Team:TU Munich/Project/Vector Design

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Vector Design

Responsible: Ingmar Polte

What is the use of DNA sequences coding for valuable enzymes without the possibility to express them and analyze the protein activity?

As we planned to detect the enzymes from our biosynthetic pathways via Strep-tag II, a RFC25 compatible backbone was necessary. As no such backbone was availabe for yeast in the PartsRegistry, an important task at the beginning of our project was the design of an expression vector for yeast which is compatible to the iGEM cloning principles and standards. Based on the commercially available pYES2 vector, manufactured by Invitrogen, we created an vector containing:

  • a RFC 25 compatible multiple cloning site
  • a strep-tag II
  • a 2µ origin for high copy number replication in Saccharomyces cerevisae
  • the URA3 gene to use the uracil prototrophy of S. cerevisiae INVSc' as a selection marker for transfection
  • a pUC origin for high copy number replication in E.coli
  • the ß-lactamase coding gene to use ampicillin as a selection marker for cloning applications in E.coli
  • a galacotsoe inducible PGal 1 promoter
  • a CYC1 transcription terminator

Furthermore we designed several vectors containing our constitutive promoters Tef1, Tef2 and ADH and different additional terminators. The different versions of the vector have been successfully applied and tested in all other subprojects.

Design

The project work began with the replacement of the original multiple cloning side for a completely new designed multiple cloning site containing the typical RFC10/25 pre- and suffixes. Furthermore we integrated a gene sequence coding for a Strep-tag II in front of the suffix. This facilitates the purification and detection via Western Blot of expressed enzymes dramatically. The new multiple cloning side was constructed of four desoxyribooligonucleotides via oligonucleotide hybridization and ligation into the original pYES vector restricted with the outermost restriction enzymes of the old MCS.

In a further step all forbidden restriction sides of the enzymes used in the RFC10/25 standard in the vector backbone were deleted via side directed mutagenesis. The picture above shows different vector samples of this successive process which have been restricted with NgoMIV, PstI and SpeI: The resulting fragments decrease with each step leading to the pure vector backbone which is linearized by cutting in the multiple cloning side. Hence our first expression vector containing the original galactose inducible pGal 1 promoter was ready for cloning with iGEM standards!

A second step of this subproject was the exclusion of the f1 origin of replication for the phage λ and of the pGAL1 promoter. The resulting vector pTUM100 can used as powerful basis to integrate a wide variety of user defined promoters, genes and terminators. In our case we integrated the constitutive promoters Tef1, Tef2 and ADH in order to characterize them.


Results

Explanations on the figure: Figure A shows the commercial available pYES from Invitrogen on the left. The gel picture in the middle shows the successive deletion of forbidden restriction sides in the vector backbone. The colures of the annotations of the bonds in the gel picture are visualized in the graphic on the right by colored dived circles.

The picture above shows the deletion of forbidden restriction sites in the vector backbone and the introduction of the new MCS. All side directed mutagnesis experiments have been verified by analytical restriction digests and DNA sequencing.

Explanations on the figure: Figure A shows the new multiple cloning site (MCS) containing the RFC 10/25 restriction sides and the DNA sequence coding for the Strep-tag II. Figure B gives an overview of all important functional elements located on the vector backbone. Upstream to the new MCS lies a T7 promoter primer binding site allowing easy forward sequencing of integrated gene constructs using the standard T7 primer. The URA 3 gene is a prototrophy marker used for the selection of transfected cells. Figure C to E present the successfully designed BioBricks: * pTUM100 simply contains the new MCS, the transcription terminator and further elements required for cloning and transfection. * pTUM102 to pTUM104 contain in addition the constitutive promoters pTef1, pTef2 and ADH. * pTUM104 keeps the galactose inducible promoter pGAL1.

Figure B gives an opinion of all important elements located on the vector backbone:

  • The T7 promoter primer binding site allows easy forward sequencing of integrated gene constructs using the standard T7 primer.
  • The new RFC 10/25 compatible multiple cloning site enables straightforward cloning operations according to the iGEM standards.
  • The strep-tag II serves as a powerfull tool to purify and detect expressed proteins
  • The CYC1 transcription terminator is an efficient and S. cerevisiae INVSc adjusted terminator of the transcription.
  • The pUC origin permits high copy number replication in E.coli and therefore a simplified cloning.
  • The ß-lactamase coding gene allows to use ampicillin as a selection marker for cloning applications in E.coli
  • The URA3 gene renders possible to use the uracil prototrophy of the stem S. cerevisiae INVSc as a selection marker for transfection.
  • The 2µ Origin of replication offers the opportnity of high copy number replication in S. cerevisiae INVSc



BioBricks backbones

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801000 BBa_K801000] pTUM100 yeast shuttle vector without promoter

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801001 BBa_K801001] pTUM101 yeast shuttle vector with pTEF1 promoter

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801002 BBa_K801002] pTUM102 yeast shuttle vector with pTEF2 promoter

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801003 BBa_K801003] pTUM103 yeast shuttle vector with pADH1 promoter

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 BBa_K801004] pTUM104 yeast shuttle vector with pGal 1 promoter

Explanations on the figure: Figure A shows the new multiple cloning site (MCS) containing the RFC 10/25 restriction sides and the DNA sequence coding for the Strep-tag II. Figure B gives an overview of all important functional elements located on the vector backbone. Upstream to the new MCS lies a T7 promoter primer binding site allowing easy forward sequencing of integrated gene constructs using the standard T7 primer. The URA 3 gene is a prototrophy marker used for the selection of transfected cells. Figure C to E present the successfully designed BioBricks: pTUM100 simply contains the new MCS, the transcription terminator and further elements required for cloning and transfection. pTUM102 to pTUM104 contain in addition the constitutive promoters pTef1, pTef2 and ADH. On pTUM104 is the galactose inducible promoter pGAL1 located.


Gel pictures of finished constructs

Characterization

Induction assays with galactose

Characterization of constitutive promoters

Reference

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