darmstadt/Protocols/SDS-Page SDS PAGE]. ''E.coli'' Top10 is transformed with BBa-K808032 and induced at an OD<sub>600</sub>=0.5 with the arabinose concentrations of 0.02%, 0.2%, 0.5% and no arabinose at all (serving as a negative control). Incubation occured at 30°C over night. Afterwards the expressed chimeric protein gets [http://2012.igem.org/Team:TU_Darmstadt/Protocols/Proteinpurification purified]. The resulting supernatant is loaded but the cell debris pellet is treated with an special detergent (n-Dodecyl β-D-maltoside) ,in order to solve our membrane bound protein, and is loaded as well.
 +
[[Image:C:\Users\Arne Wehling\Dropbox\igem\Labor\Team I - Cutinase\Fotos\120918 Coomassie for Part page.png]]
 +
* 2: 0.5% arabinose supernatant, 3: 0.5% arabinose treated pellet, 4: 0.2% arabinose supernatant, 5: 0.2% arabinose treated pellet, 7: 0.02% arabinose supernatant, 8: 0.02% arabinose treated pellet, 9: no arabinose supernatant, 10: no arabinose treated pellet
 +
 +
=====Western blot=====
 +
In order to be sure of our protein being expressed we
 +
  
−
<!-- -->
 
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K808032 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K808032 SequenceAndFeatures</partinfo>

Revision as of 15:37, 23 September 2012

Arabinose regulated RBS-PhoA-His6tag-pNBEst13-Myctag-EstA

This is a composite of 2 different BioBricks formed by a standard assembly, forming a scar in between. It contains the following units: AraC-AraO2-AraO1-AraPromo(PBAD-RBS-PhoA-His6tag-pNBEst13-Myctag-EstA which is similiar to BBa_K808000 & BBa_K808030 It is an operon, being induced by adding 0.02% to 0.5% of L-Arabinose. It is made for regulating the cell surface expression of our PET cleaving enzyme pNB-Est13, which is anchored C-terminal to EstA.


Usage and Biology

This part regulates the expression of our chimeric protein consisting of the following parts:

  • PhoA signal sequence (BBa_K808028), which leads to periplasmatic expression.
  • pNB-Est13 (BBa_K808026) is an enzyme that is able to hydrolyse PET
  • EstA (BBa_K808027) is used as a C-terminal membrane anchor, being able to express even large enzymes on the bacterial surface

In order to get a more information about our composite part BBa_K808030 please take a look at its own registry page.


This site is about its expression rate and its activity in three different E.coli strains:

  • [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10]
  • [http://ecoliwiki.net/colipedia/index.php/DH5_alpha DH5alpha]
  • [http://ecoliwiki.net/colipedia/index.php/MG1655 Mg1655]

Functionality

The functionality of BBa_K808032 is shown by the following methods:

  • SDS PAGE
  • Western blot
  • flow cytometry (after antibody staining)
  • screening for hydrolysis by bacterial colonies using Tributyrin agar plates
  • pNP-Assay with living cells using para-Nitrophenylbutyrate
SDS PAGE

Of course the first step for showing any kind of functionality is to perform a [http://2012.igem.org/Team:TU_Darmstadt/Protocols/SDS-Page SDS PAGE]. E.coli Top10 is transformed with BBa-K808032 and induced at an OD600=0.5 with the arabinose concentrations of 0.02%, 0.2%, 0.5% and no arabinose at all (serving as a negative control). Incubation occured at 30°C over night. Afterwards the expressed chimeric protein gets [http://2012.igem.org/Team:TU_Darmstadt/Protocols/Proteinpurification purified]. The resulting supernatant is loaded but the cell debris pellet is treated with an special detergent (n-Dodecyl β-D-maltoside) ,in order to solve our membrane bound protein, and is loaded as well. File:C:\Users\Arne Wehling\Dropbox\igem\Labor\Team I - Cutinase\Fotos\120918 Coomassie for Part page.png

  • 2: 0.5% arabinose supernatant, 3: 0.5% arabinose treated pellet, 4: 0.2% arabinose supernatant, 5: 0.2% arabinose treated pellet, 7: 0.02% arabinose supernatant, 8: 0.02% arabinose treated pellet, 9: no arabinose supernatant, 10: no arabinose treated pellet
Western blot
In order to be sure of our protein being expressed we


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2392
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2918
    Illegal NgoMIV site found at 3056
    Illegal NgoMIV site found at 3596
    Illegal NgoMIV site found at 3965
    Illegal AgeI site found at 979
    Illegal AgeI site found at 2463
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961