An Integrated Platform Based on Bacterial Microcompartment(BMC) for - - PowerPoint PPT Presentation

An Integrated Platform Based on Bacterial Microcompartment(BMC) for de novo Proteinaceous Artificial Organelles

USTC iGEM 2010

• Bacteria will be harder to stand this

circumstance with the concentration of the chemical products.

• The host cell’s matabolic pathway may

interfere the inducted reactions.

• Difficult to enrich those products.

About our ideas

Problems in chemical production

About our ideas

Artificial Organelle in Procaryote Host-Cell-Free Reaction Chamber

* From MOLECULSR EXPRESSIONSTM

*

Brief introduction of our presentation

Background Introduction BMC Assembly Procedure BBF RFC 53 Modeling Conclusion & Future Tasks

What is BMC?

• Polyhedron-Shaped
• 80 to 200 nm
• Comprising of several

Thousands protein and enzyme submits

*

* Todd O. Yeates,Bacterial Microcompartment Organelles: Protein Shell Structure and Evolution, Annu. Rev.

• Biophys. 2010. 39:185–205

What for?

* Todd O. Yeates,Bacterial Microcompartment Organelles: Protein Shell Structure and Evolution, Annu. Rev.

• Biophys. 2010. 39:185–205

*

• Ecapsulate many

sequentially acting enzymes.

• Enhance the

efficiency or protect the cell from toxic intermediates.

BMC Diversity

*

* Todd O. Yeates,Bacterial Microcompartment Organelles: Protein Shell Structure and Evolution, Annu. Rev.

• Biophys. 2010. 39:185–205
• Provide diverse

metabolic functions.

• Share an evolutionary

related shell, which is defined by a related protein domain.

The Pdu Microcompartment

* Joshua B. Parsons, Synthesis of Empty Bacterial Microcompartments, Directed Organelle Protein Incorporation, and Evidence of Filament-Associated Organelle Movement , DOI 10.1016/j.molcel.2010.04.008

*

• Fully understood
• Complicated in

structure, with high potential in future uses

• Not strictly regular in

structure

A B J K N T U

Genes and Protein Fusion

*

* Joshua B. Parsons, Synthesis of Empty Bacterial Microcompartments, Directed Organelle Protein Incorporation, and Evidence of Filament-Associated Organelle Movement , DOI 10.1016/j.molcel.2010.04.008

• Construction of the BMC shell in vivo.
• Encapsulation our target enzymes into
• ur microcompartment.

Our Goal

Genomic DNA from Citrobactor.freundii.

Procedure

Altogether over 15 primers & 14 BioBricks.

Tests Result

• Figure. SDS-PAGE of composite BBa_K371019

TEM Result

+1.0 mM IPTG/16°C

• IPTG

150nm

The motivation to design BBF RFC 53

Standard

Scar RFC 10

TAC TAG AG (frameshifted)

RFC 25

ACC TCC (Thr-Gly)

RFC 37

Thr-Arg, Ser-Gly, etc.

… … Shortcomings:

• Frameshifted

scar

• Limited linkers
• Incompatibility

with RFC 10 …

Existing Standards:

An Introduction to BBF RFC 53

The prefix and suffix of BBF RFC 53

An Introduction to BBF RFC 53

Meta- Prefix Meta- Suffix

Construct BioBrick parts in RFC 53

• Long parts
• - PCR
• Short parts : RBS, linker, tag, ect.
• - de novo DNA synthesis

Oligo 1 Oligo 2

Assembly in RFC 53

What’s D - part & L - part

D - part L - part

Assembly in RFC 53

Concept : D - part & L - part

D - part L - part D - part

RFC 53 assembly and fusion

• five fusion methods:MetaFusion, HeadFusion,

TailFusion, HXTFusion, and FullFusion.

• http://bbf.openwetware.org/RFC.html

SF MPF PF MSF SF PF MSF MPF SF PF MSF MPF SF MSF PF MPF SF PF MSF MPF SF PF MSF MPF

SapI SacI SacI EarI

AGCTCTTCA ATG TCGAGAAGTTAC AGGAGCT CTTC TCC TCGAGAAC AGGAGCT CTTC TCC TCGAGAAC

SF PF MSF MPF SF PF MSF MPF

GGTTGAAGAGAT CCA ACTTCTCTA

B A

Vector Insert

A*B

Verifiation RFC 53

• P1-64fuse with GFP

Confocal microscope result

Assembly BMC BioBrick under RFC 53 standard

Highlights of RFC 53

• Compatible with RFC 10 standard;
• Support protein fusion;
• The assembly scar is at most 3 bp
• Assembly strategy is ranther flexible;
• Enzymes used is not too expensive ;
• Can be used as a de novo DNA synthesis

method and a site-directed mutagenesis method;

• ...

Modeling

Goal: To evaluate the probability for pdu microcompartment to be used as a reaction chamber for different uses

Hypothesis:

PduN is the vertex. Hexamers form the facets Icosahedral with a regular hexagon of same size

1.modeling 2.co-evolution analysis

Step 1: Modeling

Volume: ~32000 nm3 About 1/1500 of a single bacterium Rubisco: ~103nm3

Step 2: Co-evolution analysis

N-terminal sequence of inside components shell components PduK PduA PduN PduB PduU PduJ PduT

PduO PduL PduG PduH PduC PduQ PduE PduD PduP

Future--- Fusion Protein

Improved Efficiency + Reaction chain + Toxicity reduction inside surface Reac acti tion Ch n Cham amber

Our microcompartment:

Pollution Treatment Drug delivery

• utside surface

Future---

BMC as a unit in novel construction

• Material
• Molecular selection

Summary

• Succeed in constructing functional in vivo

pdu microcompartment constructed in BBF RFC 10.

• Establish an efficient and highly

compatible novel standard for fusion proteins.

• Manage in finding bioinformatic evidence of

pdu microcompartment as a reaction chamber with diverse application potentials.

Acknowledgement

• Instructors

Hao Jiang Zhaofeng Luo Jiong Hong

• Advisors
• Prof. Haiyan Liu
• Prof. Jiarui Wu
• Sponsor

USTC Initiative Foundation Graduate School, USTC Alumni Foundation, USTC School of Life Science, Physics, Computer Science, Math, USTC

Our Team

Supplementary

• Figure 1| Flow chart of the process of co-evolution computational of

pdu microcompartment.