gTOW6000 project

This website provides information about gTOW6000 clones distributed by NBRP-yeast.

Search our gTOW6000 Datasheet (e.g. by gene name)

What is gTOW6000?†

• gTOW6000 is a plasmid collection covering the whole genes (about 5,700) in Saccharomyces cerevisiae. We constructed those plasmids to analyze the upper limit of copy number for each gene by gTOW method.
• Each gene has been cloned into the plasmid vector pTOWug2-836 and transformed into S. cerevisiae BY4741 strain for long-term storage.
• We store two clones per each gene. The gene coverage is >90% and can be browsed in gTOW6000 Datasheet.
• The insert size of each gene has been checked by PCR (NOT by restriction digest and/or sequencing).
• Reference
   

To whom wish to use our resource†

• Individual information for each gene can be found in gTOW6000 Datasheet.
• gTOW6000 is available via NBRP-yeast.
• Please mote:
  • NBRP manages each gene by the location on a plate e.g. BYgTOW00A01 means plate #0, position #A01.
  • There may be several clones lost during storage process, even though it certainly exists in the datasheet. Inquire NBRP for details.
  • There may be some genes successfully cloned after the datasheet was made (so it appears negative in the datasheet). Contact Hisao Moriya for details.

• Contact us if you would like to use primers or gene products in the gTOW6000 project (we may offer a collaborative studies).

Correspondence: Hisao Moriya, Okayama University | hisaom'at'cc.okayama-u.ac.jp (replace 'at' to @ upon e-mailing)

Schemes for constructing gTOW6000†

1. Design primers which cover promotor and terminator regions of individual gene on genome
2. Amplify target gene by PCR
3-4. Clone PCR product into plasmid pTOWug2-836 in S. cerevisiae BY4741 strain by Gap-Repair Cloning, and transform cells on 96-well plate
5. Pick up two colonies per each gene
6. Static culture cells in two different liquid media
7. Extract DNA individually from both media
8. Verify insert size by PCR
9. Glean unsuccessful genes in case the verification at step 8 fails to confirm expected size of insert; re-design primers, recover colonies and repeat steps 2-6

→ Cells containing plasmid with expected size (verified by PCR) have been considered to be gTOW6000 clones and stored.

gTOW6000 Datasheet†

Datasheet legends†

• Serial# (1-5806)：consecutive number given for ORFs upon cloning
• Locus：code corresponds to 'Systematic Name' in SGD
• Gene：name corresponds to 'Standard Name' in SGD
• Plate# (0-60)：consecutive number where the strain with gTOW plasmid containing a target gene ORF is stored
• Row：position of freeze stock (horizontal direction)
• Column：position of freeze stock (vertical direction)
• Well#：position of freeze stock (each well of a 96-well plate is numbered e.g. A1=1, H1=8; consecutively numbered through H12)
• Chr：designated number of chromosome where the ORF rides
• Primer name_Up_：name of upstream primer
• Up Primer：sequence of upstream primer
• Primer name_Down_：name of downstream primer
• Down Primer：sequence of downstream primer
• Size：expected size (in bp) of PCR product
• 1stPCR evaluation：amplification status of target gene, scored from A (good) to G (poor)
• Clone1：insert verification result of clone 1 by PCR (P: positive, N: negative)
• Clone2：insert verification result of clone 2 by PCR (P: positive, N: negative)

• gTOW6000 has been designed and developed by the following research groups;
  • Moriya Lab., Research Core for Interdisciplinary Sciences (RCIS), Okayama University
  • Division of Systems Biology, Cancer Institute
  • The Systems Biology Institute

• gTOW6000 project was supported by research grants below;
  • ERATO-SORST Kitano Symbiotic Systems Project, Japan Science and Technology Agency
  • PRESTO, Japan Science and Technology Agency

English/Japanese

Contact：
hisaom at cc.okayama-u.ac.jp