Team:TU Munich/Project/Light Switchable Promoter

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A light-switchable promoter system for switching on/off gene expression or switching gene-expression of product A to product B and back.

Contents

Background and principles

This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of the transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes.

This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (Pif3), which only interacts together when the PhyB is in it's active conformer (Pfr-form). PhyB, a phytochrome containing a essential chromophore phycocyanobilin B (PCB), is naturally synthesized in it's inactive form (Pr; r stand for red-light sensitive form). So it cannot bound to Pif3 once synthesized. PhyB first has to be activated by red light (λ = 660 nm) which causes a Z-E conformer isomerization to the active form Pfr (fr stands for far-red light senstive form). Relaxation (E-Z conformation change) is induced by far-red light (λ = 750 nm); the high energy form Pfr has a half-life of 30–60 min resulting that after this time half of the Pfr species is spontanously relaxed into the low-energy form Pr. This allows us to generate time-stable light-switchable promoter systems. It is also possible not only have a discret genetic switch. By varying freqrency of red/far-red light pulses one should be able to control the strength of gene-expression or the strength-ratio between two expressed genes.

Principle of light-dependent switching of gene-expression.
Modification of the original system by replacing the GAL4-BD with the LexA-BD and the LexA-recognition site.

Idea

The idea behind the light-switchable system is to create a master-switch in a gene-expression network. The light-switchable promoter-system only controls the biosynthesis of trancription factors (e. g. GAL4-TF) and repressors (e. g. LacI-mutants and TrpR-mutants). These trancription factors and repressors subsequently controls the gene-expression of the end-products.

The first idea behind a synthetical light-switchable regulon system.
The second idea synthetical light-switchable regulon system.
Idea

General remarks and issues

  • A yeast strain with GAL4/GAL80 deletion is required to avoid interference by endogenous GAL4 and GAL80 proteins. Disadvantage of GAL4/GAL80 deletion is that the cells are growing more slowly compared to strains with the wildtype alleles of these genes. Use of prokaryotic LexA instead of the DNA-binding domain of GAL4 avoids that. In this case the UAS (upstream activation sequence) of GAL1 has to be replaced by a prokaryotic upstream LexA operator.
  • I am not sure about the right sequence sequence for the GAL1/UAS or the LexA operator. Someone should help me =).
  • For a perfect light-switchable system I need an idea for inactivating/degrading LacI fastly because the half-life of LacI is about 10 min. Is it fast enough?
  • Attention: There are some problems for the parts from the registry involved in this system. This was one of the main reasons our project from last year didnt play ou that well. We need to double-check wether the sequences and the respective submitted DNA are correct. (Fabian)
    • I don't geht the point. Can you please refer to what you mean exactly? As I see, the only similarity is the synthesis of the chromophor. And you used a operon system from the registry for this which can't be used in a yeast system anyway.
    • I checked one of the parts and saw that it was a part from UTAustin 2004 and I knew Edinburgh 2010 needed to fix some of their parts so I figured there might be other problems with the parts from UTAustin 2004. But as far as I can tell Edinburgh recovered cph8 by PCR from a composite part but then messed things up later on. But since the UTAustin 2004 project worked and BBa_I5008 has the correct sequence, everything should be fine. (Fabian)
  • Check for avaible Nuclear Localization Signals (NLS) for nuclear localized proteins!
  • Better we synthesize the DNA of some promoters including UAS and operators.
  • The first ~650 N-terminal amino acids are only needed for a functional PhyB.
  • The first ~100 N-terminal amino acids are only needed for a fucntional Pif3.
  • I asked Roman, if he can get all the elements for Yeast-two-hybrid-system including the GAL4/GAL80 deletion strain from Professor Schwab. May be he also has the modifed Yeast-two-hybrid-sytem with LexA.

Biobricks and sequences

Synthesis of chromophore phycocyanobilin B (PCB)

As described phycocyanobilin is a essential chromophore for a functional phytochrome B. PCB can be synthesized in two steps from heme. First, heme is converted to biliverdin IXα (BV) and second, BV is converted to 3Z-phycocyanobilin (PCB).

Biosynthesis of phycocyanobilin and phytochromobilin. Heme oxygenase (HO1) catalyzes the conversion of heme to biliverdin IXα (BV). Subsequently, BV is reduced by phycocyanobilin/ferredoxin oxidoreductase (PcyA) in cyanobacteria or PΦB synthase (HY2) in plants to produce PCB or PΦB, respectively. ApoCph1 is capable of autocatalytically binding either of these chromophores to form a holoCph1 protein in the red-light-absorbing Pr form.[http://www.pnas.org/content/98/19/10566/F1.expansion.html]

Converting heme to biliverdin IXα

HO1[http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008] from Synechocystis oxidizes the heme group using a ferredoxin cofactor, generating biliverdin IXα. 

       1 atgagtgtca acttagcttc ccagttgcgg gaagggacga aaaaatccca ctccatggcg
      61 gagaacgtcg gctttgtcaa atgcttcctc aagggcgttg tcgagaaaaa ttcctaccgt
     121 aagctggttg gcaatctcta ctttgtctac agtgccatgg aagaggaaat ggcaaaattt
     181 aaggaccatc ccatcctcag ccacatttac ttccccgaac tcaaccgcaa acaaagccta
     241 gagcaagacc tgcaattcta ttacggctcc aactggcggc aagaagtgaa aatttctgcc
     301 gctggccaag cctatgtgga ccgagtccgg caagtggccg ctacggcccc tgaattgttg
     361 gtggcccatt cctacacccg ttacctgggg gatctttccg gcggtcaaat tctcaagaaa
     421 attgcccaaa atgccatgaa tctccacgat ggtggcacag ctttctatga atttgccgac
     481 attgatgacg aaaaggcttt taaaaatacc taccgtcaag ctatgaatga tctgcccatt
     541 gaccaagcca ccgccgaacg gattgtggat gaagccaatg acgcctttgc catgaacatg
     601 aaaatgttca acgaacttga aggcaacctg atcaaggcga tcggcattat ggtgttcaac
     661 agcctcaccc gtcgccgcag tcaaggcagc accgaagttg gcctcgccac ctccgaaggc
     721 taataa

Name Length RFC10 RFC25 Codon Usage NCBI
Heme Oxygenase 1 726bp ok ok 1AS<10% [http://www.ncbi.nlm.nih.gov/nuccore/4105612 AF048758.1] 
Available in Registry: BBa_I15008


HMX1[http://www.yeastgenome.org/cgi-bin/locus.fpl?dbid=S000004195], an endogenous ER localized heme oxygenase, is only expressed under iron starvation and oxidative stress; relocates to the perinuclear region in the presence of oxidants. Forbidded restriction sites: EcoRI (1x) Two possibile solution for introducing silent mutations:

  • Mutation of GAA (codon usage: 45.6%) to GAG (codon usage: 19.2%), both coding synonymously for gluatmic acid.
  • Mutation of TTC (codon usage: 28.4%) to TTT (codon usage: 26.1%), both conding synonymousy for phenylalanin.

