Universal Network Design and Assembly Introduction DNA Assembly - - PowerPoint PPT Presentation

A Comprehensive Approach to Universal Network Design and Assembly

Introduction

DNA Assembly

• This year, we improved upon our BrickMason assembly

method from the 2011 season

• 4-step: PCR, Digestion, Ligation, PCR

Figure 1. 2011 uOttawa BrickMason assembly method. Gene dimers were created and connected via PCR.

DNA Assembly

• Adapted the DNA assembler method from Shao et al.

DNA Assembly

• Saves time by offering a two-step procedure for construction
• f large DNA pieces that is entirely PCR-based
• Avoids the use of restriction enzymes and insertion of

restriction sites between genes

• Avoids the need for ligation
• Not limited to dimers

Co-transformation

• Combined Gietz & Schiestl LiAc/SS carrier DNA/PEG method with

the DNA assembler method by Shao et al.

Co-transformation

• Saves time by decreasing the amount of PCR cycles needed to

stitch together DNA constructs

• Reduces error that arises from multiple rounds of PCR
• Saves money by using the LiAc/SS carrier DNA/PEG

transformation method instead of electroporation

• Allows two-fold screening for the desired final product –

recombination success and proper integration can be screened for all at once via drug/color selection (ADE2/ADE4 cassettes)

Mating

• Took advantage of S.cerevisiae’s haploid and diploid states

Mating

• Saves time by reducing the number of transformations needed to

construct a complex gene network

• Combinatorial nature of the procedure allows for simple

construction of various gene combinations

• Multiple testing platforms are created – the constructs can be

tested in both the haploid and diploid state

Overall Workflow

• One day
• Two-step PCR

amplification of genes of interest

DNA Assembly Co-transformation Mating

• Three days
• Co-transformation
• f homologous

multimers

• Two days
• Combinatorial

mating of haploid strains

Goals of Characterization Project

• Increase the predictability of genetic components
• Test whether systems in haploids can be transferred to diploid

systems

• Effects of reporter tagging

Characterization of the Tet Repressor

BY4742 (α) Gal4::GEV Ade2::pGal-Tet-BFP Ade4::pGalTX-GFP Strain A Tagged Tet-Repressor Haploid

BY4741 (a) Ade2::pGal-Tet-BFP Ade4::GEV BY4742 (α) Ade2::URA3 Ade4::pGalTX-GFP Strain B Tagged Tet-Repressor Diploid

BY4741 (a) Ade2::pGal-Tet-BFP Ade4::GEV BY4742 (α) Ade2::pGal-BFP Ade4::pGalTX-GFP Strain C Un-Tagged Tet-Repressor Diploid

• All three strains have been built
• Strains A and C are being confirmed
• Strain B (Tagged-Tet, Diploid) has been tested

Results

Figure 6. Background BFP and GFP Expression from Wildtype BY4743 Diploid Yeast Cells Figure 7. Basal BFP and GFP expression from the Tagged-Tet Diploid Testing Strain

Figure 8. Confirmation of the the Tet-Responsive

• promoter. Cells were induced with [200nmol] β

estradiol and [100ng/ul] aTc to prevent binding of the Tet repressor to the Tet-Responsive promoter

Characterization of the Tet-Repressor.

Figure 9. Cells were induced with [200nmol] β estradiol to activate the gene network.

Characterization of the Tet Repressor in Diploid Yeast Cells

Figure 10. Quantitative Flow Cytometry analysis.

• Characterization of the Tet repressor in Diploid yeast cells
• Proof of concept of the Assembly Method
• Continue with the testing of the other characterization strains

Remarks

• Lack of inducible systems in yeast
• Copper and Galactose inducible systems
• Gal1 promoter is almost ideal for synthetic networks

Promoter Design

Limitations of the Gal1 Promoter

• Dependent on galactose which is also a source of energy for

yeast

• Galactose is not optimal for yeast growth

Figure 11. Full Gal1 promoter sequence. Elena Frovola, Mark Johnston, and John Majors (1999). Binding of the glucose-dependent Mig1p repressor to the GAL1 and GAL4 promoters. Nucleic Acids Research Vol. 27, No.5

Gal1 Promoter

Figure 12. Gal4 binding sites found in the Gal1 promoter. Elena Frovola, Mark Johnston, and John Majors (1999). Binding of the glucose-dependent Mig1p repressor to the GAL1 and GAL4 promoters. Nucleic Acids Research Vol. 27, No.5

Gal1 Promoter

Gal4 Binding sites Foreign Operator sites a) Gal1 Promoter b) Desired Gal1 Promoter

Figure 13. A visual representation of replacing the Gal4 binding sites with foreign operator sites.

Gal1 Promoter

DBD BFP VP16

Figure 14. A protein fused to the activation domain VP16 would theoretically be able to bind and activate the new Gal1 promoter.

Desired Gal1 Promoter

Activation using VP16

Gal4 Binding sites Tet Operator sites a) Gal1 Promoter b) New Gal1 Promoter

Figure 15. A visual representation of replacing the Gal4 binding sites with Tet operator sites.

Introduce Tet operator sites

Assembly by Overlapping Nucleotides – Adapted from Gibson (2009)

Figure 16. Re-circularization of a linear plasmid with multiple overlapping oligonucleotides with the ends homologous to the plasmid.

• Linearize pRS vector
• Co-transform plasmid and oligonucleotides into yeast
• Extract plasmid DNA from yeast
• Transform plasmid DNA into E. coli
• Extract plasmid DNA
• Sequence or PCR confirm

Assembly by overlapping nucleotides

Figure 17. Sequence of 1 out of 9 E. coli plasmids that were

• sequenced. The red highlighted

nucleotides correspond to the Tet operator sites.

• 9 Plasmids sequenced
• 33% success rate

Sequencing results

• Replacing Gal4 sites with LexA and LacI operator

sequences

• Induction of the new Gal1 promoter with TetR-VP16
• Observe and quantify any change in expression of the

new Gal1 promoter

Future Directions

Human Practices

• Full length documentary produced with the aid of

multiple professors and researchers in the University of Ottawa.

• Highlights the issues and hurdles synthetic biology is

expected to overcome in order to mature as a field, along with regulatory suggestions.

• Watch it!

Conclusion

• Utilizing unique characteristics of yeast, we improved

previous assembly methods

• Diploid strain was tested as a characterization platform
• Modified Gal1 promoter
• Created the Nature of Synthetic Biology documentary

Thank You