Difference between revisions of "Part:BBa K3159001"

Revision as of 05:54, 17 October 2019

NFX1

1 Usage and Biology

MHC(Major Histocompatibility Complex) Class II cells are crucial in immune cell communication. It informs other cells of the impeding danger so that they can be prepared and build necessary defense against the virus, restraining the virus from spreading. The gene expression of MHC Class II cells are primarily controlled by transcription factors that bind to X and Y boxes. NFX1 is one of the transcriptional factor. This sequence codes for a transcriptional repressor protein, which binds to MHC Class II genes on the highly conserved x-box motif in vitro to regulate gene expression. Malfunctioning of this protein may decrease the sensitivity of MHC Class II cells in their inflammatory immune response induced by Interferon-gamma, shortening the period in which defense can be built against antigens.

2 Plasmid construction

2.1 pLEX-MCS Vector

pLEX is a DNA molecule that is capable of self-replication. This genetic vector is often used in genetic programming to transact the gene of interest into a cell, resulting in an over-expression of the targeted gene. In other words, it’s an indispensable pathway that our gene of interest has to take in order to be expressed in a cell. Thus, constructing plasmids is a necessary step for us to get the protein coded by our inserted gene. In our experiment, the pLEX being used for all targeted gene is the pLEX-MCS Empty Vector. One of the benefits of this artificial vector is that it contains multiple restriction enzyme cutting sites, which is ideal for the design of primers for target gene, transfection, and subsequent transduction in E. coli.

Figure 1: stricture of pLEX-MCS used

2.2 Primer Design (PCR & Ribosome Cutting)

Regarding our experiment design of co-transformation, involving EGFP and NFX1, we used a double digestion technique in our construction of primers. We selected Xho1 and Mlu1 as EGFP cutting sites, and Spe1 and Xho1 as NF-X1 cutting sites.

tm value is calculated using Snapgene, which helps set up PCR. Once amplified, both genes can be inserted into pLEX-MCS using DNA ligase, creating our plasmids of interest.

2.3 Bacterial Transformation & Selection

To isolate plasmids, we transform the plasmids into DH5-alpha cells. The reason we pick DH5-alpha cells is because those E.coli cells are specifically engineered to maximize transformation efficiency. The cells are conserved in -80 degree Celsius environment, and undergo reviving process before used. Together with our constructed plasmids of interest, they are placed in 800 ul liquid lysogeny broth without ampicillin in incubation shaker for an hour at the rate of 220 rpm, 37 degree Celsius. This step allows the bacteria to ingest the plasmid and express the gene that codes for ampicillin resistance. After, centrifugation is used to filter out the waste and obtain bacteria that potentially contain our plasmid of interest. To further select the bacteria that have taken in the plasmid containing antibiotic resistance, we cultured the bacteria on solid LB medium with ampicillin overnight. From the grown bacteria colonies, we selected a few and extracted the plasmids. To further confirm they are the plasmid of interest, we send those for DNA sequencing.

3 Cell Cultivation

3.1 HeLa Cells

In our experiment investigating cervical cancer, we chose to use HeLa cells, which is the oldest and most commonly used cell line in scientific research. These immortal cells were taken from Henrietta Lacks, a cancer patient in 1951.

3.2 Thawing & Cell Culture

The cells are stored in solid nitrogen at -196 degree Celsius. When taken out, they are based in 37 degree Celsius water for 2 minutes and added into 4-inch petri dishes with 10 ml of cell culture media made with 500ml DMEM + 50ml 10%FBS+5.5ml Streptomycin/Penicillin(to avoid growth of bacteria). We used 18 of 4-inch petri dishes for three groups of experiments, 6 dishes each. During the process of cell growth, metabolism of cells requires medium exchange at least every 24 hours.

3.4 Cell Passaging

Roughly every three days, the cells will reach a certain density in the dish that they will be better off being split into different dishes. First we used PBS for cleansing purposes, and added 1ml Trypsin (a serine protease) to degrade the protein that attaches the cells to the walls of the petri dishes, so that the cells will be in spherical forms, granted with mobility. After 60 seconds, DMEM must be added to stop degrading of proteins, and all liquid in the dish will be transported into EP tubes for centrifugation (1000g 1min). Supernatant is gathered and redistributed into three separate petri dishes.

3.5 Transduction

Solution concentration: opti-MEM 20%, Lip 3000 1:3 (6ul lipofect-transfection regent used to boost the efficiency of the transduction process)

Total system: 6ml in 4-inch petri-dish

Note that cell culture medium is changed to DMEM only, no nutrient contained for the cells, promoting endocytosis of vectors into the cells.

After roughly 48 hours, the protein coded by the gene inserted into the cell will be expressed completely, and further experiments can be carried out.

4 SDS-Page Analysis (Western Blot)

Figure 2: SDS-Page