Latter one should be performed as a result of condon usage!

Coding sequence: 

>YLR205C  (954 bp)
       1 atggaggaca gtagcaatac aatcataccc tcacccactg acgtgggggc gctagcaaac
      61 agaatcaact ttcaaaccag agatgcccac aataaaatca ataccttcat gggcataaag
     121 atggccatcg ccatgagaca tggctttata tacagacagg gtattctggc gtactattat
     181 gtgttcgatg ccatcgagca agagatagat cgcctactga atgaccccgt aacggaggag
     241 gagctgcaaa cttcgaccat tctgaagcag ttttggctcg aagattttag aagatctacg
     301 cagatctata aggacctgaa gctgctatac tcaaacacgt ttaaaagcac agaatcatta
     361 aacgaattcc tggctacgtt ccagaagcca ccgctactac agcagtttat caataacatc
     421 cacgaaaaca tacacaagga gccatgcacc attctttctt actgtcacgt tctgtacttg
     481 gcgcttttcg ccggcggcaa gctaatacga tcgaatttgt acagaagact ggggctcttc
     541 cccaacttcg agaagctatc acagaaggaa ctggtcaaaa agggcacaaa cttcttcacc
     601 ttcagcgatc tgggtcccac tgaagaaaca cgcttgaaat gggaatacaa gaagaactat
     661 gagctggcca ccaggacgga attgaccgaa gcacaaaagt tgcagatcat tagcgtcgca
     721 gaaggcattt ttgattggaa cttcaacatc gttgcagaaa ttggagagtt gaatcgtcgc
     781 gagttaatgg gcaagttcag cttcaagtgt attacgtact tgtacgaaga atggatgttc
     841 aacaaggatt ctgctactag aagagcactc cacacggtca tgctgctggt gctttctatt
     901 atcgcgatct gggttcttta cttcttggta aagagttttc ttagcatagt ataa

Name Length RFC10 RFC25 Codon Usage NCBI
Heme-binding protein HMX1 954 bp 1x EcoRI (364-370) 1x NgoMIV (490-496) 0AS<10% [http://www.ncbi.nlm.nih.gov/nuccore/296146742 NM_001182092.1]

Converting biliverdin IXα to phycocyanobilin B

Codon optimized PcyA[http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 ] (derived from cyanobacteria) which converts biliverdin IXα (BV) into 3Z-phycocyanobilin B(PCB). 

>BBa_K181000 Part-only sequence (750 bp)
atggccgttaccgatttgagtttgaccaattcctccttgatgccaaccttaaaccctatgattcaacaattggctttggctattgctgcttcctggcaat
ctttgcctttgaaaccatatcaattgcctgaagatttgggttatgtcgaaggtagattagaaggtgaaaaattggttatcgaaaacagatgctatcaaac
cccacaattcagaaaaatgcacttggaattggctaaagtcggtaaaggtttagacatcttacactgtgtcatgttccctgaaccattgtatggtttacca
ttattcggttgtgacatcgttgctggtcctggtggtgtctctgctgccattgccgatttgtctccaacacaatccgatagacaattgcctgctgcctatc
aaaaatccttggccgaattgggtcaaccagaatttgaacaacaaagagaattgcctccttggggtgaaattttctccgaatattgtttgttcattagacc
atccaacgtcaccgaagaagaaagattcgtccaaagagttgtcgacttcttacaaatccactgccaccaatccatcgtagccgaaccattatccgaagct
caaacattggaacacagacaaggtcaaatccattattgccaacaacaacaaaaaaacgacaagactagaagagttttggaaaaggctttcggtgaagctt
gggccgaaagatatatgtcccaagttttattcgacgtcattcaatgatga

Name Length RFC10 RFC25 Codon Usage NCBI
Ferredoxin-dependent bilin reductase 750 bp ok ok 0AS<10% [http://www.ncbi.nlm.nih.gov/protein/ABW30269.1 ABW30269.1] 
Unverified in Registry

HO-pcyA operon (prokaryotic organisms only?!)

In the registry there is a biobrick BBa_K098010 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098010|BBa_K098010] containing a operon which encodes for two enzymes for converting heme to PCB. The problem is that only few eukaryotic systems are capable to handle operon systems, e. g. nematodes, but not yeast. I don't know if there is any chance to express a operon system in yeast (We have to ask a expert for yeast genetics! 

