Team:Shanghai-United/Design

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Design

Design:

We designed our experiment to compare two groups – control group and test group with over-expressed NFX1 gene. The EGFP can help us better locate NFX1 protein. To ensure the experiment have a higher possibility of success, we designed two plans in the comparing two groups.

Plan A (fusion)

Control Group	Experimental Group
pLex-EGFP	pLex-EGFP-NFX1
pLex-GFP	pLex-GFP-NFX1
pLex-amilGFP	pLex-amilGFP-NFX1
pLex-mCherry	pLex-mCherry-NFX1

By examining normal cell protein expression and NFX1 over-expressed protein expression in cells, we may be able to identify NFX1 related proteins by analyzing and comparing the amount of various proteins in both group and identify differential proteins. The attached EGFP is intended to better locate NFX1 protein.
However, our team recognized that because the NFX1 gene has a fairly large size, fusion protein experiment has a relatively lower chance of success. Therefore, we designed plan B as a backup.

Plan B (co-transformation)

Control Group	Experimental Group
pLex-MCS	pLex-NFX1

Both pLex in each group will be inserted into the same cell during transduction, which means that a cell will express genes on both of the plasmids. Each group will have 3 dishes of cells for each cell experiments. As we have 3 cell experiments, we will need 18 dishes of HeLa cells in total.

To confirm the over-expression of NFX1 in cells, we planned to run Western Blot to see if there is a high concentration of NFX1 protein in experimental group. To find the differential proteins, we planned to use IP-MS (Immunoprecipitation-Mass Spectrum) and FASP.

For IP-MS, we will add new flag to NFX1 protein, which can attach to the agarose beads we add later. NFX1 protein along with proteins that are interrelated with NFX1 will be attached to the agarose beads, other unrelated protein can be washed away. Then we can run SDS-Page, comparing the proteins left on the beads and total protein. In-gel enzymatic hydrolysis shall be done to break the proteins in the gel into amino acids, which can be used for Mass Spectrum.

For FASP, we want to gather all proteins' concentration in both groups. We plan to use SDT for cell lysis, and then use trypsin to break up protein into amino acid chains. They can then be sent to Mass Spectrum to evaluate concentration for different proteins. Comparing the statistics, we may filter out the proteins that differ in concentration significantly in control and experimental groups.

In the last phase, we will compare our results to past studies and researches, further consolidating certain protein's correlation with NFX1.