07/01-07/07
1.Making and sterilizing lysogeny broth, preparing agar plates
2.PCR amplification of SUMO-IL-18BPa-Fc, SUMO-IL-18BPc-Fc

Figure 1. The IL18 signal pathway diagram17
07/07-07/15
1. PCR amplification of SUMO-IL-18BPa-Fc, SUMO-IL-18BPc-Fc
2. pET28a blank plasmid extraction and PCR amplification
3. Enzyme digestion of pET28a and verification that it has been cut
4. Gel-electrophoresis verification of pET28a, SUMO-IL-18BPa-Fc,SUMO-IL-18BPc-Fc
5. DNA extraction of pET28a, SUMO-IL-18BPa-Fc, SUMO-IL-18BPc-Fc from gel
07/15-07/30
1. Restriction digestion and ligation to create recombinant plasmids
2. Transformation of recombinant plasmids into DH5a E. coli

08/01-08/07
1. Selection and verification of monoclonal colonies
2. Send monoclonal colonies' plasmids for gene sequencing for further verification
3. Amplification of DH5a with desired plasmids
08/08-08/15
1. Transformation into BL21 E. coli
2. Selection and verification of monoclonal colonies
3. cultured bacteria

08/16-08/22
1. IPTG induction of protein synthesis
2. Extraction and lysis of BL21 bacteria
3. His-tag purification
08/23-08/31

1. SDS-PAGE verification of extracted proteins
2. BCA Calorimetric method to quantify proteins
3. Western blot for further protein verification
4. Store proteins at different temperatures for 24 hours

08/31-09/04
1.Cell incubation with proteins (stored at different temperatures) and inflammatory factors
2.ELISA of cell supernatant to detect IFN-gamma levels