>BBa_K098010 Part-only sequence (1531 bp)
ctgatggctagctcagtcctagggattatgctagctactagagtcacacaggaaaggtgcacatggaagaggaaatggcaaaatttaaggaccatcccat
cctcagccacatttacttccccgaactcaaccgcaaacaaagcctagagcaagacctgcaattctattacggctccaactggcggcaagaagtgaaaatt
tctgccgctggccaagcctatgtggaccgagtccggcaagtggccgctacggcccctgaattgttggtggcccattcctacacccgttacctgggggatc
tttccggcggtcaaattctcaagaaaattgcccaaaatgccatgaatctccacgatggtggcacagctttctatgaatttgccgacattgatgacgaaaa
ggcttttaaaaatacctaccgtcaagctatgaatgatctgcccattgaccaagccaccgccgaacggattgtggatgaagccaatgacgcctttgccatg
aacatgaaaatgttcaacgaacttgaaggcaacctgatcaaggcgatcggcattatggtgttcaacagcctcacccgtcgccgcagtcaaggcagcaccg
aagttggcctcgccacctccgaaggctagttaaagaggagaaaggatccatggccgtcactgatttaagtttgaccaattcttccctgatgcctacgttg
aacccgatgattcaacagttggccctggcgatcgccgctagttggcaaagtttacccctcaagccctatcaattgccggaggatttgggctacgtagaag
gccgcctggaaggggaaaagttagtgattgaaaatcggtgctaccaaacgccccagtttcgcaaaatgcatttggagttggccaaggtgggcaaagggtt
ggatattctccactgtgtaatgtttcctgagcctttatacggtctacctttgtttggctgtgacattgtggccggccccggtggagtaagtgcggctatt
gcggatctatcccccacccaaagcgatcgccaattgcccgcagcgtaccaaaaatcattggcagagctaggccagccagaatttgagcaacaacgggaat
tgcccccctggggagaaatattttctgaatattgtttattcatccgtcccagcaatgtcactgaagaagaaagatttgtacaaagggtagtggacttttt
gcaaattcattgtcaccaatccatcgttgccgaacccttgtctgaagctcaaactttggagcaccgtcaggggcaaattcattactgccaacaacaacag
aaaaatgataaaacccgtcgggtactggaaaaagcttttggggaagcttgggcggaacggtatatgagccaagtcttatttgatgttatccaataaggta
ccccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggc
tcaccttcgggtgggcctttctgcgtttata


 Bad Part in Registry

PhyB-GAL4BD/PhyB-LexA

PhyB-GAL4BD

The chimeric PhyB-GAL4BD[http://partsregistry.org/Part:BBa_K207001] (nuclear localization signal included) contains the light-dependant PhyB part, performing a (far-)red-light induced (Z/E)E/Z-isomerization, and the DNA-binding domain of the transcriptionfactor GAL4. Active conformer of PhyB binds to Pif3 which is fused to the transcription activation domain of GAL4. As a result transcription is started by red-light and stopped by far-red light. 

>BBa_K207001 Part-only sequence (2303 bp)
atggtttccggagtcgggggtagtggcggtggccgtggcggtggccgtggcggagaagaagaaccgtcgtcaagtcacactcctaataaccgaagaggag
gagaacaagctcaatcgtcgggaacgaaatctctcagaccaagaagcaacactgaatcaatgagcaaagcaattcaacagtacaccgtcgacgcaagact
ccacgccgttttcgaacaatccggcgaatcagggaaatcattcgactactcacaatcactcaaaacgacgacgtacggttcctctgtacctgagcaacag
atcacagcttatctctctcgaatccagcgaggtggttacattcagcctttcggatgtatgatcgccgtcgatgaatccagtttccggatcatcggttaca
gtgaaaacgccagagaaatgttagggattatgcctcaatctgttcctactcttgagaaacctgagattctagctatgggaactgatgtgagatctttgtt
cacttcttcgagctcgattctactcgagcgtgctttcgttgctcgagagattaccttgttaaatccggtttggatccattccaagaatactggtaaaccg
ttttacgccattcttcataggattgatgttggtgttgttattgatttagagccagctagaactgaagatcctgcgctttctattgctggtgctgttcaat
cgcagaaactcgcggttcgtgcgatttctcagttacaggctcttcctggtggagatattaagcttttgtgtgacactgtcgtggaaagtgtgagggactt
gactggttatgatcgtgttatggtttataagtttcatgaagatgagcatggagaagttgtagctgagagtaaacgagacgatttagagccttatattgga
ctgcattatcctgctactgatattcctcaagcgtcaaggttcttgtttaagcagaaccgtgtccgaatgatagtagattgcaatgccacacctgttcttg
tggtccaggacgataggctaactcagtctatgtgcttggttggttctactcttagggctcctcatggttgtcactctcagtatatggctaacatgggatc
tattgcgtctttagcaatggcggttataatcaatggaaatgaagatgatgggagcaatgtagctagtggaagaagctcgatgaggctttggggtttggtt
gtttgccatcacacttcttctcgctgcataccgtttccgctaaggtatgcttgtgagtttttgatgcaggctttcggtttacagttaaacatggaattgc
agttagctttgcaaatgtcagagaaacgcgttttgagaacgcagacactgttatgtgatatgcttctgcgtgactcgcctgctggaattgttacacagag
tcccagtatcatggacttagtgaaatgtgacggtgcagcatttctttaccacgggaagtattacccgttgggtgttgctcctagtgaagttcagataaaa
gatgttgtggagtggttgcttgcgaatcatgcggattcaaccggattaagcactgatagtttaggcgatgcggggtatcccggtgcagctgcgttagggg
atgctgtgtgcggtatggcagttgcatatatcacaaaaagagactttcttttttggtttcgatctcacactgcgaaagaaatcaaatggggaggcgctaa
gcatcatccggaggataaagatgatgggcaacgaatgcatcctcgttcgtcctttcaggcttttcttgaagttgttaagagccggagtcagccatgggaa
actgcggaaatggatgcgattcactcgctccagcttattctgagagactcttttaaagaatct

end of PhyB match, begin of GAL4 match 

                                                               atgaagctactgtcttctatcgaacaagcatgcgata
tttgccgacttaaaaagctcaagtgctccaaagaaaaaccgaagtgcgccaagtgtctgaagaacaactgggagtgtcgctactctcccaaaaccaaaag
gtctccgctgactagggcacatctgacagaagtggaatcaaggctagaaagactggaacagctatttctactgatttttcctcgagaagaccttgacatg
attttgaaaatggattctttacaggatataaaagcattgttaacaggattatttgtacaagataatgtgaataaagatgccgtcacagatagattggctt
cagtggagactgatatgcctctaacattgagacagcatagaataagtgcgacatcatcatcggaagagagtagtaacaaaggtcaaagacagttgactgt
atc

Name Length RFC10 RFC25 Codon Usage NCBI
PhyB-DBD Fusion 2303bp ok ok 2AS < 10 % PhyB:[http://www.ncbi.nlm.nih.gov/nuccore/X17342.1 X17342.1] (partial match) GAL4:[http://www.ncbi.nlm.nih.gov/nuccore/NM_001184062.1 NM_001184062.1] (partial match) 
 Unverified in Registry, '''NOT DIVISIBLE BY 3'''

PhyB

It seems that only the ~650 N-terminal amino acids are essential for the function of the light-sensitive phytochrome B.

