Transcription attenuation for metabolic control by engineering intrinsic terminators

2008 Virginia Genetically Engineered Machine TeamBrandon Freshcorn 1, Patrick Gildea 2, Eyad Lababidi 3, Dan Tarjan4, and George Washington2

Departments of 1Biomedical Engineering, 2Chemical Engineering, 3Electrical Engineering, and 4Biology, University of Virginia, Charlottesville, Virginia, USA

A main challenge in constructing synthetic biological systems is the inability to precisely regulate gene expression using artificial means. Tightly-regulated control of any given set of related transcriptional, translational

and posttranslational events will likely require a combination of powerful strategies. Therefore, the 2008 Virginia iGEM team is developing a library of transcriptional terminators intentionally redesigned to be functionally

inefficient. Well-characterized, standardized terminators of various efficiencies should allow finely-tuned transcription attenuation and represents yet another step toward global biological control. This work complements

other gene expression control methods that focus on initiation of transcription. The desired result is quantitative control of transcript levels, which is often necessary to balance flux through a synthetic metabolic

pathway. To demonstrate its potential for real-world application, the team is planning to employ this approach to control the expression of a heterologous pathway in E. coli for the biosynthesis of polyhydroxybutyrate

(PHB), a biodegradable polyester plastic.

Genetic AttenuatorsBioBrick Placeholders

Each BioBrick Placeholder contains a pair of restriction sites

within a BioBrick. These sites in turn are compatible with

the BioBrick standard.

EX - ApoI (NotI) AvrII - SP

EX - ApoI (NotI) NheI - SP

EX - ApoI (NotI) NsiI - SP

EX - ApoI (NotI) SbfI - SP

EX - MfeI (NotI) AvrII - SP

EX - MfeI (NotI) NheI - SP

EX - MfeI (NotI) Nsil - SP

EX - MfeI (NotI) SbfI - SP

PHB Pathway

A naturally synthesized bioplastic

Sustainable

Biodegradable

Medically useful

Sutures

have been

made from

PHB

Save a place for your insert

Citations1. Kremers, G.-J. et al., Biochemistry, 46, 12, 3775 -

3783, 2007

2. Manual of Clinical Enzyme Measurements.

Worthington Biochemical Corporation, 1972.

3. Pohlmann et al. Nature Biotechnology - 24, 1257 -

1262 (2006)

4. Shishatskaya et al., Tissue response to the

implantation of biodegradable polyhydroxyalkanoate

sutures. Journal of Materials Science: Materials in

Medicine, June 2004 , pp. 719-728(10)

Genetic Attenuators exploit intrinsic terminators to control

the relative amounts of gene transcript produced in a

metabolic system.

CoA CoA

CoA

OH

PhaA

PhaB1

PhaC1

To test each gene in the pathway we had to construct an expression vector. What if we had a

modular vector to streamline repetitive assembly?

Substrate concentration affects reaction velocity

Reaction velocity affects downstream substrate concentration

By controlling the relative enzyme

ratios it is possible to control the

relative velocities that make up a

metabolic system. This enables

optimization of product synthesis.

OFP SBFP2

Streptomycin

resistance

Adding another

mechanism for

selection

means another

tool can be

applied in your

experiments

Reporters & Selection

Traditional pathway engineering measures enzyme

and intermediate concentrations with techniques such

as SDS-PAGE, HPLC and GC/MS respectively.

These techniques are non-trivial and time intensive.

Background noise obscures data as in our SDS-

PAGE result. Fluorescence allows for faster, more

direct observation of the systems in question.

(left to right)

6kDa

16kDa

30kDa

36kDa

50kDa

64kDa

98kDa

1.Marker

2.Control

3.PhaA - 40.5 kDa

4.PhaB1 - 26.3 kDa

5.PhaC1 - 64.3 kDa

Enzyme Assay Pathway Optimization

A NEW TECHNICAL STANDARD!

More reporters in the Registry allows the simultaneous imaging of

many factors of a complex system

Pro

ble

ms

Solu

tions

Acknowledgements

Simplifying how we measure protein presence allows for more rapid research. Is there

a faster, more accurate way to look for our enzymes?

Metabolic systems are complex and involve many interdependent factors. How these factors correlate

affects the efficiency of the pathway. How can we optimize pathway output via enzyme level control?

Time (days)

[PH

B] norm

aliz

ed

Modeling the effects of non-

optimal substrate levels lets us

estimate how varying enzyme

levels will affect product

formation. Small changes in

these variables can alter the

pathway’s efficiency

negatively. This may make the

difference between the system

being industrially viable or not.

Position on DNA strand

Am

ount of T

ranscript

Genetic

Attenuator Gene 2Gene 11. Insert BioBrick Placeholder like a normal part2. Cut your vector with the internal restriction sites and

your BioBrick insert with the compatible sites

3. Ligate and enjoy!

1

2

3!

EcoRI - ApoI & MfeI

X & S - AvrII, NheI

PstI - NsiI, SbfI

Sponsors

Erik Fernandez - Chemical Engineering

Jason Papin - Biomedical Engineering

Michael Timko - Biology

Keith Kozminski – Biology

Kay Christopher

Brianne Ray

George McArthur

Office of the Vice President for Research

School of Engineering and Applied Sciences

Department of Chemical Engineering

Department of Biomedical Engineering

Department of Biology

Terminators cause polymerases on a

DNA strand to disengage. By

engineering terminators to specific

efficiencies, it is possible to cause a

percentage of polymerases to arrest

transcription. We have exploited this

phenomenon to create Genetic

Attenuators.

[3]

[1]

[4]

[2]