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From 2010.igem.org

5/7
Membership meeting
New participant of CBNU-KOREA team.
Introduced what is the iGEM, what we should do if we join the competition.
5/15
Concept meeting
Decided 2010 CBNU-KOREA iGEM team’s project(almost 4hr)
5/18
Researching
Finding related articles, paper and so on.
Essential gene data search
5/25
Strategy about database
Planned how to re-construct essential gene database.
5/27
Gathering
Gathering the essential genes data from DEG, NCBI.
Starting to lean about experimental technique.
6/1
Clean up the lab room. Too dirty…
Computer engineering department meeting about project
6/24
Summer iGEM Workshop Asia
7/19
Plasmid Culture
· Pick up a single colony from fresh cultured LB agar plate.
· Our established media and inoculate the cell into the 5ml of fresh LB liquid media (contains antibiotics) and our established media at 37℃ with shaking for 12hr.
· plasmid : pSB1C3, pSB1A3, pSB1T3, pSB1K3 (contain J54450)
7/20
Plasmid extract
· Uses kit and extracted plasmid. (pSB1C3, pSB1A3, pSB1T3, pSB1K3)
· Confirmed size by electrophoresis.
7/23
Plasmid Digest
· Digest of 4 plasmid.
· Used EcoRⅠ-PstⅠ restriction endonuclease.
· Confirmed size by electrophoresis.
7/24
Primer Design
· Designed Vibrio cholerae's oriC2vc, parA, parB, parS, dif gene primer using ‘Ginko primer’.
7/28
DNA Amplification
· Amplified ori, parA, parB, parS, dif gene of vibrio cholerae.
· Confirmed size by electrophoresis. And that’s correct.
Concentration
· Because concentration of parS, dif gene was low, it was hard to confirm result by electrophoresis.
· Concentrated gene sample
7/29
DNA(gene) Digest
· Digest of 4 plasmid.
· Used EcoRⅠ-PstⅠ restriction endonuclease.
· Confirmed size by electrophoresis.
7/30
Prepared Compentent cell
· E.coli DH10B, DH5α, JM109
8/2
DNA Ligation/Transformation
· Ligation inserting gene in vector
(parA+pSB1A3, parS+pSB1C3, ori+pSB1K3, dif+pSB1A3)
· Transformation (Competent cell : E.coli DH10B)
· Spreading on plate (each antibiotics contained LB agar media)
8/3
Cell Culture
· Pick up a single colony from fresh cultured LB agar plate.
· Our established media and inoculate the cell into the 5ml of fresh LB liquid media (contains antibiotics) and our established media at 37℃ with shaking for 12hr.
· vector : parA+pSB1A3, parS+pSB1C3, ori+pSB1K3, dif+pSB1A3
8/4
Vector extract
· Uses kit and extracted plasmid.
(parA+pSB1A3, parS+pSB1C3, ori+pSB1K3, dif+pSB1A3)
· Confirmed size by electrophoresis.
8/9
Plasmid Digest
· Digest of 4 vector.
· ori+pSB1K3, parA+pSB1A3, parS+pSB1C3 digests by EcoRⅠ-SpeⅠ restriction endonuclease and dif+pSB1A3 gene digested by XbaⅠ-PstⅠ restriction endonuclease.
· Confirmed size by electrophoresis.
· Came out as size of ori+pSB1K3 gene is smaller.
8/10
Sequencing Order
· Analyzed plasmid DNA extraction (cultured cell)
8/16
Primer Again design
· According to analysis result, primer of ori gene knew wrong fact.
· Designed primer of ori gene again.
· Primer of I51020 and p1003 designed.
8/20
DNA Amplification
· Amplified I51020, p1003
· Confirmed size by electrophoresis.
8/23
Plasmid Digest
· I51020 digests by EcoRⅠ-PstⅠ restriction endonuclease.
· p1003 digests by XbaⅠ-PstⅠ restriction endonuclease.
· Confirmed size by electrophoresis.
8/24
DNA Ligation/Transformation
· Ligated inserting gene in vector
(Inserted ori gene and p1003 antibiotic resistance cassette in I51020 vector, inserted parS gene and dif gene in pSB1A3 vector)
· Transformation (Competent cell : E.coli DH10B)
· Spreading on plate (each antibiotics contained LB agar media)

8/25
I51020 transformation failed
Plan to design Cn vector
Ready to P1003, ori miniprep

9/2

Primer design for Cn vector. May be it won’t work….

All parts re-transformation for selection and miniprep.

 

9/7

Cn vector primer arrived.
Ready to PSB1C3 plasmid miniprep
Ori X/S digestion.

 

9/10

Cn vector PCR, purification, ligation.
Cn vector PCR Product electroporhesis.
9/15
Cn vector transformation failed.
Cn vercor PCR condition re arrangement
PrctB promoter primer design

 

9/25
Cn vector + ori(X-S) ligation, transformation.
PrctB primer arrived , PCR

9/27
Cn vector colony appear. But not red, may be failure.
PrctB + rctB ligation.
Cn vector transformation

9/30
Cn vector colony appear again. Not red too. Ready to miniprep.
parB + GFP fusion

10/3
parA, parS, dif ligation PSB1C3 and PSB1A3
computer engineering student meeting,

 

10/8
Ori PCR and miniprep
Ori digestion EP/XP/XS

 

10/10

parS, dif parts complete.
parA re PCR.

10/15
All members of CBNU-KOREA team meeting

10/21
Parts making start.

Ori function is not checked.

10/25

Parts sending
Prepare Team wiki freeze