PhyB (First 908 N-terminal residues)[http://partsregistry.org/Part:BBa_K365002] 

>BBa_K365002 Part-only sequence (2724 bp)
atggtttccggagtcgggggtagtggcggtggccgtggcggtggccgtggcggagaagaagaaccgtcgtcaagtcacactcctaataaccgaagaggag
gagaacaagctcaatcgtcgggaacgaaatctctcagaccaagaagcaacactgaatcaatgagcaaagcaattcaacagtacaccgtcgacgcaagact
ccacgccgttttcgaacaatccggcgaatcagggaaatcattcgactactcacaatcactcaaaacgacgacgtacggttcctctgtacctgagcaacag
atcacagcttatctctctcgaatccagcgaggtggttacattcagcctttcggatgtatgatcgccgtcgatgaatccagtttccggatcatcggttaca
gtgaaaacgccagagaaatgttagggattatgcctcaatctgttcctactcttgagaaacctgagattctagctatgggaactgatgtgagatctttgtt
cacttcttcgagctcgattctactcgagcgtgctttcgttgctcgagagattaccttgttaaatccggtttggatccattccaagaatactggtaaaccg
ttttacgccattcttcataggattgatgttggtgttgttattgatttagagccagctagaactgaagatcctgcgctttctattgctggtgctgttcaat
cgcagaaactcgcggttcgtgcgatttctcagttacaggctcttcctggtggagatattaagcttttgtgtgacactgtcgtggaaagtgtgagggactt
gactggttatgatcgtgttatggtttataagtttcatgaagatgagcatggagaagttgtagctgagagtaaacgagacgatttagagccttatattgga
ctgcattatcctgctactgatattcctcaagcgtcaaggttcttgtttaagcagaaccgtgtccgaatgatagtagattgcaatgccacacctgttcttg
tggtccaggacgataggctaactcagtctatgtgcttggttggttctactcttagggctcctcatggttgtcactctcagtatatggctaacatgggatc
tattgcgtctttagcaatggcggttataatcaatggaaatgaagatgatgggagcaatgtagctagtggaagaagctcgatgaggctttggggtttggtt
gtttgccatcacacttcttctcgctgcataccgtttccgctaaggtatgcttgtgagtttttgatgcaggctttcggtttacagttaaacatggaattgc
agttagctttgcaaatgtcagagaaacgcgttttgagaacgcagacactgttatgtgatatgcttctgcgtgactcgcctgctggaattgttacacagag
tcccagtatcatggacttagtgaaatgtgacggtgcagcatttctttaccacgggaagtattacccgttgggtgttgctcctagtgaagttcagataaaa
gatgttgtggagtggttgcttgcgaatcatgcggattcaaccggattaagcactgatagtttaggcgatgcggggtatcccggtgcagctgcgttagggg
atgctgtgtgcggtatggcagttgcatatatcacaaaaagagactttcttttttggtttcgatctcacactgcgaaagaaatcaaatggggaggcgctaa
gcatcatccggaggataaagatgatgggcaacgaatgcatcctcgttcgtcctttcaggcttttcttgaagttgttaagagccggagtcagccatgggaa
actgcggaaatggatgcgattcactcgctccagcttattctgagagactcttttaaagaatctgaggcggctatgaactctaaagttgtggatggtgtgg
ttcagccatgtagggatatggcgggggaacaggggattgatgagttaggtgcagttgcaagagagatggttaggctcattgagactgcaactgttcctat
attcgctgtggatgccggaggctgcatcaatggatggaacgctaagattgcagagttgacaggtctctcagttgaagaagctatggggaagtctctggtt
tctgatttaatatacaaagagaatgaagcaactgtcaataagcttctttctcgtgctttgagaggggacgaggaaaagaatgtggaggttaagctgaaaa
ctttcagccccgaactacaagggaaagcagtttttgtggttgtgaatgcttgttccagcaaggactacttgaacaacattgtcggcgtttgttttgttgg
acaagacgttacgagtcagaaaatcgtaatggataagttcatcaacatacaaggagattacaaggctattgtacatagcccaaaccctctaatcccgcca
atttttgctgctgacgagaacacgtgctgcctggaatggaacatggcgatggaaaagcttacgggttggtctcgcagtgaagtgattgggaaaatgattg
tcggggaagtgtttgggagctgttgcatgctaaagggtcctgatgctttaaccaagttcatgattgtattgcataatgcgattggtggccaagatacgga
taagttccctttcccattctttgaccgcaatgggaagtttgttcaggctctattgactgcaaacaagcgggttagcctcgagggaaaggttattggggct
ttctgtttcttgcaaatcccgagc

Name Length RFC10 RFC25 Codon Usage NCBI
PhyB part 2724bp ok ok 3AS<10% [http://www.ncbi.nlm.nih.gov/nuccore/X17342.1 X17342.1] (partial match)

PhyB (First 642 N-terminal residues)[http://partsregistry.org/Part:BBa_K365003] 

