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DNA Cloning and Expression of HMGB1 in E. Coli
Liam Arnade-Colwill, Carbone/Yang
Background on HMGB1
• HMGB1 is released by damaged mesothelial cells in response to asbestos exposure
• Initiates the inflammatory response that can lead to mesotheliomadevelopment
Project Objectives
• To express HMGB1 in E. Coli in order to obtain enough samples of the protein to conduct further laboratory experiments investigating the link between HMGB1 and mesothelioma
• To learn important tools and techniques to work with protein and DNA in the future and to gain invaluable laboratory experience
Project Outline
• Phase One: DNA cloning
– Design gene-of-interest
– Ligate into PGEMT plasmid
– Transformation of DH5-a cells
– Screening
– Cell Growth
– Plasmid Extraction
– Restriction Digestion
• Phase Two: Expression
– Ligate GOI into PET14B expression vector
– Transformation of BL21 cells
– Cell Growth
– Induction of gene of expression with IPTG
– Protein purification
Protein Expression
• DNA is transcribed by RNA polymerase in the nucleus to form pre-mRNA
• Pre-mRNA is processed to form mRNA• mRNA leaves the nucleus and binds to ribosomes,
beginning translation• tRNA builds a polypeptide chain (protein)
DNA
Transcription
mRNA
Translation
Protein
Factors that affect Gene Expression
Rare Codons in HMGB1 Sequence
• 25/216 codons are rare for E. Coli
DNA Cloning
• 1) Add “A” overhangs to the the 3’ ends of our gene block
• 2) Ligate our gene-of-interest into PGEMT plasmids
• 3) Induce transformation of DH5-alpha cells through heath shock
• 4) Allow cell culture to grow on LB-Amp plates
Advantages of the PGEMT Vector
• “Copy Number” refers to the ability of DNA to replicate
• PGEMT is a high copy number plasmid, which means it yields a large amount of DNA
Blue/White Screening
• If gene-of-interest is absent from PGEMT, the lacz gene is activated and a blue color is expressed
• If the gene-of-interest is present in PGEMT, the lacz gene is inactivated and blue is no longer expressed
Colony PCR
• If gene-of-interest is present in PGEMT, a longer band of DNA is amplified, resulting in a single band of DNA
• If gene-of-interest is also absent, small and large bands of DNA are amplified, resulting in multiple bands of DNA
L 1 2 3 4 5 6 7 8
700 bp
Growth, Plasmid Extraction and Restriction Digestion
• After isolating the colonies with recombinant plasmids, we prepared a glycerol stock of our cells to grow them and make multiple copies of our gene
• Next, we performed plasmid extraction to re-obtain our PGEMT vector
• From there, we performed restriction digestion and ran a gel to isolate our gene-of-interest, then conducted gel extraction to obtain a pure sample of our replicated DNA
Expression
• 1) Ligate replicated DNA into PET14B expression vector
• 2) Induce transformation of BL21 cells through heat shock
• 3) Induce expression through addition of IPTG
Advantages of BL21 and PET14B
• HMGB1 can be toxic in high quantities to E. Coli, resulting in cell death
• PET14B is a low copy number plasmid, meaning that is does not replicate as easily. This results in less protein expression
• BL21 is protease deficient, protecting protein
• PLysS, a plasmid found in BL21, blocks gene expression until the addition of IPGT to sustain the viability of the cell
Results/Conclusion
• Five strong bands of protein in induced samples, no band of desired protein in un-induced sample (negative control)
• Protein is 25kDa, which indicates it is HMGB1
IPTG - + + + + +
25kDa
Next Steps
• Re-induce expression of HMGB1 in BL21 cells with IPTG
• Extract protein through protein purification process
• Quantify data with micro-plate system
Acknowledgments
• Dr. Carbone
• Dr. Yang
• Sandra Pastorino
• Vishal Negi
• Karin Koga
• All the other interns!
• UH Cancer Center