Bioengineering a red fluorescent protein tag from cyanobacteriochromes
1. Bioengineering a red fluorescent protein tag from cyanobacteriochromes found in Thermosynechococcus elongatus: transfection and visualization using advanced microscopes Mills CBST and Barrett Research Program 2011 Presented by Rosa Meza-Acevedo and Tianling Ou
2. Our Research Goal Bioengineer a new red fluorescent tag Red illumination of the cytoskeleton Images modified from: cbst.ucdavis.edu
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.