>BBa_K365003 Part-only sequence (1926 bp)
atggtttccggagtcgggggtagtggcggtggccgtggcggtggccgtggcggagaagaagaaccgtcgtcaagtcacactcctaataaccgaagaggag
gagaacaagctcaatcgtcgggaacgaaatctctcagaccaagaagcaacactgaatcaatgagcaaagcaattcaacagtacaccgtcgacgcaagact
ccacgccgttttcgaacaatccggcgaatcagggaaatcattcgactactcacaatcactcaaaacgacgacgtacggttcctctgtacctgagcaacag
atcacagcttatctctctcgaatccagcgaggtggttacattcagcctttcggatgtatgatcgccgtcgatgaatccagtttccggatcatcggttaca
gtgaaaacgccagagaaatgttagggattatgcctcaatctgttcctactcttgagaaacctgagattctagctatgggaactgatgtgagatctttgtt
cacttcttcgagctcgattctactcgagcgtgctttcgttgctcgagagattaccttgttaaatccggtttggatccattccaagaatactggtaaaccg
ttttacgccattcttcataggattgatgttggtgttgttattgatttagagccagctagaactgaagatcctgcgctttctattgctggtgctgttcaat
cgcagaaactcgcggttcgtgcgatttctcagttacaggctcttcctggtggagatattaagcttttgtgtgacactgtcgtggaaagtgtgagggactt
gactggttatgatcgtgttatggtttataagtttcatgaagatgagcatggagaagttgtagctgagagtaaacgagatgatttagagccttatattgga
ctgcattatcctgctactgatattcctcaagcgtcaaggttcttgtttaagcagaaccgtgtccgaatgatagtagattgcaatgccacacctgttcttg
tggtccaggacgataggctaactcagtctatgtgcttggttggttctactcttagggctcctcatggttgtcactctcagtatatggctaacatgggatc
tattgcgtctttagcaatggcggttataatcaatggaaatgaagatgatgggagcaatgtagctagtggaagaagctcgatgaggctttggggtttggtt
gtttgccatcacacttcttctcgctgcataccgtttccgctaaggtatgcttgtgagtttttgatgcaggctttcggtttacagttaaacatggaattgc
agttagctttgcaaatgtcagagaaacgcgttttgagaacgcagacactgttatgtgatatgcttctgcgtgactcgcctgctggaattgttacacagag
tcccagtatcatggacttagtgaaatgtgacggtgcagcatttctttaccacgggaagtattacccgttgggtgttgctcctagtgaagttcagataaaa
gatgttgtggagtggttgcttgcgaatcatgcggattcaaccggattaagcactgatagtttaggcgatgcggggtatcccggtgcagctgcgttagggg
atgctgtgtgcggtatggcagttgcatatatcacaaaaagagactttcttttttggtttcgatctcacactgcgaaagaaatcaaatggggaggcgctaa
gcatcatccggaggataaagatgatgggcaacgaatgcatcctcgttcgtcctttcaggcttttcttgaagttgttaagagccggagtcagccatgggaa
actgcggaaatggatgcgattcactcgctccagcttattctgagagactcttttaaagaatctgaggcggctatgaactctaaagttgtggatggtgtgg
ttcagccatgtagggatatggcgggg

Name Length RFC10 RFC25 Codon Usage NCBI
PhyB essential 1926bp ok ok 2AS<10% [http://www.ncbi.nlm.nih.gov/nuccore/X17342.1 X17342.1] (partial match)

GAL4BD

The DNA-binding domain of GAL4 (GAL4BD)[http://partsregistry.org/Part:BBa_J176020][http://partsregistry.org/Part:BBa_K364319] recognizes a special upstream activating sequence (UAS). By fusing this protein to Pif3, GAL4BD can be indirectly recruited by PhyB-GAL4BD after red light exposition. 

>BBa_J176020 Part-only sequence (459 bp)
atgaagctactgtcttctatcgaacaagcatgcgatatttgccgacttaaaaagctcaagtgctccaaagaaaaaccgaagtgcgccaagtgtctgaaga
acaactgggagtgtcgctactctcccaaaaccaaaaggtctccgctgactagggcacatctgacagaagtggaatcaaggctagaaagactggaacagct
atttctactgatttttcctcgagaagaccttgacatgattttgaaaatggattctttacaggatataaaagcattgttaacaggattatttgtacaagat
aatgtgaataaagatgccgtcacagatagattggcttcagtggagactgatatgcctctaacattgagacagcatagaataagtgcgacatcatcatcgg
aagagagtagtaacaaaggtcaaagacagttgactgtatcgccggaatttccggggatc

Name Length RFC10 RFC25 Codon Usage NCBI
Binding-domain of DNA-binding transcription factor GAL4 459bp ok ok 0 AS < 10% [http://www.ncbi.nlm.nih.gov/nuccore/NM_001184062.1 NM_001184062.1] (partial match) 
 Unavailable in Registry 


>BBa_K364319 Part-only sequence (441 bp)
gccaagctactgtcttctatcgaacaagcatgcgatatttgccgacttaaaaagctcaagtgctccaaagaaaaaccgaagtgcgccaagtgtctgaaga
acaactgggagtgtcgctactctcccaaaaccaaaaggtctccgctgactagggcacatctgacagaagtggaatcaaggctagaaagactggaacagct
atttctactgatttttcctcgagaagaccttgacatgattttgaaaatggattctttacaggatataaaagcattgttaacaggattatttgtacaagat
aatgtgaataaagatgccgtcacagatagattggcttcagtggagactgatatgcctctaacattgagacagcatagaataagtgcgacatcatcatcgg
aagagagtagtaacaaaggtcaaagacagttgactgtatcg

Name Length RFC10 RFC25 Codon Usage NCBI
GAL4??? 441bp ok ok 0 AS < 10% [http://www.ncbi.nlm.nih.gov/nuccore/NM_001184062.1 NM_001184062.1] (partial match) 
 Unverified in Registry

This is majorly confusing ... the difference between the sequences is that BBa_K364319 starts with gcc instead of atg and is shorter. But the one description states its the UAS (=Promoter?) and the other states its the Transcription factor. Is this correct? (User:Fabian)

LexA

LexA[http://partsregistry.org/Part:BBa_K105005] is a prokaryotic transcription activator which binds a specific lexA recognition site. LexA can be used in fusion proteins as a DNA-binding domain in eukaryotic systems due to the natural lack of prokaryotic transcription factors. So no interferences with other expression systems are expected. 

