Difference between revisions of "Part:BBa K3939998"

Revision as of 08:36, 21 October 2021

Cas9 with flipped promoter

Brief introduction

This part is constructed by two LoxP sequences and a reversed pGal promoter. the pGal promoter is not a tightly regulated promoter. thus,we used estrogen and galactose to induce Cas9 expression, allowing this element to be regulated more precisely. In our project, we use it to cut chromosomes to make chromosome-free eukaryotic cell-- CREATE. More details about this part, please click Measurement. Sequence and Features

Characterization

Use fluorescent microscope to test the function of part

1.Experimental principle & purpose：
Nucleic acid dyes made it possible to visualize the degradation of chromosomes. Hoechest is a membrane-permeable nucleic acid dye that binds to DNA and generates a strong fluorescent signal. We stained the cells with the dye and photographed them under a fluorescent microscope to directly verify whether the cells' DNA was lost.
2. Experimental design：
Control group: Saccharomyces cerevisiae 4742 nfGFP
Experimental group: Saccharomyces cerevisiae 4742 nfGFP with 7flip plasmid+Cre plasmid ( cleavage system 2.0)

We placed the experimental and control groups’cells in the corresponding induction medium on the 96 well microtiter plate. Then, the cells were stained with Hoechest dye and placed under a fluorescent microscope lens for a fixed field of view for continuous filming to observe changes in intracellular DNA in the same field of view. According to the hypothesis, if it is indeed CREATE, the DNA will be progressively degraded, and its nuclear fluorescence signal will disappear, while the control group will remain unchanged. We will directly characterize the disappearance of DNA by the results of the dye staining shots.
3. Experimental operation
(1) Add samples: The cells were directly picked from the solid medium and added to the corresponding medium on the 96 well microtiter plate. We place 200μl sample of the medium in each well. We set blank reference (water), control groups and experimental groups in three parallel groups when adding samples.
(2) Staining: Cells were stained with Hoechst dye for 30 min according to the protocol, in the proportion of 1μl dye/200μl sample.
(3) Filming: To ensure a fixed field of view of the cells, after the suspended cells have settled and are motionless, they are placed under the fluorescence microscope lens for continuous fixed filming. According to the dye's protocol, the fluorescence microscope's channel settings can be the same as those of the commonly used nucleic acid dye DAPI. Each photograph was taken every 30 min for 18h. Since only one lens was available simultaneously, the experimental group was photographed continuously, while the control group was photographed only twice at the beginning and the end.
4.Results analysis
Hoechest is a nucleic acid dye that may bind to the RNA in the cell, resulting in a diffuse blue fluorescence inside the cell, while the very bright dots (indicated by arrows) in the cell indicate the location of the nucleus. This phenomenon can be identified in the pictures of the control group, which indicates that our dye is working correctly. The following two figures show the changes in the control group (Saccharomyces cerevisiae 4742 nfGFP) after 18h of staining with Hoechst dye.