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 12:39, 24 September 2012 by Arne (Talk | contribs) (→‎SDS PAGE)

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 site is about the expression rate of BBa_K808032 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]

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


Our part BBa_K808032 regulates the expression of a chimeric protein (BBa_K808030) 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

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.

Figure 1. 2: supernatant 0.5% arabinose, 3: treated pellet 0.5% arabinose, 4: supernatant 0.2% arabinose, 5: treated pellet 0.2% arabinose, 7: supernatant 0.02% arabinose, 8: treated pellet 0.02% arabinose, 9: supernatant no arabinose, 10: treated pellet no arabinose
  • 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

To be sure that our protein is 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 was performed with supernatant and pellet being treated with a detergent.

Westernblot 2 auf Gesamt konstrukt aus DH5 alpha 5111.png

  • 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
Flow Cytometry

Flow cytometry is good for quantifiying the rate of cell surface expression. Our construct contains a myc tag, allowing us to perform an [http://2012.igem.org/Team:TU_Darmstadt/Protocols/Antibody_staining antibody staining]. The second antibody is biotinylated and can be conjugated to streptavidin, being labeld with a fluorescent marker. We used Streptavidin, R-phycoerythrin conjugate (SAPE). The relative absorbance is measured and detected by our flow cytometer (Accuri C6 flow cytometer). We transformed E.coli Top10 with BBa_K808032 and induced at an OD600=0.5 with an arabinose concentration of 0.02%, 0.2%, 0.5% and no arabinose at all, serving as a negative control. The antibody staining was performed after 3 h of incubation at 30°C.

Flow cytometry 002 ara.png Flow cytometry 02 ara.png Flow cytometry 05 ara.png Flow cytometry alle.png

Tributyrin agar plates

[http://2012.igem.org/Team:TU_Darmstadt/Materials/LB_Tributyrin#About Tributyrin agar plates] are a common way to detect bacteria secreting hydrolases. Hydrolyis results in significant lysis areas around the respective colony.

We transfromed the E.coli strains [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10], [http://ecoliwiki.net/colipedia/index.php/DH5_alpha DH5alpha] and [http://ecoliwiki.net/colipedia/index.php/MG1655 Mg1655] with BBa_K808032 and [http://2012.igem.org/Team:TU_Darmstadt/Protocols/Activity_assay inoculated] Tributyrin agar plates. The concentration of arabinose used for induction was around 0.5%. Incubation occured at 30°C over night.

120920 k808032 k808030 dh5a top10 mg1655.png

Of course a longer incubation results in bigger lysis areas, the plate beneath was incubated one night at 30°C and 2 days at room temperature. It contains DH5 alpha, carrying BBa_K808032. Colony 6 only carries BBa_K808030. An arabinose concentration of around 0.2% was used for induction.

120913 dh5alpha k808032 0.2 ara.png

pNP-Assay

Using Tributyrin agar plates gave us a good qualitative proof of hydrolytic acivity, but an assay was needed to quantify the enzymatic activity of our induced cells. For this reason we performed a pNP-assay using our cells (Top10) with an OD600=0.1-0.025 suspenden in PBS-buffer as catalysts and para-Nitrophenylbutyrate as a substrate. We were able to quantify the relation of substrate degradation and induction. Please take a look at the graphs.

Top ten induz (geschwindigkeiten).pngTop10 induz.png



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