Difference between revisions of "Part:BBa K808032"
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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). | 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). | ||
| − | [[Image:Flow cytometry 002 ara.png| | + | [[Image:Flow cytometry 002 ara.png|150px]] [[Image:Flow cytometry 02 ara.png|150px]] |
| − | [[Image:Flow cytometry 05 ara.png| | + | [[Image:Flow cytometry 05 ara.png|150px]] [[Image:Flow cytometry alle.png|150px]] |
<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
Revision as of 17:10, 23 September 2012
Status: 500 Content-type: text/html
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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 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.
- 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.
- 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).
Sequence and Features Status: 500 Content-type: text/html
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