>BBa_K105005 Part-only sequence (603 bp)
aaagcgttaacggccaggcaacaagaggtgtttgatctcatccgtgatcacatcagccagacaggtatgccgccgacgcgtgcggaaatcgcgcagcgtt
tggggttccgttccccaaacgcggctgaagaacatctgaaggcgctggcacgcaaaggcgttattgaaattgtttccggcgcatcacgcgggattcgtct
gttgcaggaagaggaagaagggttgccgctggtaggtcgtgtggctgccggtgaaccacttctggcgcaacagcatattgaaggtcattatcaggtcgat
ccttccttattcaagccgaatgctgatttcctgctgcgcgtcagcgggatgtcgatgaaagatatcggcattatggatggtgacttgctggcagtgcata
aaactcaggatgtacgtaacggtcaggtcgttgtcgcacgtattgatgacgaagttaccgttaagcgcctgaaaaaacagggcaataaagtcgaactgtt
gccagaaaatagcgagtttaaaccaattgtcgttgaccttcgtcagcagagcttcaccattgaagggctggcggttggggttattcgcaacggcgactgg
ctg

Name Length RFC10 RFC25 Codon Usage NCBI
LexA Transcription Factor 603bp ok ok 0 AS < 10% [http://www.ncbi.nlm.nih.gov/nucleotide/J01643 J01643] (partial match) 
 Inconsistent in Registry

Pif3-GAL4AD

Pif3

The phytochrome interacting factor 3 (Pif3)[http://partsregistry.org/Part:BBa_K365000] which interacts with the active form of phytochrome B (PhyB). Only the first 100 N-terminal residues seem to be bound by Pfr form of PhyB. 

>BBa_K365000 Part-only sequence (300 bp)
atgcctctgtttgaacttttcaggctcaccaaagctaagcttgaatctgctcaagacaggaacccttctccacctgtagatgaagttgtggagctggtgt
gggaaaatggtcagatatcaactcaaagtcagtcaagtagatcgaggaacattcctccaccacaagcaaactcttcaagagctagagagattggaaatgg
ctcaaagacgactatggtggacgagatccctatgtcagtgccatcactaatgacgggtttgagtcaagacgatgactttgttccatggttgaatcatcat

Name Length RFC10 RFC25 Codon Usage NCBI
Pif3 Transcription Factor 300bp ok ok 0 AS < 10% [http://www.ncbi.nlm.nih.gov/nucleotide/NM_179295.2 NM_179295.2] (partial match) 


 Unverified in Registry

GAL4AD

Still missing, but we receive this part from Professor Schwab (please see discussion site).

Inverters

An inverter is logic gate which implements logical negation. Typical genetic inverter systems are repressors and the genes which are dependant from the repressor, thus if the repressor is active (input = 1) the gene-expression is off (output = 0) and vice versa.

LacI IS repressor

An inverter could be used in a light-dependant system to negotiate gene-expression of one or more genes. LacI IS (IPTG unresponsive mutant to prevent interference with IPTG inducible systems) [http://partsregistry.org/Part:BBa_K142007][http://partsregistry.org/Part:BBa_K142006][http://partsregistry.org/Part:BBa_K142005][http://partsregistry.org/Part:BBa_K142004][http://partsregistry.org/Part:BBa_K142003][http://partsregistry.org/Part:BBa_K142002][http://partsregistry.org/Part:BBa_K142002][http://partsregistry.org/Part:BBa_K142001] can be used as an inverter because LacI IS is derived from bacteria and doesn't interfere with other yeast expression systems. 

>BBa_K142007 Part-only sequence (1128 bp)
atggtgaatgtgaaaccagtaacgttatacgatgtcgcagagtatgccggtgtctcttatcagaccgtttcccgcgtggtgaaccaggccagccacgttt
ctgcgaaaacgcgggaaaaagtggaagcggcgatggcggagctgaattacattcccaaccgcgtggcacaacaactggcgggcaaacagtcgttgctgat
tggcgttgccacctccagtctggccctgcacgcgccgtcgcaaattgtcgcggcgattaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcg
atggtagaacgaagcggcgtcgaagcctgtaaagcggcggtgcacaatcttctcgcgcaacgcgtcagtgggctgatcattaactatccgctggatgacc
aggatgccattgctgtggaagctgcctgcactaatgttccggcgttatttcttgatgtctctgaccagacacccatcaacagtattattttctcccatga
agacggtacgcgactgggcgtggagcatctggtcgcattgggtcaccagcaaatcgcgctgttagcgggcccattaagttctgtctcggcgcgtctgttt
ctggctggctggcataaatatctcactcgcaatcaaattcagccgatagcggaacgggaaggcgactggagtgccatgtccggttttcaacaaaccatgc
aaatgctgaatgagggcatcgttcccactgcgatgctggttgccaacgatcagatggcgctgggcgcaatgcgcgccattaccgagtccgggctgcgcgt
tggtgcggatatctcggtagtgggatacgacgattttgaagacagctcatgttatatcccgccgttaaccaccatcaaacaggattttcgcctgctgggg
caaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccctgg
cgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcaggctgcaaacga
cgaaaactacgctttagtagcttaataa

Name Length RFC10 RFC25 Codon Usage NCBI
lacI Repressor 1128bp ok ok 2 AS < 10%  ? 


 Unverified in Registry

cI repressor

cI[http://partsregistry.org/Part:BBa_C0051] is a repressor from Escherichia coli, which recognize specific DNA sequence to which it binds. 

>BBa_C0051 Part-only sequence (750 bp)
atgagcacaaaaaagaaaccattaacacaagagcagcttgaggacgcacgtcgccttaaagcaatttatgaaaaaaagaaaaatgaacttggcttatccc
aggaatctgtcgcagacaagatggggatggggcagtcaggcgttggtgctttatttaatggcatcaatgcattaaatgcttataacgccgcattgcttgc
aaaaattctcaaagttagcgttgaagaatttagcccttcaatcgccagagaaatctacgagatgtatgaagcggttagtatgcagccgtcacttagaagt
gagtatgagtaccctgttttttctcatgttcaggcagggatgttctcacctgagcttagaacctttaccaaaggtgatgcggagagatgggtaagcacaa
ccaaaaaagccagtgattctgcattctggcttgaggttgaaggtaattccatgaccgcaccaacaggctccaagccaagctttcctgacggaatgttaat
tctcgttgaccctgagcaggctgttgagccaggtgatttctgcatagccagacttgggggtgatgagtttaccttcaagaaactgatcagggatagcggt
caggtgtttttacaaccactaaacccacagtacccaatgatcccatgcaatgagagttgttccgttgtggggaaagttatcgctagtcagtggcctgaag
agacgtttggcgctgcaaacgacgaaaactacgctttagtagcttaataa

Name Length RFC10 RFC25 Codon Usage NCBI
cI Repressor 750bp ok ok 0 AS < 10%  ? 
 Confirmed in Registry

Promoter sequences

Minimal promoter

A minimal promoter is a essential DNA sequence on which the transcription machinery can bind. By adding binding sites upstream the transcription can be enhanced.

cyc100 minimal promoter

This sequence[http://partsregistry.org/Part:BBa_K105027] is the core of CYC1 promoter of S. cerevisiae. It has a basal transcription activity. This activity can be modulated by upstream activation sequences or by downstream operator sequences. 

