Part:BBa_K808032

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.

• 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.

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.

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