Revision as of 18:13, 23 September 2012

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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.

• 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.

• 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.

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.

Sequence and Features Status: 500 Content-type: text/html

Software error:

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