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 BBa_I716101 carrying P1010 insert was chosen. It is a Low Copy Ampicillin Resistance Plasmid (3165 bp) in BglBrick assembly standard. Assembly 21 was chosen due to unique restriction enzyme sites which were not present in any of our inserts' sequences. The P1010 insert (the death gene) carried by the BBa_I716101 was removed by digestion with EcoRI and XhoI restriction enzymes. The gene was then replaced by the chosen insert in a ligation process.

We have successfully ligated three components of the toggle switch "AyeSwitch" to the BBa_I716101 plasmid: Phage MS2 coat protein, Phage lambda N-peptide (and tandem) as well as B-box sequence encoding a regulatory mRNA stem loop. Parts Submitted to 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 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 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. CUP1p - [MS2-CFP])

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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