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