Week 6: Drafting my Review

Welcome back to my blog!

This week I started to draft the background section of my literature review. I also have decided to focus on looking into using CRISPR as a tool to improve agriculture. 

In the lab:

On Tuesday after a seminar, Dr. Lindsay and I used the confocal microscope to screen the N.benth (tobacco) that we infiltrated last Friday. We were looking for GFP signaling from the plasmid we inserted. As we screened the N.benth we noticed that the (see figure 1) signal was around the edges of the cell and in a circle near the edge. As we looked closer we also saw that the signal around the edges was also present as stringy lines and the middle of the circle did not have the signal present. From this, Dr. Lindsay hypothesized that the plasmid was present in the cytoplasm of the cell. This is because the circle is the nucleus, we know this because the empty space in the circle is the nucleolus and in plant cells, most of the cell is made up of the vacuole, so all the cytoplasm and the other organelles are pushed to the edge of the cell. What distinguishes that the plasmid is in the cytoplasm is the stringy lines, in the cell, the cytoplasm will connect different parts of the cell through these little bridges.

On Wednesday, Dr. Lindsay taught me how to analyze the DNA sequences on Benchling, the website we are using to take notes and analyze confocal images using a program called ImageJ2. Using Benchling I looked at the leaf and flower DNA we had sent out to get sequenced. During this process, I looked for edits in the sequence that would imply that the gene that we are trying to insert into the plant was present. 

Here is one photo that I was working on in ImageJ2:

Fig.1: Confocal image of N.benth, magenta: chlorophyll, green: GFP

On Friday after the lab meeting, Dr. Lindsay and I screened some leaf tissue and a couple of flower buds from our EYA-GFP first generation of transformed plants under the confocal microscope. We were looking for a GFP signal in the cells’ cytoplasm, but we were not able to see any clear-cut signaling. Instead, we saw a lot of autofluorescence (already present fluorescence) and some possible signaling. This was not too surprising as this is only the first generation so there is not a high chance of having a homozygous organism/ a transformed plant that will strongly express the edits.