BioBrick construction

Introduction

For the iGEM 2010 project one of the team's aim was to contribute to the iGEM community via the testing and building of Bio-brick parts using standard plasmid parts. Here, we outline the process we used to construct Biobricks that were submitted to the Registry of Parts. The process consists of three steps: vector preparation (its purification and digestion), insert preparation (its amplification and digestion) and the final ligation step.

For the first step, plasmid pSB1C3 was chosen. It is a high copy BioBrick assembly plasmid (2072 bp) compatible with assembly standard 10.

We have successfully ligated four components of the toggle switch "AyeSwitch" to the pSB1C3 plasmid: Phage MS2 coat protein, Phage lambda N-peptide (and a tandem N-peptide variant) as well as B-box sequence encoding a regulatory mRNA stem loop. Parts Submitted to Registry of Parts

Protocol

Vector Preparation

Construction plasmid: pSB1C3 (High Copy BioBrick assembly plasmid) was provided by iGEM HQ as a PCR-amplified linear piece of DNA
1) The linear vector preparation was cut with EcoRI and PstI restriction enzymes
2) Cut vector was electrophoresed on an agarose gel to estimate size, quality and quantity (the latter in comparison to known amounts of molecular mass DNA ladders)

4) Restriction enzymes heat inactivation – 20 min at 65°C then pulse spin
5) In a normal ligation, at this point the vector would be treated with alkaline phosphatase to remove the 5’ phosphate groups and prevent self ligation. However, with linear, PCR amplified vector as the starting material this was not necessary; from the Registry of Parts;

Short single stranded DNA fragments will not ligate to 4 bp overhangs. By creating a very short overhang on a PCR of a plasmid backbone, the remnant, when cut with EcoRI and PstI is sufficiently short that it will not anneal at ligation temperature, and will therefore not ligate.

RESULT: purified plasmid backbone with EcoRI and PstI cohesive ends

Insert Preparation

Selected part of the AyeSwitch such as MS2 coat protein.
1) PCR reaction to amplify the desired fragment for BioBrick construct i.e. MS2 coat protein from CUP1p - [MS2-CFP] plasmid (template) + forward and reverse primers of MS2 coat protein
2) Gel electrophoresis to assess whether desired fragment was amplified and to determine its concentration.
3) Digestion with restriction enzymes (EcoRI and PstI) to generate sticky ends
4) Restriction enzymes heat inactivation - 20 min at 65°C then pulse spin.

RESULT: purified selected insert with EcoRI and PstI cohesive ends.

Ligation Reaction

Vector + selected insert
1) Ligation in the molar ration of 1:3 (vector : insert).
Including a number of controls: a) vector alone (control for uncut vector presence) b) vector alone + ligase (control for unsuccessful alkaline phosphatase treatment) c) insert alone (control for template presence i.e. CUP1p - [MS2-CFP])

2) The ligation mix is then transformed into E. coli competent cells and grown overnight in LB plates + Chloramphenicol. It would be expected to see E. coli growing colonies only on vector backbone + insert plates.
3) PCR of E. coli colonies to amplify chosen fragment after successful ligation.
4) Gel electrophoresis to verify the lengths of fragments after successful ligation.
5) Getting DNA sequenced – final verification.
6) BioBrick submission.

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