Wild Type LLP~Project Bravo~

Clinical and Business ReportMonday, June 6, 2009

Lucy He, Danko Krstevski. Jason Kwan, Jing Li, Miki Stanikic

Presentation Overview

• Objectives

• Results and Discussion

• Applications of GFP

• Business & Financial Reports http://www.marcomm.ca/images/CorpOverview.JPG

Preface and Objectives

• Project Bravo

• DNA template contained ~250 bp flanking ends

• 27 kDa protein

• Seven milestones and timeframe

http://www.beacon.nc.gov/training/wbt/images/knowledge_check_all.jpg

2

4

6

5

Time Chart by MilestoneDay 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Day 1

Days 2 to 9

Day 8

Days 8 to 13

Days 12 to 13

Days 9 to 13

Days 12 to 13

1

3

7

Quality Control

Polymerase Chain Reaction

• L1: 1 μL of Fermentas® DNA Ladder

• L2: 45 ng of purified PCR product

• L3: Negative control, No DNA template

L1 L2 L3

500 bp

1031 bp

3000 bp

964 bp

• L1: 1 μL of Fermentas® DNA Ladder

• L2: 502 ng of undigested TOPO product

•L3: 738 ng of digested TOPO product by Eco RI

TOPO CloningL1 L3L2

500 bp

1031 bp

3000 bp5000 bp

957 bp

~4000 bp

Sequencing

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GFP Gene in pET-15b Vector

• L1: 1 μL of Fermentas® DNA Ladder

• L2: 45 ng of undigested vector & insert

• L3: 31 ng of MiniPrep 1: Xba I → Bam HI

• L4: 31 ng of MiniPrep 1: Bam HI → Xba I

• L5: 28 ng of MiniPrep 2: Xba I → Bam HI

• L6: 28 ng of MiniPrep 2: Bam HI → Xba I

500 bp

1031 bp

3000 bp

5000 bp 5700 bp

822 bp

L1 L2 L3 L4 L5 L6

MiniPrep 1 MiniPrep 2

Protein Expression from TOPO

• Colonies with pCR4-TOPO and GFP

• Mach1™ T1 and fluorescence

• Cultures

GFP Purification

• Ni+2-NTA Chromatography

• Nine elution fractions

• Fractions of interest were combined and purified

http://www.mbl.edu/news/features/images/gfp_test_tube.jpg

SimplyBlue Stain Gel

• L1: Invitrogen® BenchMark™ protein ladder (unstained)

• L2: GFP standard

• L3: 375 ng of purified GFP

• L4: 750 ng of purified GFP

• L5: Elution fraction 3

• L6: Elution fraction 2

• L7: Elution fraction 120 kDa25 kDa

30 kDa

50 kDa

L1 L2 L3 L4 L5 L6 L7

27 kDa

• L1: Elution fraction 1

• L2: Elution fraction 2

Protein Immunoblot

25 kDa

30 kDa

40 kDa

27 kDa

L1 L2

Protein Yield

• Three methods used for quantification• UV-Vis at 280 nm: 651 ± 88 μg/mL• BCA Assay: 739 ± 38 μg/mL • Fluorimetry: 806 ± 67 μg/mL• Average [GFP]: 731 ± 44 μg/mL

Protein Yield

• Three methods used for quantification• UV-Vis at 280 nm: 651 ± 88 μg/mL• BCA Assay: 739 ± 38 μg/mL • Fluorimetry: 806 ± 67 μg/mL• Average [GFP]: 731 ± 44 μg/mL

Protein Yield

• Three methods used for quantification• UV-Vis at 280 nm: 651 ± 88 μg/mL• BCA Assay: 739 ± 38 μg/mL • Fluorimetry: 806 ± 67 μg/mL• Average [GFP]: 731 ± 44 μg/mL

Protein Yield

• Three methods used for quantification• UV-Vis at 280 nm: 651 ± 88 μg/mL• BCA Assay: 739 ± 38 μg/mL • Fluorimetry: 806 ± 67 μg/mL• Average [GFP]: 731 ± 44 μg/mL

Protein Yield

• Three methods used for quantification• UV-Vis at 280 nm: 651 ± 88 μg/mL• BCA Assay: 739 ± 38 μg/mL • Fluorimetry: 806 ± 67 μg/mL• Average [GFP]: 731 ± 44 μg/mL

Ratio: 14710 ± 213 M-1cm-1

15330 ± 427 M-1cm-1 = 95 ± 0.91%

Significance of GFP

• Practical uses in industry• Biological marker• Fluorescence imaging• Therapeutic drug monitoring

• Functionality• Many derivatives• Easily expressed

http://brainwindows.files.wordpress.com/2008/10/image-gfp-mouse-crop-copy.jpg

Wild Type LLPIncome Statement

For the Period June 8, 2009 to June 30, 2009

REVENUES

Pre-Project Income $ 35.00

Project Milestones 150.00

Sale/lease of Materials to Industry 10.00

Sale of Inventory to D5Pharma 1960.00

Earnings for saved labour hours 1200.00

Total Revenue $ 3355.00

EXPENSES

Supplies from D5Pharma 15.00

Total Expenses $ 15.00

NET INCOME $ 3340.00

Wild Type LLPBalance Sheet as of June 30, 2009

ASSETS Current Assets Cash $ 180.00 Account Receivables 1200.00 Inventory 1960.00Total Current Assets 3340.00 Property, Plant and Equipment 0.00 Other Assets 0.00TOTAL ASSETS $ 3340.00

LIABILITIES and SHAREHOLDERS' EQUITY Current Liabilities Accounts Payable 0.00Total Current Liabilities 0.00 Long-Term Debt 0.00Total Long-Term Liabilities 0.00TOTAL LIABILITIES $ 0.00

Shareholders' Equity Common Stock 3340.00TOTAL SHAREHOLDERS' EQUITY $ 3340.00TOTAL LIABILITIES and SHAREHOLDERS' EQUITY $ 3340.00

Conclusion

• Mission Statement• Efficiency• Quality• Accuracy

• The bottom line

Acknowledgements

Thank you to D5Pharma specifically, Mr. Leigh, Veronica, Anja, Waldi and Windsor for their

support and expertise throughout our project

References

[1] Lissemore JL, Bayes J, Calvey M, Reineke L, Colagiavanni A, Tscheiner M, Mascotti. Green fluorescent protein is superior to blue fluorescent protein as a quantitative reporter of promoter activity in E. coli. Mol Bio Rep 2009, 36: 1107-1112

[2] Tsien R. The Green Fluorescent Protein. Annual Review of Biochemistry 1998, 67: 509–44.

[3] Yang F, Moss L, Phillips G. The molecular structure of green fluorescent protein. Nature and Biotechnology 1996, 14(10): 1246–51.

[4] Chalfie M, Kain S: Green fluorescent protein: properties, applications, and protocols. John Wiley and Sons 2005.

[5] Yuste R. Fluorescence microscopy today. National Methods 2005, 2(12): 902–4.

[6] Park SH, Raines RT: Green fluorescent protein as a signal for protein-protein interactions. Protein Science 1997 6(11):2344-9

Supplementary Slides

Western (Purified Samples)

25 kDa

30 kDa

40 kDa

27 kDa

2

2

5

6

1 3 7

4

Milestone Map

Team B

Team A