>BBa_K105027 Part-only sequence (103 bp)
gcatgtgctctgtatgtatataaaactcttgttttcttcttttctctaaatattctttccttatacattaggacctttgcagcataaattactatacttc
tat

Name Length RFC10 RFC25 Codon Usage NCBI
Minimal yeast promoter 103bp ok ok Promoter  ?
cyc70 minimal promoter

It's a variation[http://partsregistry.org/Part:BBa_K105028] of cyc100 minimal promoter with a point mutation which reduces the basal activity to 70%. 

>BBa_K105028 Part-only sequence (103 bp)
gcatgtgctctgtatgtatataacactcttgttttcttcttttctctaaatattctttccttatacattaggacctttgcagcataaattactatacttc
tat

Name Length RFC10 RFC25 Codon Usage NCBI
Minimal yeast promoter 103bp ok ok Promoter  ?
cyc43 minimal promoter

Another derivative[http://partsregistry.org/Part:BBa%20K105029] of cyc100 minimal promoter. This minimal promoter has a 43% transcription activity of cyc100 minimal promoter. 

>BBa_K105029 Part-only sequence (103 bp)
gcatgtgctctgtatgtatatagaactcttgttttcttcttttctctaaatattctttccttatacattaggacctttgcagcataaattactatacttc
tat

Name Length RFC10 RFC25 Codon Usage NCBI
Minimal yeast promoter 103bp ok ok Promoter  ?
cyc28 minimal promoter

Another derivative[http://partsregistry.org/Part:BBa%20K105030] of cyc100 minimal promoter. This minimal promoter has a 28% transcription activity of cyc100 minimal promoter. 

>BBa_K105030 Part-only sequence (103 bp)
gcatgtgctctgtatgtatattaaactcttgttttcttcttttctctaaatattctttccttatacattaggacctttgcagcataaattactatacttc
tat

Name Length RFC10 RFC25 Codon Usage NCBI
Minimal yeast promoter 103bp ok ok Promoter  ?
cyc16 minimal promoter

Another derivative[http://partsregistry.org/Part:BBa%20K105031] of cyc100 minimal promoter. This minimal promoter has a 16% transcription activity of cyc100 minimal promoter. 

>BBa_K105031 Part-only sequence (103 bp)
gcatgtgctctgtatgtatatgaaactcttgttttcttcttttctctaaatattctttccttatacattaggacctttgcagcataaattactatacttc
tat

Name Length RFC10 RFC25 Codon Usage NCBI
Minimal yeast promoter 103bp ok ok Promoter  ?
mCYC1 minimal promoter

Another biobrick[http://partsregistry.org/wiki/index.php?title=Part:BBa_K165016] for a minimal yeast promoter for designing new promoters. 

>BBa_K165016 Part-only sequence (245 bp)
cagatccgccaggcgtgtatatatagcgtggatggccaggcaactttagtgctgacacatacaggcatatatatatgtgtgcgacgacacatgatcatat
ggcatgcatgtgctctgtatgtatataaaactcttgttttcttcttttctctaaatattctttccttatacattaggacctttgcagcataaattactat
acttctatagacacgcaaacacaaatacacacactaaattaataa

Name Length RFC10 RFC25 Codon Usage NCBI
Minimal yeast promoter 245bp ok ok Promoter  ?

Gal1 promoter sequence/GAL4 target sequence

These promoters[http://partsregistry.org/wiki/index.php/Part:BBa_K517000][http://partsregistry.org/wiki/index.php?title=Part:BBa_J63006] can be induced by galactose. Does this means that the sequence contains a upstream activating region (UAS) to which GAL4BD can bind? 

>BBa_K517000 Part-only sequence (340 bp)
gcgccgcactgctccgaacaataaagattctacaatactagcttttatggttatgaagaggaaaaattggcagtaaccgggccccacaaaccttcaaatg
aacgaatcaaattaacaaccataggatgataatgcgattagttttttagccttatttctggggtaattaatcagcgaagcgatgatttttgatctattaa
cagatatataaatgcaaaaactgcataaccactttaactaatactttcaacattttcggtttgtattacttcttattcaaatgtaataaaagtatcaaca
aaaaattgttaatatacctctatactttaacgtcaaggag

Name Length RFC10 RFC25 Codon Usage NCBI
Galactose inducible Promoter 340bp ok ok Promoter  ? 
 Confirmed in Registry 

>BBa_J63006 Part-only sequence (549 bp)
ccccattatcttagcctaaaaaaaccttctctttggaactttcagtaatacgcttaactgctcattgctatattgaagtacggattagaagccgccgagc
gggtgacagccctccgaaggaagactctcctccgtgcgtcctcgtcttcaccggtcgcgttcctgaaacgcagatgtgcctcgcgccgcactgctccgaa
caataaagattctacaatactagcttttatggttatgaagaggaaaaattggcagtaacctggccccacaaaccttcaaatgaacgaatcaaattaacaa
ccataggatgataatgcgattagttttttagccttatttctggggtaattaatcagcgaagcgatgatttttgatctattaacagatatataaatgcaaa
aactgcataaccactttaactaatactttcaacattttcggtttgtattacttcttattcaaatgtaataaaagtatcaacaaaaaattgttaatatacc
tctatactttaacgtcaaggaggaaactagacccgccgccaccatggag

Name Length RFC10 RFC25 Codon Usage NCBI
Galactose inducible Promoter 549bp ok ok Promoter  ? 
 Confirmed in Registry

Target sequence for the GAL4 DNA binding domain [http://partsregistry.org/Part:BBa_J176019]. 

