Construction of Plasmids In Vivo using Yeast Homologous Recombination

Introduction

For the iGEM 2010 project the yeast shuttle vectors pRS414 and pRS415 were used to express the main constructs that form the AyeSwitch. In addition to this, in vivo modifications to these plasmids were also made to allow troubleshooting and testing of Bio-Brick parts. See Results Section

To carry out these modifications, in vivo homologous recombination was used to do this as it utilised the natural processes of the yeast involved whilst simultaneously transforming it with the modified plasmid.

Protocol

1.) The shuttle vector is linearised by cutting at unique restriction sites. In the case of pRS414 and pRS415 this can be done by checking its DNA sequence and the construct it carries to ensure there are no identical restriction sites.

2.) To check the desired cut has been obtained, the restriction cut products can be run on a gel to detect for the correct bands.

3.) In parallel, Primers are designed for the inserts that have to be homologously recombined. The essential feature is that the primers must also include 45bp from the end of the restriction cut site of the shuttle vector as part of the primer sequence. This applies for both forward and reverse primers and is essential to allow homologous recombination of insert into shuttle vector.

4.) The primers are then used to PCR amplify the desired insert. Again, gel electrophoresis may be used to check the correct length of PCR product has been obtained.

5.) A transformation is carried out using yeast, the cut shuttle vector and PCR amplified products from previously. Transformed yeast is selectively cultured with suitable controls.

6.) To check that the appropriate modified plasmids have been transformed, the yeast can screened by using Colony PCR Screening.

7.) Colony PCR Screening involved taking a smallest amount of yeast from an individual colony, digesting its cell wall to release its DNA content. PCR primers for the homologous insert used previously are then used to see if any PCR amplification occurs.

The presence of the desired PCR product indicates that the desired modified shuttle vector has been made and transformed.

References

Hinnen A, Hicks JB and Fink GR. Transformation of yeast. Proc Natl Acad Sci, 75 1978 (1929-33)