TTE REU Final presentation
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Transcript of TTE REU Final presentation
ENGINEERING SIGNAL SEQUENCES ON THE 1,2-PROPANEDIOL UTILIZATION
MICROCOMPARTMENT
Kamaria Ashley KermahMentor: Chris JakobsonDanielle Tullman-Ercek
Department of Chemical and Biomolecular Engineering
July 28, 2015
Purified Microcompartment
ENGINEERING BACTERIAL CELLS
• Why?
o Ubiquitous
o Easily Replicated
o DNA Easily Manipulated
•How?
o Manipulating the genetic code
1,2-PROPANEDIOL UTILIZATION MICROCOMPARTMENT (PDU MCP)
• Proteinacious Organelle
• Protection and Efficiency
1,2-propanediol
Carbon
METABOLIC PATHWAY
• Salmonella enterica Pathogenicity in
human gut 1,2-propanediol
propionaldehyde
propionyl-CoA
PduCDE
PduP
PduW
propionateATP
ADP
PDU CDE COMPLEX
• Enzyme localization
• N-terminus of PduD
• Alpha helix
• Hydrophobic and hydrophilic
faces
6
HOMOLOGOUS RECOMBINATIONHOW DOES IT WORK?
B.
7
METHODSGrowth on 1,2-
propanediol
[G. Cannon 2001. Microcompartments in Prokaryotes doI: 10.1128/AEM.67.12.5351-5361.2001]
Homologous Recombination
Sequencing Reaction
TEM Microscopy
RESULTS AND ANALYSIS
Sucrose Sensitivity Confirmation of gene size
~5kb
Amplification of pduD gene
1kb
0.5kb
Sucrose Resistance
FUTURE APPLICATIONS
• Engineering microcompartments for industrial use
o Nanobioreactors
o Antibiotics Replace PduD N-terminal with PduP
signal peptide
Increasing size of microcompartments and number of enzymes to increase titer
Localization of co-factors and heterologous enzymes into MCP
MCP formation in yeast