>BBa_J176019 Part-only sequence (93 bp)
cggagtactgtcctccgagcggagtactgtcctccgagcggagtactgtcctccgagcggagtactgtcctccgagcggagtactgtcctccg

Name Length RFC10 RFC25 Codon Usage NCBI
5x GAL target sequence 93bp ok ok Target Sequence  ? 
 Unavailable in Registry

LexA operator

DNA recognition site[http://partsregistry.org/Part:BBa_K105022][http://partsregistry.org/Part:BBa_K079048][http://partsregistry.org/wiki/index.php?title=Part:BBa_K079039][http://partsregistry.org/wiki/index.php?title=Part:BBa_K079040][http://partsregistry.org/Part:BBa_K106686] which can be bound by LexA and can be used for recruiting transcription activators/repressors by fusing it to them. 

>BBa_K105022 Part-only sequence (31 bp)
tttacactggttttatatacagcagtacgta

Name Length RFC10 RFC25 Codon Usage NCBI
lexA Binding Domain 31bp ok ok Binding Domain  ? 
 Partially Confirmed in Registry 

>BBa_K079048 Part-only sequence (40 bp)
ctgtatatatatacagtactagagctgtatgagcatacag

Name Length RFC10 RFC25 Codon Usage NCBI
2 lexA Binding Domains 40 bp ok ok Binding Domain  ? 
 Unavailable in Registry

LacI regulated promoter/operator

A promoter sequence[http://partsregistry.org/wiki/index.php?title=Part:BBa_R0011][http://partsregistry.org/wiki/index.php?title=Part:BBa_K079017][http://partsregistry.org/wiki/index.php?title=Part:BBa_K079018][http://partsregistry.org/wiki/index.php?title=Part:BBa_K079019]which is recognized by LacI. LacI prevents RNA-polymerase from transcription of downstream regulated genes. 

>BBa_R0011 Part-only sequence (55 bp)
aattgtgagcggataacaattgacattgtgagcggataacaagatactgagcaca

Name Length RFC10 RFC25 Codon Usage NCBI
lacI repressible Promoter 55 bp ok ok Promoter  ? 
 Confirmed in Registry

cI operator

DNA motif recognized by cI repressor.[http://partsregistry.org/wiki/index.php?title=Part:BBa_K079047][http://partsregistry.org/wiki/index.php?title=Part:BBa_K079041][http://partsregistry.org/wiki/index.php?title=Part:BBa_K079042][http://partsregistry.org/wiki/index.php?title=Part:BBa_K079043] 

>BBa_K079047 Part-only sequence (67 bp)
tatcaccgccagaggtatactagagtaacaccgtgcgtgttgtactagagtatcaccgcaagggata

Protein-protein-interaction based FRET-sensor

FRET-fluorophores

EYFP

[http://partsregistry.org/Part:BBa_E2030] This is an enhanced version of EYFP. It is yeast codon-optimized, it is more chloride- and pH-resistant than EYFP and has a faster chromophore maturation than EYFP and YFP Citrine 

>BBa_E2030 Part-only sequence (753 bp)
atggcaactagcggcatggttagtaaaggagaagaacttttcactggagttgtcccaattttagttgaactagatggcgacgtgaacggtcataagttca
gtgtctccggcgaaggtgagggtgatgcaacgtacggtaagttaactttgaagttaatatgtacaaccggcaagctgcctgttccctggcctaccctggt
gacaacgttaggttatgggttgatgtgctttgctagatacccagatcacatgaaaaggcatgacttctttaaatctgcaatgccagaaggttacgtccaa
gaacgtactattttctttaaagatgacggtaattataaaactagggctgaagttaaattcgaaggtgacacacttgtaaatcgaatagagttaaagggga
ttgatttcaaagaggatggtaatattctaggccataaacttgaatataactataattcacacaacgtttacattaccgccgacaagcagaagaatggaat
caaagccaattttaagattagacacaatattgaggatggtggagtacagcttgctgatcattaccaacaaaataccccgatcggtgatggaccagttttg
ctacccgataaccattatctgtcctatcaaagcaaattgtcaaaagatcctaacgaaaaaagagaccacatggtactcttggaatttgtaacagctgctg
ggattacacatggcatggatgaactatacaaaggttctggtaccgcataataa

Name Length RFC10 RFC25 Codon Usage NCBI
Venus EYFP 753 bp ok ok 0AS<10% [http://www.ncbi.nlm.nih.gov/nucleotide/AB634497.1 AB634497.1] 
 Confirmed in Registry
ECFP

Enhanced version of ECFP[http://partsregistry.org/Part:BBa_E2020], yeast-optimized CFP, codon-optimized for yeast. Derived from ECFP. Has single exponential decay kinetics, is at least 2X brighter than ECFP, and is more resistant to photobleaching. 

>BBa_E2020 Part-only sequence (753 bp)
atggcaactagcggcatggttagtaaaggagaagaacttttcactggagttgtcccaattctggttgaattggatggcgatgtcaatggccataagtttt
cggttagtggtgaaggagagggcgacgctacctatgggaagttaactttaaagttcatttgtactaccggtaagttaccagttccttggcctactttggt
cacaacccttacatggggggtgcagtgctttgccagatatccggatcacatgaaacaacacgattttttcaaatccgctatgcctgaaggatatgtacaa
gaaagaaccatatttttcaaggatgacggcaactacaaaactagagccgaagttaaattcgaaggtgacacattggtaaatcgaattgagctcaaaggaa
tagattttaaggaagatggtaacatccttggtcataagttagagtataatgcaatttctgataacgtctacataactgcggataaacagaaaaatggtat
taaagccaattttaaaattaggcataacatcgaagatgggagtgttcaacttgcagaccactaccaacaaaatacacccataggagacggtcccgtactg
ttgccagataaccattatctgtctacacaatctaaattaagcaaagatccaaatgaaaagcgtgaccacatggtgttgctagagtttgtaacagctgctg
ggattacacatggcatggatgaactatacaaaggttctggtaccgcataataa

Name Length RFC10 RFC25 Codon Usage NCBI
ECFP 753 bp ok 1 x AgeI (Position 166) 0AS<10% several 
 Confirmed in Registry

Protein-protein-interaction partners

References

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Retrieved from "http://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_Promoter"