Team:Bielefeld-Germany/Results/S-Layer/Guide/3b

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=Expression of S-layer protein under control of T7 promoter in a bioreactor=
=Expression of S-layer protein under control of T7 promoter in a bioreactor=
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[Image:IGEM-Bielefeld2011-Bioreaktor.JPG|400px|thumb|center|Inocculating a [http://www.bioengineering-inc.com/standard-reactors.php?id=2.1 Bioengineering NLF22 7 L] with Bioengineering DCU.]
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[[Image:IGEM-Bielefeld2011-Bioreaktor.JPG|400px|thumb|center|Inocculating a [http://www.bioengineering-inc.com/standard-reactors.php?id=2.1 Bioengineering NLF22 7 L] with Bioengineering DCU.]]
The expression of S-layer proteins is stressful for ''Escherichia coli''. So using ''E. coli'' [http://www.promega.com/products/cloning-and-dna-markers/cloning-tools-and-competent-cells/bacterial-strains-and-competent-cells/single-step-_krx_-competent-cells/ KRX] to express genes under the control of a T7 promoter is an easy way to overexpress your proteins and seperate growth and production phase. This strain carries a T7 polymerase gene under the control of a rhamnose promoter in its genome and it is also optimized for cloning so you do not have to transform your plasmids after assembly in e.g. TOP10, isolate them and bring them in an expression strain like BL21(DE3) - you can go with a single transformation step from assembly to production.
The expression of S-layer proteins is stressful for ''Escherichia coli''. So using ''E. coli'' [http://www.promega.com/products/cloning-and-dna-markers/cloning-tools-and-competent-cells/bacterial-strains-and-competent-cells/single-step-_krx_-competent-cells/ KRX] to express genes under the control of a T7 promoter is an easy way to overexpress your proteins and seperate growth and production phase. This strain carries a T7 polymerase gene under the control of a rhamnose promoter in its genome and it is also optimized for cloning so you do not have to transform your plasmids after assembly in e.g. TOP10, isolate them and bring them in an expression strain like BL21(DE3) - you can go with a single transformation step from assembly to production.
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The advantage of a bioreactor over shaking flasks is the possibility to control cultivation parameters like temperature, pH or dissolved oxygen (DO).
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The advantage of a bioreactor over shaking flasks is the possibility to control cultivation parameters like temperature, pH or dissolved oxygen (DO). When cultivating ''E. coli'' in shaking flasks the cells are often growing oxygen limited because of a oxygen transmission rate (normally k<sub>L</sub>a = 40 - 80 h<sup>-1</sup> in shaking flasks but up to 600 h<sup>-1</sup> in a bioreactor). The production of acetate and lactate leeds to a pH shift in a shaking flask. In a bioreactor pH and DO is steady during the whole cultivation leeding to higher growth rates and yields of biomass.
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For our cultivations we used a [http://www.bioengineering-inc.com/standard-reactors.php?id=2.1 Bioengineering NLF22 7 L] with Bioengineering DCU. We implemented a sequencer which automatically pumped an inducer solution after 4 h cultivation time to start protein expression. Other parameters were:
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* Medium: [[Team:Bielefeld-Germany/Protocols/Materials#HSG_medium | HSG medium]] with 20 mg L<sup>-1</sup> chloramphenicol or 100 mg L<sup>-1</sup> ampicillin
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* Culture volume: 4 L
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* Starting OD<sub>600</sub>: 0.2
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* DO: 40 % airsaturation (controlled with stirrer cascade starting with 200 rpm)
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* pH: 7.0 (controlled with 20 % phosphoric acid and 2 M NaOH)
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* Antifoam: BASF pluronic XXX

Revision as of 07:07, 28 October 2011

Expression of S-layer protein under control of T7 promoter in a bioreactor

Inocculating a [http://www.bioengineering-inc.com/standard-reactors.php?id=2.1 Bioengineering NLF22 7 L] with Bioengineering DCU.


The expression of S-layer proteins is stressful for Escherichia coli. So using E. coli [http://www.promega.com/products/cloning-and-dna-markers/cloning-tools-and-competent-cells/bacterial-strains-and-competent-cells/single-step-_krx_-competent-cells/ KRX] to express genes under the control of a T7 promoter is an easy way to overexpress your proteins and seperate growth and production phase. This strain carries a T7 polymerase gene under the control of a rhamnose promoter in its genome and it is also optimized for cloning so you do not have to transform your plasmids after assembly in e.g. TOP10, isolate them and bring them in an expression strain like BL21(DE3) - you can go with a single transformation step from assembly to production.

The advantage of a bioreactor over shaking flasks is the possibility to control cultivation parameters like temperature, pH or dissolved oxygen (DO). When cultivating E. coli in shaking flasks the cells are often growing oxygen limited because of a oxygen transmission rate (normally kLa = 40 - 80 h-1 in shaking flasks but up to 600 h-1 in a bioreactor). The production of acetate and lactate leeds to a pH shift in a shaking flask. In a bioreactor pH and DO is steady during the whole cultivation leeding to higher growth rates and yields of biomass.

For our cultivations we used a [http://www.bioengineering-inc.com/standard-reactors.php?id=2.1 Bioengineering NLF22 7 L] with Bioengineering DCU. We implemented a sequencer which automatically pumped an inducer solution after 4 h cultivation time to start protein expression. Other parameters were:

  • Medium: HSG medium with 20 mg L-1 chloramphenicol or 100 mg L-1 ampicillin
  • Culture volume: 4 L
  • Starting OD600: 0.2
  • DO: 40 % airsaturation (controlled with stirrer cascade starting with 200 rpm)
  • pH: 7.0 (controlled with 20 % phosphoric acid and 2 M NaOH)
  • Antifoam: BASF pluronic XXX
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