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 construct Biobricks that were submitted to the Registry of Parts.

Protocol

Vector Preparation

Construction plasmid: BBa_ I716101 (Low Copy Ampicillin Resistance Plasmid) containing BBa_P1010 (ccdB cell death gene)
1) Inoculate E. coli containing the construction plasmid overnight in LB medium + Amp (100 μg / mL)
2) Plasmid purification (mini-prep). Follow “The QIAprep Spin MiniPrep” protocol.
3) Plasmid restriction digest (EcoRI and XhoI) – to separate ccdB gene and plasmid backbone. Follow the “Restriction digestion of plasmid DNA” protocol.

4) Gel electrophoresis to assess whether there is a successful cut and to determine the plasmid backbone concentration
5) Restriction enzymes heat inactivation – 20 min at 65°C then pulse spin
6) Alkaline phosphatase treatment – to remove the 5’ phosphate groups to prevent self ligation. Follow the protocol for “Antarctic Phosphatase”.
7) Alkaline phosphatase heat inactivation.
RESULT: purified plasmid backbone with EcoRI and XhoI cohesive ends, without 5’ phosphate groups

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 pRS414 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 XhoI) to generate sticky ends
4) Restriction enzymes heat inactivation - 20 min at 65°C then pulse spin.
RESULT: purified selected insert with EcoRI and XhoI 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. pRS414)

2) The ligation mix is then transformed into E. coli competent cells and grown overnight in LB plates + Amp. 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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