Expression control UNIVERSITY OF SOUTHERN DENMARK Bacteriorganic Rubber Team: SDU-Denmark. Please contact us at: [email protected] Patrick R. Andreassen, Mattias P. D. Horan, A. Sofie F. Jørgensen, Heidi W. Jørgensen, Andreas Kjær, Maria H. Knudsen, Thøger J. Krogh, Nicky C. Mattsson, Sissel I. Schmidt Future labwork • Further tests using head-space GC are needed to confirm that overexpression of Dxs produces more IPP/DMAPP. • Tests need to be done to determine the amount of rubber produced (rubber purification yield) and the polymer length (MALDI-ToF or TLC). • The H 1 -NMR needs to be repeated to validate our rubber findings. Using our GFP based reporter system for our dxs construct, we validated our design using FACS. It was proven that we could control expression of dxs and that natural lacI was superior to the lacI:LVA BioBrick (which in turn was better than the plasmid without a Plac repressor). Design As dxs from B. subtilis has proven more effec- tive than intrinsic E. coli dxs, we used dxs from this species and put dxs under the control of the IPTG indu- cible lac promotor (Plac). Figure 5: Basic design of our dxs plasmid – Plac is repressed by the lacI enzyme Figure 6: Basic design of our HRT2 plasmid – Para is repressed by the araC enzyme Northern blot analysis proved that our construct was controllable using arabinose since mRNA concentration rises 16-fold after induction. Any effect of AraC was not visible after 15 minutes of induction. References: Y. Zhao et al. Biosynthesis of isoprene in Escherichia Coli via methylerythritol phosphate (MEP) pathway The polyprenyltransferease HRT2 from Hevea Brasiliensis is the en- zyme which polymerizes IPP and DMAPP to rubber. We placed HRT2 under the control of the arabinose inducable arabi- nose promotor (Para). Introduction Rising global demand for quality rubber calls for innovative ways of satisfying needs without compromising the environment. Natural rubber is produced by draining the rubber tree, and the establishment of new rubber plantations causes deforestation of rainforest and occupation of arable land. We introduced rubber-producing abilities from the rubber tree to Escherichia coli. Moreover, we tested whether the manipulated bacteria were capable of producing natural rubber. Human Outreach • We helped the Edinburgh team with modeling. • We made a Brick of Knowledge video to help future iGEM teams as part of the UNIK team’s project. • We hosted a Jamboree preparing meet-up for the Danish iGEM teams. • We spread the word about iGEM by lecturing students in grade and high schools. Figure 9: From lecture at grade school Modelling the MEP pathway Figure 4: The Methylerythritol Phosphate Pathway Figure 2: Model showing Dxs being the first rate- limiting step. Figure 3: The outflux to Vitamin B1. The precursors of rubber biosynthesis (IPP and DMAPP) come from the intrinsic MEP pathway. We modelled pathway and the results were two-fold: •Dxs, IspG, and IspF are the rate-limiting steps of the MEP pathway, in that order •The exit term to vitamin B1 is negligible until increased velocity of 100-200% We chose to introduce dxs into E. coli in order to increase output of IPP and DMAPP. Discover our interactive wiki tour! Rubber production Figure 10: H 1 -NMR of our strain (A), WT+polyisoprene (B), WT E. coli (C) and standard polyisoprene solution (D). Conclusion Preliminary results suggest that our bacteria produce rubber. This needs validation by further H 1 -NMR experiments. The ability to control our constructs allows us to grow bacteria to a desirable concentration before induction, ensuring optimum rubber yield. For the H 1 -NMR analysis, rubber was purified from the bacteria by washing with acetone and extracted using hexane. Peaks with a recognizable splitting pattern at 5.12ppm in an H 1 -NMR spectrum provide proof of the presence of rubber. Attributions Laboratory support: Jakob M. Jensen & The Microbiology group, The Department of Biochemistry and Molecular Biology, Simon Rose, Anders Boysen, Mikkel Jørgensen, Eva Lillebæk, Paul Stein , Lennart B. Nielsen, Pernille Lassen, Lise J. Nielsen , Tine D. Schrøder, Lars P. Christensen, Hanne V. Hemmingsen, Dorthe Lillesø, Brian Hermansen. Technical support: Kristian Debrabant, Kenneth H. Jensen, Claus A. Lykkebo. Special thanks to: Ann Zahle, Kirstine Jacobsen, and Tina Kronborg For more information visit the attributions page of our wiki. These peaks, while lacking in the wild type, are present in the standard solution of polyisoprene, in the positive control, and in our strain! Figure 7: FACS showing more dxs-GFP expression in natural LacI construct than in LacI:LVA concstruct. Figure 8: A) Intensity of HRT2 mRNA using intensity of 5S rRNA as reference. B) Diagram reflecting Northern blot results. Figure 1: Traditional harvesting of rubber D
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Expression control
UNIVERSITY OF SOUTHERN DENMARK
Bacteriorganic Rubber Team: SDU-Denmark. Please contact us at: [email protected]
Patrick R. Andreassen, Mattias P. D. Horan, A. Sofie F. Jørgensen, Heidi W. Jørgensen, Andreas Kjær, Maria H. Knudsen, Thøger J. Krogh, Nicky C. Mattsson, Sissel I. Schmidt
Future labwork • Further tests using head-space GC are needed to confirm that overexpression of Dxs produces more IPP/DMAPP.
