Composite

Part:BBa_K808032

Designed by: Marie Burghard, Jascha Diemer, Philipp Rottmann, Rene Sahm, Arne Wehling   Group: iGEM12_TU_Darmstadt   (2012-09-04)
Revision as of 15:52, 23 September 2012 by Arne (Talk | contribs)

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\SDS Page of BBa K808030.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 performed a [http://2012.igem.org/Team:TU_Darmstadt/Protocols/Western_Blot Western blot]. Our construct


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]
    Unknown
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961


[edit]
Categories
//cds/enzyme
Parameters
control
protein