pPCK1

BBa_K2030001 Version 1

Component

Source:
http://parts.igem.org/Part:BBa_K2030001
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: John Hellgren
Date created: 2016-08-01 11:00:00
Date modified: 2016-10-17 01:02:59

pPCK1 S. cerevisiae promoter



Types
DnaRegion

Roles
Regulatory

promoter

Sequences BBa_K2030001_sequence (Version 1)

Description

The upstream regulatory sequence to the gene PCK1, coding for Phosphoenolpyruvate carboxykinase. This promoter is induced by glycerol, acetate and ethanol (10x) [1].

Notes

The complete sequence of Saccharomyces cerevisiae S288c chromosome XI (Genbank accession number BK006944) was used to design primers for amplification of this promoter.


FW: gccgcttctagagTCGTTCGTTGTACGTACATTTAC

RV: ttcttcctgcagcggccgctactagtaGTTGTTATTTTATTATGGAATAATTAGTTGC


The primers were designed to anneal at 63 degrees (although PCR worked better at 61), using Phusion High-Fidelity DNA Polymerase.


The uppercase nucleotides hybridizes with the template and the lowercase nucleotides contains the overhang. The overhang in the FW primer contains the last 13 bp of the prefix, which includes 6 protective bases before the XbaI site to ensure efficient cutting. The overhang in the RV primer contains the suffix with additional 6 protective bases to ensure efficient cutting with PstI.


For assembly of this biobrick, the vector pSB1C3 (BBa_J04450) was cut with XbaI and PstI and purified from gel. The PCR product of pPCK1 was cut with XbaI and PstI and spin column purified. The two pieces was ligated and chemically transformed into E. coli DH5alpha and plated on LB + chloramphenicol. False positive colonies are red. White colonies were restreaked on LB + chloramphenicol and their plasmids extracted using miniprep. The plasmids were verified by digestion with XbaI and PstI, resulting in two bands: 2044 (vector) and 797 (pPCK1). The biobrick was then sent for sequencing for further verification.

Source

Genomic DNA from Saccharomyces cerevisiae CEN.PK113-5D.

Sequence Annotation Location Component / Role(s)
pPCK1
1,770
promoter feature/promoter
igem#experience
None
 
igem#status
Planning
 
synbiohub#ownedBy
user/james
 
synbiohub#ownedBy
user/myers
 
synbiohub#topLevel
BBa_K2030001/1