University of Copenhagen iGEM 2012 Copenhagen in numbers: 2463 GWh - - PowerPoint PPT Presentation

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University of Copenhagen iGEM 2012 Copenhagen in numbers: 2463 GWh - - PowerPoint PPT Presentation

Jul 14, 2023 789 likes •1.16k views

University of Copenhagen iGEM 2012 Copenhagen in numbers: 2463 GWh a year in electricity (2011) 782.000 t CO 2 emitted a year (2011) 550kg CO 2 every year pr. citizen Circadian Clock Day-and-night rhythm influences gene

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University of Copenhagen iGEM 2012

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Copenhagen in numbers:

ž 2463 GWh a year in electricity (2011) ž 782.000 t CO2 emitted a year (2011) ž 550kg CO2 every year pr. citizen

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ž Circadian Clock – Day-and-night rhythm influences gene

expression

ž Performs photosynthesis

6 CO2 + 6 H2O + sun light à C6H12O6 + 6 O2

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ž Night induced promotor lrtA ž Self-sufficient: Need no nutrient in media ž CO2 consuming, sustainable, light source

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Bioluminescence

The LuxCDABE cassette

From Photorhabdus luminescens Kindly provided by T. Knight

Ref (2) Ref (1) C D A B E

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A

(Fre) FMN Reductase Transferase Synthetase Reductase

C D E B

Luciferase

Green light emission: λ 480 nm

C D A B E

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ž Strategy 1:

A light-regulated histidine kinase system

ž Strategy 2:

A cyanobacterial light-sensitive promoter

Expression of luxCDABE under the control of:

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Based on the ”pDUSK”-system which has earlier been shown to work in E. coli (3) YF1, FixJ, and FixK2 biobricks kindly provided by Team Uppsala iGEM 2011

Light

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Strategy 2: Light-sensitive cyanobacterial promoter

lrtA: Cyanobacterial promoter (Synechocystis sp. PCC 6803) Night: Photosynthesis inactive à lrtA turned on

lrtA biobrick kindly provided by team Utah State iGEM 2010

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Construct 1: Histidine kinase-controlled luciferase expression Construct 2: Cyanobacterial promoter-controlled luciferase expression

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Uracil-Specific Excision Reaction

USER Standard assembly Assembly of how many parts? Up to 6 in one go 2 at a time Time? Fast Slow How? Custom-made overhangs Restriction enzymes, ligation Illegal restriction sites? No Yes Scarring? No Yes

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A B Part A Part B Target vector

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ATCGCATT AGGCTTGdU dUTCGTACGA A B ATCGCATdU AAGCATGCT TCCGAACA AGGCTTGT dUCCGAACA TAGCGTAA dUAGCGTAA Part A Part B Target vector AAGCATGCdU TTCGTACGA

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ATCGCATT AGGCTTG A B ATCGCAT TCCGAACA AGGCTTGT CCGAACA TAGCGTAA AGCGTAA Part A Part B TCGTACGA AAGCATGCT AAGCATGC TTCGTACGA

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ATCGCATT A B TCCGAACA AGGCTTGT CCGAACA TAGCGTAA Part A Part B AAGCATGCT A A G C A T G C TTCGTACGA

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ATCGCATT A B TCCGAACA AGGCTTGT TAGCGTAA Part A Part B AAGCATGCT TTCGTACGA

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ATCGCATT A B TCCGAACA AGGCTTGT TAGCGTAA AAGCATGCT TTCGTACGA

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But…..

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ž PCR-Amplification of target genes with appropriate

USER ends

ž Direct primer annealing for short regulatory sequences

ProC lrtA YF1 FixJ FixK2 Terminator pSB1C3

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Biobrick Bba_K770000: Assembly of ProC, YF1 and pSB1C3 by USER cloning.

BBa_K770000

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How much CO2 is consumed? How swiftly does the system respond to darkness and light? How much light is emitted? For how long can the bacteria sustain a high light output? Let’s try to make a mathematical model of the system! Can the emitted light inhibit the system, creating oscillations?

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  • Biobricks submitted by the DTU 2011 iGEM

team

  • USER assembly was used with succesful results
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Two at a time starting with ProC-YF1

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ž DNA pieces have to be equimolar to each other ž DNA pieces that are different in length do not

contain the same number of mol within the same volume.

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Large pieces in general are easier to assemble with USER Speculation on trend ProC = 35 bp LrtA = 38 bp LuxCDABE = 5798 bp YF1 = 1131 bp pSB1C3 = 2070 bp Smaller pieces have a larger area, in which, by chance, to hit the correct USER-site

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Hindered area (HA) Surface Area Surface Area

Some possible coil behavior in large DNA HA non accessible by larger DNA Increased chance of correct impact HA more Accessible by smaller DNA Decreased chance of correct impact A B C

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CO2 consuming, sustainable, light source

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REMOVE CO2 REDUCE ELECTRICITY CONSUMPTION USE

We will light your way

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Sponsors iGEM teams iGEM Chile iGEM Valencia iGEM Uppsala

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ž

(1): en.wikipedia.org/wiki/biolumniscence (2): http://www.pnas.org/content/97/18.cover-expansion

ž

(3) Ohlendorf, R., Vidavski, R., Eldar, A., Moffat, K., and Möglich, A. 2012. From dusk till dawn: one-plasmid systems for light-regulated gene expression. J. Mol.

  • Biol. 416:534-42.
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External members: Caroline Have – Practical applications Charlotte Varslev-Pedersen – Practical applications Thomas Holst-Hansen – Mathematical models Lea Hansen - Ethics Supervisors Johan Ranberg-Andersen, Peter Naur Lab work performed at: Department of Plant Biology and Biotechnology, Section for Plant Biochemistry Faculty of Life Sciences, University of Copenhagen Parts provided Uppsala University YF1 (BBa_K592004), FixJ (BBa_K592005), FixK2 (BBa_K592006) Thomas Knight luxCDABE gene casette Charles Miller, Utah University lrtA promotor (BBa_K390008) Sponsors Novozymes Novo Nordisk