Synthetic Minimal Chromosome 2010 CBNU-KOREA team genetic - - PowerPoint PPT Presentation

Synthetic Minimal Chromosome

2010 CBNU-KOREA team

• The minimal chromosome contains the minimal set of

genetic information necessary and sufficient to sustain a functioning chromosome.

• It is a self-replicating chromosome compatible with a host

chromosome.

• It includes more than one essential gene for viability.

WHAT is a Minimal Chromosome?

Ⅲ

Ⅰ

Ⅱ

Genome = Chr I + Chr II + Chr III Minimal Genome = MC I + MC II + MC III

Ⅰ Ⅱ Ⅲ Theoretically, the smallest minimal chromosome contains self replicating system and one essential gene for viability.

Minimal Chromosome vs. Minimal Genome

Minimal Chromosome vs. Minichromosome

Minichromosome

• riC

Bottom up

plasmid More than one essential gene for other function Essential genes for chromosome replication & segregation

• riC

Minimal Chromosome

Minimal Chromosome Minimal Genome Minimal Cell Normal Cell

Biomedicine Diagnostics Therapeutic Drug Vaccines Bioremediation Food Ingredients Fine Chemicals Bio-fuels Biomaterials Biosensor

Synthetic Biology

Construction of a Minimal Chromosome will be a very important foundational advance in Synthetic Biology

Minimal Genome Minimal Chromosome

Design and Synthesis of the Minimal Genome is difficult.

Types of gene gens arrangement gene order codon usage direction of gene genome structure metabolic pathway metabolites interaction genome stability Statistically analysis Environmental effect RNA structure RNA interaction

WHY Minimal Chromosome?

So, we need

& DESIGN SYNTHESIS

DB & PROGRAM

Concept

Concept

• Used NCBI and DEG Information.
• This Program Based Artemis.
• Show Gene's Essential-Gene.

– Mapping DEG E-Gene and Custom’s Genebank Data(.GB) – Artemis used showing tools

Principle-①

CBNU_KOREA DATABASE Gene Bank Format File E-Gene Information

Loading GB File

Gene information 04 05 06 07 08 09 10

Principle-②

CBNU_KOREA DATABASE Gene Bank Format File E-Gene Information

Loading DB

E-GeneTable 01 M 05 L 10 A 13 C 40 H 69 D 80 D Gene information 04 05 06 07 08 09 10

COG Code

Principle-③

CBNU_KOREA DATABASE Gene Bank Format File E-Gene Information

DB&GB Matching

E-GeneTable 01 M 05 L 10 A 13 C 40 H 69 D 80 D Gene information 04 05 06 07 08 09 10

Principle-④

CBNU_KOREA DATABASE Gene Bank Format File E-Gene Information

Apply Color

E-GeneTable 01 M 05 L 10 A 13 C 40 H 69 D 80 D Gene information 04 05 06 07 08 09 10

4 5 6 7 8 9 10

Principle-⑤

Drawing Diagram

Database

• Based DEG and NCBI
• Mapping by GI number
• Schema is right follows.
• Schema designed for

extendible.

• Surface, function parsing

Database

DEG MATCHING

• Make list of

DEG’s Number

• Add DEG data

NCBI MATCHING

• Matching DEG list

NCBI

• Add NCBI data

Store MySQL Server

• All information

Strore Database

If you want to see it, you access website http://synb.chungbuk.ac.kr

Original Artemis

I want essential gene and color of essential gene! uu…

Genome Designer

How about it?

WOW !!! It ‘s Good !! Wonderful !! Thank you^^

Genome Designer

• We implemented this feature using

preciously described database and GB.

• This feature is added to the Artemis

program.

• Genome Designer show essential gene

and color of essential gene.

Flow Chart(Overall)

Next GB data Block Print START

Yes END

GB data

Matching?

Essential Gene DB Represent

End of GB data?

Yes No No

Making Parts

rctB

DnaAbox

rctA PrctA PrctB 14-mer 11-mers 12-mer IHF site AT-rich parB - gfp par A Promoter BBa_J61100

dif parS

Chloramphenicol resistance

PrctB(syn)

Minimal Chromosome

• V. cholerae chromosome II

rctB parB parA parS dif

Replication system Partition system Termination system

rctA

• riVIIvc

BioBrick vector and parts

BioBrick for Replication

rctB rctA

• riVIIvc

rctA-ori BBa_K307107 rctB BBa_307105 PrctB syn BBa_307104

PrctB(syn)

E X S P

12-mer

DnaAbox

rctA PrctA PrctB 14-mer 11-mers IHF site AT-rich

E X S P

Primer Primer

rctB

E X S P

Primer Primer

rctB

DnaAbo x

rctA PrctA 14-mer 11- mers 12-mer IHF site AT-rich

PrctB(syn)

• V. cholerae chromosome II replication system

rctB

DnaAbox

rctA PrctA PrctB 14-mer 11-mers 12-mer IHF site AT-rich

• riCIIvc

In V. cholerae

rctB

DnaAbox

rctA PrctA PrctB 14-mer 11-mers 12-mer IHF site AT-rich

In E. coli

• riCIIvc

PrctB was not strong for working in E. coli

rctB

DnaAbox

rctA PrctA 14-mer 11-mers 12-mer IHF site AT-rich

PrctB ATTTTTCTTTATTTATGATCTCTTTTTCTTTATTCTCTTGGAACTATAGTGATATTACGGTAAGTGTGATACGGATC

• 35 R13 -10

RBS PrctB(syn) ATTTTTCTTGACATATACTATGATCTCTTTTTCTTTATAATCTTGGAACTATAGTGATATTACGGTAAGTGTAAGGAGGTGAGGATC

• 35 R13 -10

RBS

PrctB(Syn) Synthesis PrctB with storng promoter and strong ribosome binding site.

BioBrick for Partition

parA BBa_307103 parB BBa_307106 parS BBa_307101

parB parA parS

Promoter BBa_J61100 BBa_K125500

gfp

parB - gfp parA

X S P

Promoter BBa_J61100

E

parB

E X S P

Primer Primer

parA

E X S P

Primer Primer

parS

E X S P

Primer Primer

Partition System of a Minimal Chromosome

parS site

parB

parB - gfp

ParB-parS complex ParB-GFP fusion protein

…GTTTACAGTGTAAAT…

ParB

Chloramphenicol resistance

Rep (pMB1)

BBa_J _J04450 4450

PCR Primer

NheI

PCR Primer

NheI

E X SP

pSB1C3

BioBrick Vector

Chloramphenicol resistance

BBa_J _J04450 4450

E X SP

NheI NheI

CN

Modified Vector

Modified Vector

Future Work

Minimal Chromosome for iGEM2011