Download - GFP (Green Fluorescent Protein) Presentation
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
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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
