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Metabolic Engineering for Robust Natural Product Biosynthesis in C. glutamicum
Jason Ronstadt, Chemical EngineeringMentor: Arul M. Varman, Assistant Professor
School for Engineering of Matter, Transport & Energy
Methodology
Current Results
pAJL01
• Resulted in many
background colonies
• Will assemble again once pAJL04
is successfully transformed
pAJL02
• Successfully assembled
and transformed
• Currently being sequenced
Future Work
Acknowledgements
• Finish assembling, transforming, and
sequencing all plasmids
• Determine baseline naringenin production
• Manipulate expression of ACC subunits and
measure change in naringenin production
Arren Liu- Graduate Mentor
Arul Varman- Principal Investigator
David Nielsen- Committee Member
Varman Lab
Objective• To engineer a pathway within C. glutamicum
that would allow the cell to produce naringenin.
• Significance: Naringenin is a natural product
that has pharmaceutical and agricultural
applications. This method will allow naringenin
to be produced more sustainably.
PCR to
amplify
GOIs
Purify
fragments
Gibson Assembly
Transform to cells
Colony PCR
Sequencing
Expected:> 5kb and > 3kb
Experimental Design
pAJL01
pAJL02
pAJL03
pAJL04
4CLpc CHSat CHI
4CLpc CHI
CHI
CHI
CHSph
4CL2at CHSat
CHSph4CL2at
pAJL03
• Had trouble cloning with 4CL2at
• Will assemble once pAJL04 is
confirmed
pAJL04
• In progress; successfully
assembled
• Currently running colony PCRs
to confirm transformation
0.5kb
1 kb
3 kb
Expected: 1.6 kb
CHSat Amplicons
pc_overhang at_overhang
pY-4CL2at gradient PCR
w/ old and new primers
Expected: 1.2 kb
3 kb
1 kb
0.5kb
4CL2at Amplicon
pAJL01
pAJL04
3 kb
1 kb
0.5kb
RD of pAJL02 plasmids w/ SacI
RD of pAJL01 & 02 plasmids w/ NdeI