Constitutive Promoters

Background and principles

Georg

Jie Sun et al. describes 14 constitutive promoters from Saccharomyces cerevisiae, and its reaction at different culture conditions, oxy(-)/glu(+),oxy(+)/glu(+). 2 Where described as higher strength promoters TEF1p, TPI1p, and 7 as medium strenth pr. GPM1p, GPDp (TDH3p), FBA1p, PDC1p, ENO2p, PYK1p, and TEF2p. With exception to the strongest TEF1p all exhibit nearly the same expression values with varying oxygen conditions. TEF1p was also tested for small glucose concentrations showed 100% expression levels with oxygen limitation and 70 % without oxygenlimitation after 80 hours of cell cultivation.

A.Blount et al. states that repeated use of the same biological parts is not possible since the cells recombine long stretches of homologous DNA

Siavash Partow et al. compares several constitutive Promoters during varying Glucose concentrations Media:Tum12Promoterstärken.pdf, TEF1, HXT7,PGK1 are shown to be the most suitable ones. They also show the fusion of two promoters for bidirectional control of two genes namely TEF1 and PGK1 with comparable activity in both states Media:Tum12Promoterfusion.pdf, two constructs were successfully used pSP-G1-Vector and pSP-G2.

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Media:Tum12Fusionspromoter_Vector.pdf

Elke Nevoigt et al. generated a library of TEF1 promotermutants for finetuning of Expression Media:Tum12 TEF-Promoter-mutants.pdf

Idea

General remarks and issues

TEF1p guarantues relative strong expression values under varying conditions as it is the case for brewing.

Biobricks and sequences

TEF1 sequence : from Promoter Database for Sacharomyces cerevisiae SCPD :(http://rulai.cshl.edu/cgi-bin/SCPD/getgenelist)

RAP1

         	GCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTCCCCTCTT	-301	
         	
         	TCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAA	-251
         	
         	AAAAGAGACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAAT	-201
         	
         	TTTTATCACGTTTCTTTTTCTTGAAAATTTTTTTTTTTGATTTTTTTCTC	-151
         	
         	TTTCGATGACCTCCCATTGATATTTAAGTTAATAAACGGTCTTCAATTTC	-101
         	
         	TCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCT	-51
         	
         	TGCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGTTTTAATTACAAA

TPI1 Promotorsequenz

               CTACTTATTCCCTTCGAGATTATATCTAGGAACCCATCAGGTTGGTGGAA	-401
         	                         GCR1             RAP1    
         	GATTACCCGTTCTAAGACTTTTCAGCTTCCTCTATTGATGTTACACCTGG	-351
         	RAP1           GCR1                               
         	ACACCCCTTTTCTGGCATCCAGTTTTTAATCTTCAGTGGCATGTGAGATT	-301
         	                                                  
         	CTCCGAAATTAATTAAAGCAATCACACAATTCTCTCGGATACCACCTCGG	-251
         	                                                  
         	TTGAAACTGACAGGTGGTTTGTTACGCATGCTAATGCAAAGGAGCCTATA	-201
         	                                                  
         	TACCTTTGGCTCGGCTGCTGTAACAGGGAATATAAAGGGCAGCATAATTT	-151
         	                                                  
         	AGGAGTTTAGTGAACTTGCAACATTTACTATTTTCCCTTCTTACGTAAAT	-101
         	                                                  
         	ATTTTTCTTTTTAATTCTAAATCAATCTTTTTCAATTTTTTGTTTGTATT	-51
         	                                                  
         	CTTTTCTTGCTTAAATCTATAACTACAAAAAACACATACATAAACTAAAA	-1

PGK1-Promoter

               CGATTTGGGCGCGAATCCTTTATTTTGGCTTCACCCTCATACTATTATCA	-601
         	                                       REB1    ABF1
         	GGGCCAGAAAAAGGAAGTGTTTCCCTCCTTCTTGAATTGATGTTACCCTC	-551
         	                            ABF1
         	ATAAAGCACGTGGCCTCTTATCGAGAAAGAAATTACCGTCGCTCGTGATT	-501
         	                            RAP1          GCR1
         	TGTTTGCAAAAAGAACAAAACTGAAAAAACCCAGACACGCTCGACTTCCT	-451
         	
         	GTCTTCCTATTGATTGCAGCTTCCAATTTCGTCACACAACAAGGTCCTAG	-401
         	
         	CGACGGCTCACAGGTTTTGTAACAAGCAATCGAAGGTTCTGGAATGGCGG	-351
         	
         	GAAAGGGTTTAGTACCACATGCTATGATGCCCACTGTGATCTCCAGAGCA	-301
         	
         	AAGTTCGTTCGATCGTACTGTTACTCTCTCTCTTTCAAACAGAATTGTCC	-251
         	
         	GAATCGTGTGACAACAACAGCCTGTTCTCACACACTCTTTTCTTCTAACC	-201
         	
         	AAGGGGGTGGTTTAGTTTAGTAGAACCTCGTGAAACTTACATTTACATAT	-151
         	
         	ATATAAACTTGCATAAATTGGTCAATGCAAGAAATACATATTTGGTCTTT	-101
         	
         	TCTAATTCGTAGTTTTTCAAGTTCTTAGATGCTTTCTTTTTCTCTTTTTT	-51
         	
         	ACAGATCATCAAGGAAGTAATTATCTACTTTTTACAACAAATATAAAACA	-1

For Yeast genes there is also another database calles yeast promoter atlas, the sequences however differ from the first one (http://ypa.ee.ncku.edu.tw/) Here the sequences from YPA Media:Tum12Promotersequenzen.pdf‎, Media:Tum12 Promoterliste.pdf

Vectorsystems for Yeast

A common commercial Vector system for Yeast transformation is the pESC lineage from Stratagene

a broad range of Protocols for yeast transformation and for the making of competent cells can be found on [1] from several laboratories that work with yeast.

Plasmids from Publications as well as from companies can be viewed at[2] and can also be ordered there, as far as I could see they are far less expensive than those from companies.