• Tests need to be done to determine the amount of rubber produced (rubber purification yield) and the polymer length (MALDI-ToF or TLC).
• The H1-NMR needs to be repeated to validate our rubber findings.
Using our GFP based reporter system for our dxs construct, we validated our design using FACS. It was proven that we could control expression of dxs and that natural lacI was superior to the lacI:LVA BioBrick (which in turn was better than the plasmid without a Plac repressor).
Design As dxs from B. subtilis
has proven more effec- tive than intrinsic E. coli
dxs, we used dxs from this species and put dxs under
the control of the IPTG indu- cible lac promotor (Plac).
Figure 5: Basic design of our dxs plasmid – Plac is repressed by the lacI enzyme
Figure 6: Basic design of our HRT2 plasmid – Para is repressed by the araC enzyme
Northern blot analysis proved that our construct was controllable using arabinose since mRNA concentration rises 16-fold after induction. Any effect of AraC was not visible after 15 minutes of induction.
References: Y. Zhao et al. Biosynthesis of isoprene in Escherichia Coli via methylerythritol phosphate (MEP) pathway
The polyprenyltransferease HRT2 from Hevea Brasiliensis is the en-
zyme which polymerizes IPP and DMAPP to rubber. We placed
HRT2 under the control of the arabinose inducable arabi-
nose promotor (Para).
Introduction Rising global demand for quality rubber calls for
innovative ways of satisfying needs without compromising the environment. Natural rubber is produced by draining the rubber tree, and the establishment of new rubber plantations
causes deforestation of rainforest and occupation of arable land.
We introduced rubber-producing abilities from the rubber tree to Escherichia coli. Moreover, we tested whether the manipulated bacteria were capable of producing natural rubber.
Human Outreach • We helped the Edinburgh team
with modeling.
• We made a Brick of Knowledge video to help future iGEM teams as part of the UNIK team’s project.
• We hosted a Jamboree preparing meet-up for the Danish iGEM teams.
• We spread the word about iGEM by lecturing students in grade and high schools.
Figure 9: From lecture at grade school
Modelling the MEP pathway
Figure 4: The Methylerythritol Phosphate Pathway
Figure 2: Model showing Dxs being the first rate-limiting step. Figure 3: The outflux to Vitamin B1.
The precursors of rubber biosynthesis (IPP and DMAPP) come from the intrinsic MEP pathway. We modelled pathway and the results were two-fold:
•Dxs, IspG, and IspF are the rate-limiting steps of the MEP pathway, in that order •The exit term to vitamin B1 is negligible until increased velocity of 100-200% We chose to introduce dxs into E. coli in order to increase output of IPP and DMAPP.
Discover our interactive wiki tour!
Rubber production
Figure 10: H1-NMR of our strain (A), WT+polyisoprene (B), WT E. coli (C)
and standard polyisoprene solution (D).
Conclusion Preliminary results suggest that our bacteria produce rubber. This needs validation by further H1-NMR experiments.
The ability to control our constructs allows us to grow bacteria to a desirable concentration before induction, ensuring optimum rubber yield.
For the H1-NMR analysis, rubber was purified from the bacteria by washing
with acetone and extracted using hexane.
Peaks with a recognizable splitting pattern at 5.12ppm in an H1-NMR spectrum provide
proof of the presence of rubber. Attributions Laboratory support: Jakob M. Jensen & The Microbiology group, The Department of Biochemistry and Molecular Biology, Simon Rose, Anders Boysen, Mikkel Jørgensen, Eva Lillebæk, Paul Stein , Lennart B. Nielsen, Pernille Lassen, Lise J. Nielsen , Tine D. Schrøder, Lars P. Christensen, Hanne V. Hemmingsen, Dorthe Lillesø, Brian Hermansen. Technical support: Kristian Debrabant, Kenneth H. Jensen, Claus A. Lykkebo.
Special thanks to: Ann Zahle, Kirstine Jacobsen, and Tina Kronborg
For more information visit the attributions page of our wiki.
These peaks, while lacking in the wild type, are present in the
standard solution of polyisoprene, in the positive
control, and in our strain!
Figure 7: FACS showing more dxs-GFP expression in natural LacI construct than in LacI:LVA concstruct.
Figure 8: A) Intensity of HRT2 mRNA using intensity of 5S rRNA as reference. B) Diagram reflecting Northern blot results.
Figure 1: Traditional harvesting of rubber
D
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