Team:Baltimore BioCrew/Results

Successful Cloning of the Cerastocytin and Russel Viper Snake Venom Genes

We were able to successfully clone five of our six Clot Maker constructs:
1. Cerastocytin with a His tag and the Lac promoter
2. Cerastocytin with a constitutive promoter
3. Russell Viper Snake Venom with a constitutive promoter
4. Russell Viper Snake Venom with a His tag and the Lac promoter
5. Russell Viper Snake Venom with a His tag
We were not able to clone Cerastocytin with a His tag and constitutive promoter

We were able to determine that we successfully cloned the genes by colony-screening PCR using VF2 and VR primers. We also purified plasmids and sent them for sequencing.

Successful Expression of the Cerastocytin and Russel Viper Snake Venoms

We were able to successfully express five of our six Clot Maker constructs in E. coli cells. We grew the cells overnight, diluted them in the morning and then diluted and grew them to an OD of 0.6. We lysed the cells and ran them on SDS-PAGE gels.

Lane 1: Cerastocytin with a His tag and the Lac promoter induced with IPTG
Lane 2: Cerastocytin with a His tag and the Lac promoter not induced
Lane 3: Cerastocytin with a constitutive promoter induced with IPTG
Lane 4: Cerastocytin with a constitutive promoter not induced
Lane 5: Cerastocytin with a constitutive promoter induced with IPTG
Lane 6: Cerastocytin with a constitutive promoter not induced
Lane 7: Russell Viper Snake Venom with a constitutive promoter induced with IPTG
Lane 8: Russell Viper Snake Venom with a constitutive promoter not induced
Lane 9: Russell Viper Snake Venom with a constitutive promoter induced with IPTG
Lane 10: Russell Viper Snake Venom with a constitutive promoter not induced
Lane 11: Russell Viper Snake Venom with a His tag and the Lac promote r induced with IPTG
Lane 12: Russell Viper Snake Venom with a His tag and the Lac promoter not induced
Lane 13: Russell Viper Snake Venom with a His tag induced with IPTG
Lane 14: Russell Viper Snake Venom with a His tag not induced
Lane 15: Ladder



We saw bands on our gel, but we were not sure if they were our protein or just a normal protein in E. coli. So we re-ran our gel with a control from E. coli without a plasmid.
Lane 1: Cerastocytin with a His tag and the Lac promoter induced with IPTG
Lane 2: Cerastocytin with a His tag and the Lac promoter not induced
Lane 3: Cerastocytin with a constitutive promoter induced with IPTG
Lane 4: Cerastocytin with a constitutive promoter not induced
Lane 5: Cerastocytin with a constitutive promoter induced with IPTG
Lane 6: Cerastocytin with a constitutive promoter not induced
Lane 7: Russell Viper Snake Venom with a constitutive promoter induced with IPTG
Lane 8: Russell Viper Snake Venom with a constitutive promoter not induced
Lane 9: Russell Viper Snake Venom with a His tag and the Lac promote r induced with IPTG
Lane 10: Russell Viper Snake Venom with a His tag and the Lac promoter not induced
Lane 11: Russell Viper Snake Venom with a His tag induced with IPTG
Lane 12: Russell Viper Snake Venom with a His tag not induced
Lane 13: Control no plasmid
Lane 15: Ladder


Since there was no band in our control lane, the band(s) we see in the other lanes are our protein and our gene is working properly and expressing the protein, even if we don't induce it.