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Week 7: Writing

May 19, 2022

Welcome back to my blog!

 

This week I continued to draft my literature review and read up on the applications of CRISPR in agriculture [1, 2, 3]. 

 

In the lab:

On Tuesday, My mentor and I dissected some buds from our 35S-EYA-GFP mutants and screened them under the confocal microscope for the GFP (green fluorescent protein) signal. Unfortunately, we were not able to see any clear signaling, there was some auto-fluorescence. 

 

On Wednesday,  I set up an overnight culture with the agrobacterium I transformed a few weeks ago for Friday’s floral dip [4]. The purpose of this overnight culture is to allow the culture, i.e. the agrobacteria, to grow. The purpose of the floral did is to quickly transform some Arabidopsis with our agrobacterium. My mentor and I also visited the farm to collect leaf tissue from our CRISPR mutants for a different DNA extraction protocol. This protocol had way fewer steps than the protocol that required the TissueLyser. For this protocol, all I had to do was crush the tissue samples in water and then use that water as the DNA template for the PCR reaction. The DNA extraction was successful and my mentor sent the samples in for sequencing.

 

 On Friday, we performed the floral dip  [4] with some wild-type Arabidopsis and our agrobacterium. The process for the floral dip was very simple. I added the agrobacterium solution to a sucrose solution and then added a surfactant to the mix. The surfactant helps the agrobacterium coat and stick to the plant. After making the mixture we prepared the plants by cutting off the already formed siliques (where the seeds develop). Lastly, we inverted the plants and dipped them into the agrobacterium mixture. Now we wait for them to develop new siliques and for the time to harvest the seeds from these plants.

 

Sources:

  1. Liu G, Lin Q, Jin S, Gao C. The CRISPR-Cas toolbox and gene editing technologies. Mol Cell. 2022 Jan 20;82(2):333-347. doi: 10.1016/j.molcel.2021.12.002. Epub 2021 Dec 29. PMID: 34968414.
  2. Pan C, Qi Y. CRISPR-Act3.0-Based Highly Efficient Multiplexed Gene Activation in Plants. Curr Protoc. 2022 Feb;2(2):e365. doi: 10.1002/cpz1.365. PMID: 35157372.
  3. Naik BJ, Shimoga G, Kim SC, Manjulatha M, Subramanyam Reddy C, Palem RR, Kumar M, Kim SY, Lee SH. CRISPR/Cas9 and Nanotechnology Pertinence in Agricultural Crop Refinement. Front Plant Sci. 2022 Apr 8;13:843575. doi: 10.3389/fpls.2022.843575. PMID: 35463432; PMCID: PMC9024397.
  4. Zhang, X., Henriques, R., Lin, S.-S., Niu, Q.-W., & Chua, N.-H. (2006). Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nature Protocols, 1(2), 641–646. https://doi.org/10.1038/nprot.2006.97

One Reply to “Week 7: Writing”

  1. Courtney H. says:

    Sounds like a fun week filled with a lot of lab work! I look forward to hearing next week about how your literature review is going and what sources you will be citing.

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