Design of a gene expression system for Rhdbhodobacter … · 2015. 8. 10. · 2010 IBE Annual Conference Inui, 2003. Isolation and molecular characterization of pMG160, a cryptic

1DESIGN OF A GENE EXPRESSION SYSTEM FOR RHODOBACTER SPHAEROIDESJunlingJunling HUO HUO

Design of a gene expression f h d bsystem for Rhodobacter

sphaeroidessphaeroides

Junling Huo

Ronald C. Sims

H. Scott Hinton

2010 IBE Annual Conference


Unless otherwise specified, pmost parts in the Registry work in Escherichia coli



OutlineI t d ti• Introduction

• Design a gene expression system for R. sphaeroides– Cloning vectorCloning vector– Promoters – Ribosome Binding Sitesg– Terminator



Rhodobacter Sphaeroides• Gram‐negative: do not retain

crystal violet dye in the gram staining protocol.

• Purple, non‐sulfur: cannot use hydrogen sulfide as electron donor for the reduction of carbon dioxide.

• Facultative: can live with or without oxygen

• Phototrophic: light as the ultimate source of energy

• An‐oxygenic: No oxygen produced • Alpha proteobacteria. http://www.nottingham.ac.uk/biology/contacts/sockett/research.php?S=&ID=128&pubID=NTE2NTIy


p p hp?S=&ID=128&pubID=NTE2NTIy


Rhodobacter sphaeroides • Aerobic respirationp• Anaerobic respiration using

Dimethyl sulfoxide (DMSO), T i th l i N idTrimethylamine N‐oxide (TMAO), NO3

• Anoxygenic photosynthesisyg p y• Fermentation• Spectral complex formation

occurs only under O2‐limiting and anaerobic conditions

30%O2

2%O2

PS DarkDMSO

D S K l U i it f T t H t


Dr. Sam Kaplan University of Texas at Houston


Absorption Spectrum850

800

850

875carotenoid

DMSO

3 W/m10 W/m

100 W/m2

2

2

30% O

400 1000900800700600500

230% O

wavelength (nm)


Dr. Sam Kaplan University of Texas at Houston


Our DreamEnergySource

NaturalP d tProducts

EssentialElements

R. sphaeroides

CarbonSource

CO2




• Design a gene expression system for R. sphaeroides– Cloning vectorCloning vector– Promoter – Ribosome Binding Sitesg– Terminator



pMG170/pMG171 vector

Inui 2003 “Isolation and molecular characterization of pMG160 a mobilizable cryptic plasmid from


Inui, 2003. Isolation and molecular characterization of pMG160, a mobilizable cryptic plasmid from Rhodobcater blasticus”. Applied and environmental microbiology 69(2):725-733.


Standard BioBrick Vector

htt // t it d /i 07/i d h /Bi b i k V t h i


http://parts.mit.edu/igem07/index.php/Biobrick_Vector_choice



http://www.qcbio.com/stratagene/210518.pdf


pMG170/pMG171 vector

Inui 2003 “Isolation and molecular characterization of pMG160 a mobilizable cryptic plasmid from


Inui, 2003. Isolation and molecular characterization of pMG160, a mobilizable cryptic plasmid from Rhodobcater blasticus”. Applied and environmental microbiology 69(2):725-733.


Standard Cloning Vectors

BBa_J73999 BBa_J73998

PstIEcoRI

NotI

SpeI

NotI

XbaI

XbaI

NotI

SpeI

NotI

ECoRIPstI




• Design a gene expression system for R. sphaeroidessphaeroides– BioBrick vectors– BioBrick Promoters– BioBrick Promoters – BioBrick Ribosome Binding Sites– BioBrick Terminator



Structure of a prokaryotic gene


Lodge, J; Lund, P; Minchin, S. 2007. Gene cloning: Principles and applications.


Promoter regions: E. coli VS R. sphaeroides

E liE. coli

R S h idR. Sphaeroides

Up: Sequences of the seven E. coli rrn P1 promoter regions (Newlands, 1993)


Bottom: Sequences of the three R. sphaeroides rrn promoter regions (Dryden and Kaplan, 1993)


Shine‐Dalgarno SequenceSD S TAAGAAGGT• SD‐Sequence: TAAGAAGGT

L f E li Rib bi di it (S h id d St h 1990)


Logo for E. coli Ribosome binding sites (Schneider and Stephens, 1990).


Ribosome Binding Sites (RBS)‐Sequence Logo



R. sphaeroides RBS sequence Logo



Potential RBS for R. sphaeroides



A typical rho‐independent terminator

Figure 14: Structure of Omega terminator (Prentki and Krisch, 1984)



Conjugation lab



Future workM Bi B i k i i i R• Measure BioBrick parts activity in R. sphaeroides – Promoter strength– RBS activity– Terminator efficiency

• Demonstrate production of a protein using designed operon.



SummaryD i d DNA (Bi B i k ) f• Designed DNA sequences (BioBricks) for gene expression system in R. sphaeroides

Vectors: BBa J73998 BBa J73999– Vectors: BBa_J73998,BBa_J73999– Promoters: BBa_K208109,BBa_K208110, BBa_K208111– Ribosome Binding Sites :BBa K208101~K208108boso e d g S tes a_ 08 0 08 08– Terminator: BBa_K208112

• Developed a conjugation protocolp j g p• Begin measurement and analysis of BioBricks



Acknowledgements• Dr. Dong Chen • Dr. Kuanchiang Cheng• Dr. Daryll DeWald• Dr. Jesus Eraso

g

• Gary and Frankie Chiu• Jaclyn Evans

• Dr. Michelle Grilley• Dean Scott Hinton

D S l K l

• Melanie Ivans• Dr. Qin Li

• Dr. Samuel Kaplan• Dr. Charles Miller• Dr Ronald Sims

• Rebeca Olsen

• Dr. Ronald Sims• Dr. Jon Takemoto• Dr. Jixun Zhan



Questions/CommentsQuestions/Comments

Thank You!


Documents

Design of a gene expression system for Rhdbhodobacter … · 2015. 8. 10. · 2010 IBE Annual Conference Inui, 2003. Isolation and molecular characterization of pMG160, a cryptic