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 16:33, 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. 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 contains a myc epitope, downstream of pNB-Est13. This myc tag is used for detecting whether our surface expression is successful or not. The expression host is E.coli DH5 alpha, transformed with BBa_K808032, and induced at a OD600=0.6 after being incubated for 20 min on ice with arabinose concentrations of 1.5%, 1% and 0.5%. The incubation on ice and the higher arabinose concentrations are crucial for using other expression hosts than Top10, due to their ability to metabolize L-arabinose. The SDS PAGE is performed with supernatant and pellet being treated with a detergent. File:120831 Westermblot 2 auf Gesamt konstrukt aus DH5 alpha 5111.tif

  • 2: 1.5% arabinose supernatant, 3: 1.5% arabinose treated pellet, 4: 1.0% arabinose supernatant, 5: 1.0% arabinose treated pellet, 6: 0.5% arabinose supernatant, 7: 0.5% arabinose treated pellet, 8: myc positive probe


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


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