Cloning of ratiometric FRET sensors using “F” series of vector backbones

Authors

Alen Piljic

Summary

A protocol for cloning of FRET reporters, using a library of backbone vectors as described in Rapid development of genetically developed FRET reporters.

Procedure

The FRET plasmid backbones described here contain different donor and acceptor fluorescent proteins and different linkers to the inserted protein.

There are two possible strategies to insert a protein into an “F” backbone:

1) If the insert does not contain internal AgeI and MluI site, then regular restriction cloning procedure can be used.

Steps:

design primers containing AgeI/MluI > PCR > restriction > ligation > transformation

Example:

FOR 5’ XXX XXX ACC GGT ATG YYY YYY YYY YYY YYY YYY YYY 3’

REV 5’ XXX XXX ACG CGT YYY YYY YYY YYY YYY YYY YYY YYY 3’

XXX XXX, overhangs for efficient cutting

ACC GGT, AgeI

ACG CGT, MluI

ATG, start codon

YYY, last codon of the inserted protein, not a stop codon!

YYY, insert sequence

2) If the insert contains internal AgeI and/or MluI sites, the following protocol can be used (restriction-independent, based on 3’>5’ exonuclease activity of T4 DNA polymerase).

Steps:

design primers that contain 5 nucleotides of the AgeI/MluI, but not the first nucleotide > PCR > treatment with T4 DNA polymerase > ligation > transformation

Example:

FOR 5’ CC GGT ATG YYY YYY YYY YYY YYY YYY YYY 3’

REV 5’ CG CGT YYY YYY YYY YYY YYY YYY YYY YYY 3’

CC GGT, AgeI

CG CGT, MluI

ATG, start codon

YYY, last codon of the inserted protein, not a stop codon!

YYY, insert sequence

Following PCR, T4 DNA polymerase can be used to create AgeI/MluI sticky ends:

10 µL PCR product (PCR product purified with QIAGEN kit and eluted in 30µL water or buffer)

2 µL T4 DNA polymerase buffer

2 µL dATP (25mM)

1 µL BSA

0.5 µL T4 DNA polymerase (NEB)

4.5 µL H2O

incubate at RT for 30’, then inactivate at 75C for 20’

Continue with ligation of inserts and transformation of bacteria using your favorite ligation/transformation protocol.

References

Rapid Development of Genetically Encoded FRET Reporters. Alen Piljić, Iñaki de Diego, Matthias Wilmanns and Carsten Schultz. ACS Chemical Biology 2011 6 (7), 685-691.

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