Ligase-Independent Cloning Ligase-Independent Cloning for BioBrick - - PowerPoint PPT Presentation

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Ligase-Independent Cloning Ligase-Independent Cloning for BioBrick - - PowerPoint PPT Presentation

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Ligase-Independent Cloning Ligase-Independent Cloning for BioBrick Preparation for BioBrick Preparation Calvin Christian School Lethbridge, Alberta, Canada November 2008 Lethbridge CCS Team Lethbridge Edmonton Calgary Lethbridge, Alberta,

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Ligase-Independent Cloning Ligase-Independent Cloning for BioBrick Preparation for BioBrick Preparation

Calvin Christian School Lethbridge, Alberta, Canada November 2008

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Lethbridge CCS Team

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Lethbridge, Alberta, Canada

Calgary Edmonton

Lethbridge

(USA)

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SLIDE 4

x

Standards

BioBrick definition

  • prefix & suffix

BioBrick composition BioBrick creation

x

EcoRI PstI SpeI XbaI

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  • Proposed new standard uses ligation-independent

cloning (LIC)

  • Reference:

Aslanidis, C. & de Jong, P.J. (1990) “ Ligation- independent cloning of PCR products (LIC- PCR).” Nucl. Acids Res. 18 :20, 6069-6074.

Ligation-Independent Cloning

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Current (Soon-to-be-Old ☺) Method

PCR

Restriction site DNA Legend Restriction enzyme

  • blunt
  • sticky

Primer

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Current (Soon-to-be-Old) Method

Restriction sites: EcoRI PstI SpeI XbaI

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PCR

LIC method - insert

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LIC method - plasmid

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LIC method: T4 polymerase treatment

T4 DNA polymerase dCTP

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LIC method: T4 polymerase treatment

+

Mix

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Result of LIC procedure

  • backbone nicks repaired in vivo by
  • E. coli ligases after transformation
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Making the LIC plasmid

PCR

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Recircularized LIC plasmid

BioBrick part: K155000

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Attempted transformation using LIC method Colonies grew, but no green fluorescence observed No time to carry out further trials…

.

GFP Trial

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EcoRV digestion of pSB_LIC

1kb Ladder Sample A Sample B Sample C Sample D 1kb Ladder Cut / Uncut Cut / Uncut Cut / Uncut Cut / Uncut

1.0 kb 1.5 kb 2.0 kb 2.5 kb

pSB_LIC: 2066 bases

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Conclusion

Proposed standard method

for BioBrick preparation

(In principle) easy to implement Readily automated

pSB_LIC now available

in the Registry...

Try it out!

x

BioBrick definition

  • prefix & suffix

BioBrick composition BioBrick creation

x x

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Future Directions

Klenow fragment of DNA polymerase I also has 3’-5’

exonuclease activity, and is cheaper than T4

Optimize conditions for T4 DNA polymerase

treatment

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Acknowledgements

Thanks to...

Nathan Puhl

  • Dr. Hans-Joachim Wieden
  • Dr. Brent Selinger

University of Lethbridge iGEM team

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Questions?

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Restriction sites: EcoRI PstI SpeI XbaI Ampicillin resistance Double transcriptional terminator Origin of replication EcoRV Restriction enzyme primer Restriction site DNA sequence

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Double terminator site EcoRI SpeI XbaI PstI Ampicillin resistance Origin of replication

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We are then our religating our plasmid. We’ll

transform the Lic vector, grow it up, to ensure that it religated properly. We’ll transform the plasmid into ecoli and grow the

  • cells. The GFP will then become visible.
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SLIDE 27

Application

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Proof of Principle

The whole process of designing it, how we did it First considered using T4 polymerase , now using

Klenow fragment of DNA Pol I, cheaper with the same effect (200x less effective as 3’-5’exonuclease)

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Paul Vanden Broek

  • NorQuest college
  • Work in care home
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Elizabeth Van Essen

  • U of L student- General Science/ Pre-Ed
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Team Members

Elizabeth Van Essen Peter Van Herk Paul Vanden Broek

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Marc Slingerland Glenda Bron

  • Dr. HJ Wieden

Nathan Puhl

Supervisors & Sponsors

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Bio-engineering

Software Hardware Computation DNA Organism Output

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Engineering approach

BioBrick standard

  • standard format
  • standard assembly