Barrett rosa meza-acevedo & lingou


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  • Add a red fluorescent tag to the cytoskeleton of a lymphocyte to …Our research goal is to bioengineer a new red fluorescent tag. We want to do it because the red fluorescent tag can be a very useful tool to help us visualize living cells under advanced microscope. Here you can see the picture. This is a cell with red labeled cytoskeleton. Cytoskeleton is the internal network of the cell. On the top, it’s HIV labeled by green fluorescence, and a healthy Jurkat cell labeled by red fluorescence. Jurkat cell is a lymphocyte cell in the immune system.
  •  PCR genomic DNA with primers to capture the GAF domain DNA sequence Insert our PCR product into a transformation vector built upon pBAD by doing restriction enzyme digest and ligation Site directed mutagenesis to change amino acid cysteine in the class II GAF motif to aspartate to make it fluorescence Transform E. coli bacteria with two plasmids pBAD + insert and pPL from Lagarias protein expression in the E. coli system using the plasmids we constructed protein purification to confirm the fluorescence (Wavelength, brightness)
  • pDsRed-Monomer-Actin Vector: This plasmid encodes DsRed-Monomer, a monomeric mutant of the Discosoma sp. red fluorescent protein DsRed. The pDsRed-Monomer-Actin fusion protein incorporates into growing actin filaments, allowing visualization of the actin cytoskeleton in living or fixed cells.This plasmid contains all the correct origins of replication and promoter regions to be active in both e.coli and mammalian cells. G418- antibiotic mixture – Neo, allowing the selection of Jurkat cells that actually incorporated the plasmids.
  • INSERT a picture of Jurkat
  • The basic mechanism of transfection (using plasmid DsRedMonomer-Actin as an example):The DNA combines with transfection reagent. The complex will then enter the cell through the transient pores. Chemical method and physical method for opening the pores
  • The difference between chemical method and electroporationUse of high-voltage electric pulse to perturb the cell membrane and form transient pores, introducing DNAHighly efficient for the introduction of plasmids, especially mammalian cells Process:Cell Counts: an accurate cell concentration is very essential for electroporaion. One million cells per millimeter is required.Cells are extremely fragile after nucleofection, because there are pores in the membrane.Sterile technique is strictly demanded
  • Our data is not sufficient enough to draw a valid conclusion. We are looking forward to a cell culture room at Mills, so that we can continue and optimize the transfection of Jurkat cells. The estimated concentrations of these plasmids may not be accurate pAcGFP1-tubulin turned out to be more concentrated when measured by nanodrop
  • You may wonder what gel quantification and nanodrop are.Gel quantification is to run a DNA gel with our plasmids and markers. You can see here the bands on the gel. We compared the brightness and position of the plasmids to the markers. Since we know the size and concentration of each band of the markers, so that we can estimate the concentration
  • The Deconvolution fluorescence microscope at CBST is used for rapid live and fixed cell fluorescence microscopy. It can capture time elapse images showing the fluorescent cytoskeleton in 2 to 3 dimensions. Dark and temperature controlled
  • This is the video from CBST of Jurkat cells that transfected by pDsRed-Monomer-Actin. The two Jurkat cells are ~15um in diameter. This video was taken on a deconvolution microscope.The grey cells that appear in the end are also Jurkat cells, but are not transfected successfully, and thus they are not fluorescent.
  • Barrett rosa meza-acevedo & lingou

    1. 1. Bioengineering a red fluorescent protein tag from cyanobacteriochromes found in Thermosynechococcus elongatus: transfection and visualization using advanced microscopes<br />Mills CBST and Barrett Research Program 2011<br />Presented by Rosa Meza-Acevedo and Tianling Ou<br />
    2. 2. Our Research Goal<br />Bioengineer a new red fluorescent tag<br />Red illumination of the <br />cytoskeleton<br />Images modified from:<br />
    3. 3. Genes from CyanobacteriochromesT. elongatus<br />
    4. 4. Review of our procedure<br />
    5. 5. Our Focus<br />
    6. 6. Transfection<br /><ul><li>Transfect Jurkat Cells with three plasmids</li></li></ul><li>Plasmids used for transfection<br />pAcGFP1-Tubulin<br />pDsRed-Monomer-Actin<br />pLifeAct-TagGFP2<br /><br /><br /><br />
    7. 7. Jurkat cells<br /><ul><li>T lymphocyte cells
    8. 8. Why Jurkat cells?
    9. 9. ideal to study HIV viral entry
    10. 10. Hard to transfect
    11. 11. Maintenance:
    12. 12. CO2 incubator
    13. 13. Optimal cell density: 30*104 cells/ml
    14. 14. Selection:
    15. 15. G418 antibiotics
    16. 16. Optimal concentration: 5 mg/ml</li></li></ul><li>Transfection<br /><ul><li>The process of introducing nucleic acids into eukaryotic cells
    17. 17. Opening transient pores in the cell membrane to allow the uptake of plasmid</li></li></ul><li>Transfection<br />Chemical Method<br />Electroporation<br />MirusIngenio Electroporation <br />with LonzaNucleofector<br />MirusTransIT-Jurkat<br />Reagent<br />ROCHE X-treme<br />GENE 9 DNA Reagent<br />
    18. 18. Electroporation<br />Process :<br /><ul><li>Collect Cells and Count: 1X106 cells/ml
    19. 19. Centrifugation and Aspiration to collect the cells in a pellet
    20. 20. Resuspend the pellet with transfection reagent, and mix with plasmids. Transfer to a special nucleofectioncuvette.
    21. 21. Electrify the cells by Nucleofector</li></li></ul><li>Comparison of different methods<br /><ul><li>MirusTransIT Jurkat Reagent and Electroporation are more efficient</li></li></ul><li>Comparison of different plasmids<br />*We just started to do transfection with pLifeAct-TagGFP2. Thus this data may not be representative.<br />pAcGFP1-Tubulin is relatively more efficient than other plasmids<br />
    22. 22. Why plasmid pAcGFP1-Tubulin is more efficient?<br /><ul><li> Optimal starting concentration of plasmid for transfection: 1.00 ug/ul
    23. 23. pAcGFP1-tubulin is more concentrated and closer to the optimal concentration </li></li></ul><li>Gel Quantification and NANodrop<br /><br />
    24. 24. Remarks from transfection<br /><ul><li>Optimal starting concentration of plasmid for transfection is 1 ug/ul
    25. 25. DNA purity
    26. 26. Plasmid DNA preparation
    27. 27. Endotoxin-free
    28. 28. Low passage numbers of Jurkat Cells
    29. 29. Proceed with transfection in a strictly sterile environment</li></li></ul><li>Visualization of transfected cells<br />Real-time, live-cell confocal fluorescence microscope (Spinning disk)<br />Deconvolution fluorescence microscope at CBST<br />
    30. 30. A video from CBST, taken on a deconvolution microscope<br />
    31. 31. GFP transfected cells<br />pLifeAct-TagGFP2 electroporation<br />pAc-GFP-Tubulinelectroporation<br />
    32. 32. Future Goals<br />Translate into mammalian code<br /><br /><ul><li>Insert our 569TM fluorescent tag into a plasmid vector
    33. 33. Optimize expression and purification protocols</li></li></ul><li><ul><li>Dr. Susan Spiller
    34. 34. Barrett Family
    35. 35. Deanna Thompson and Ana Popovic from CBST
    36. 36. UC Davis Medical Center
    37. 37. Dr. Lagarias and Nathan Rockwell
    38. 38. Mills CBST lab members
    39. 39. National Science Foundation (NSF)</li></ul>Thank you<br />