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

  • a RFC 25 compatible multiple cloning site (MCS)
  • a Strep-tag II
  • a 2µ origin for high copy number replication in Saccharomyces cerevisiae
  • 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 galactose inducible PGal1 promoter
  • a CYC1 transcription terminator

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

Design


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

Per side directed mutagenesis all forbidden restriction sides of enzymes used in RFC10/25 standard in the vector backbone were deleted. Different vector samples of this successive process are shown in the picture below. Their have been digested by NgoMIV, PstI and SpeI: The resulting fragments were decreased with each step leading to the pure vector backbone linearized by cutting in MCS. Hence our first original galactose inducible pGal1 promoter containing expression vector was ready for cloning with iGEM standards!

A second step was the exclusion of f1 origin of replication for phage λ and of pGAL1 promoter. The resulting vector pTUM100 can be used as powerful basis for integration of 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 new multiple cloning site (MCS) containing the RFC 10/25 restriction sides and the DNA sequence coding for the Strep-tag II.
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.

The figure on the right 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 powerful 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 opportunity of high copy number replication in S. cerevisiae INVSc













Based on the yeast shuttle vector without promoters called pTUM100 we created several vector versions containing constitutive promoters. This is represented in figures C to E:

  • 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.

All of these versions were submitted as Biobricks.


BioBricks backbones

BBa_K801000 pTUM100 yeast shuttle vector without promoter

BBa_K801001 pTUM101 yeast shuttle vector with pTEF1 promoter

BBa_K801002 pTUM102 yeast shuttle vector with pTEF2 promoter

BBa_K801003 pTUM103 yeast shuttle vector with pADH1 promoter

BBa_K801004 pTUM104 yeast shuttle vector with pGal 1 promoter



Induction assays with galactose

The pGAL1 promoter system is repressed in presence of glucose(West et al., 1984). Therefore maintaining cells in glucose containing medium causes the lowest basal transcription activity. Changing the carbon source to galactose de-represses the promoter so that transcription activity increases(Giniger et al., 1985). Alternatively cells can be maintained in raffinose which neither repress nor induce transcriptional activity. However the induction of transcriptional activity by galactose is accelerated by maintaining cells in raffinose. To characterize th pGAl1 promoter we cloned eGFP in a pTUM104 vector and transfected S. cerevisiae INVSc cells. A single colony of 2-3 mm diameter was used to inoculate a overnight culture in 15 ml SC-U medium containing glucose. The OD at 600 nm was measured and an appropriate volume of cells was sedimented and resuspended in galactose containing SC-U media to create an initial OD of 0.4. In the further process samples of 1 ml volume were taken hourly and the OD at 600 nm as well as the fluorescence at 488 nm were determined. The fluorescence signal was normalized by divide by the OD600 signal.

Explanations on the figure: The figures show the normalized fluorescence signal in dependence of time after induction

The figures above show that the cells need to 6-8 h to reach a stable level of promoter activity. However the obtained data may vary in dependence on the time which is necessary for a protein to fold itself correctly.











Outlook and conclusion

The data shown above as well as the numerous successful expressions of desired enzymes based on the pGAL1 promoter did prove its functionality. Nevertheless we will characterize this as well as all of the constitutive promoters via luciferase assays to consider different protein folding times. Furthermore we will design an additional vector carrying a His-tag instead of a Strep-tag II in order to extend the application range.

References


  • [West et al., 1984] West, R. W. J., Yocum, R. R., and Ptashne, M. (1984). Saccharomyces cerevisiae GAL1-GAL10 Divergent Promoter Region: Location and Function of the Upstream Activator Sequence UASG. Mol. Cell. Biol.4, 2467-2478.
  • [Giniger et al., 1985] Giniger, E., Barnum, S. M., and Ptashne, M. (1985). Specific DNA Binding of GAL4, a Positive Regulatory Protein of Yeast. Cell 40, 767-774.