Achievements


Parts


Registry number Construct name Gene lenght [nts] Protein lenght [aa] Physical DNA sent Construct type product Native host Plasmid Standard
BBa_K1459001 PmrA 669 222 yes protein Salmonella spp. pSB1C3 RFC 10
BBa_K1459016 PmrB WT (Fe3+) 1071 356 no protein Salmonella spp. pSB1C3 RFC 10
BBa_K1459010 PmrB (MUT) 1029 343 yes protein Salmonella spp. pSB1C3 RFC 10
BBa_K1459003 PmrA-PmrB 1749 222 + 356 no 2 proteins Salmonella spp. pSB1C3 RFC 10
BBa_K1459004 PmrA-PmrB(MUT)-terminator 1791 222+343(two proteins) yes proteins + transcription terminator Salmonella spp. pSB1C3 RFC 10
BBa_K1459011 PmrB N-terminus 102 34 yes protein domain Salmonella spp. pSB1C3 RFC 25
BBa_K1459009 PmrB C-terminus 882 294 yes protein domain Salmonella spp. pSB1C3 RFC 25
BBa_K1459005 PmrA-PmrB N-terminus 782 220 + 34 yes protein and protein domain Salmonella spp. pSB1C3 RFC 25
BBa_K1459006 pmrC promoter 46 - no promoter Salmonella spp. pSB1C3 RFC 10
BBa_K1459017 pmrC-GFP 1119 238 no promoter and protein Salmonella spp. pSB1C3 RFC 10
BBa_K1459008 pmrC-GFP-terminator 1166 238 yes promoter and protein and terminator Salmonella spp. pSB1C3 RFC 10
BBa_K1459012 SENG lanthanide binding tag 60 20 no peptide synthetic pSB1C3 RFC 25
BBa_K1459013 wSE3 lanthanide binding tag 51 17 no peptide synthetic pSB1C3 RFC 25
BBa_K1459014 Lanthanide Binding Tag 51 17 no peptide synthetic pSB1C3 RFC 25
BBa_K1459015 1L2Y short peptide 66 22 no peptide synthetic pSB1C3 RFC 25

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BBa_K1459001 - PmrA


Protein name:PmrA
Other names:basR, parA
Gene name:basR
Source organism for the data:Salmonella enterica subsp. enterica serovar Typhimurium str. strain LT2 / SGSC1412 / ATCC 700720
UniProtKB signature:P36556
Gene sequence RefSeq accession number:NC_003197.1
Protein sequence RefSeq accession number:NP_463157.1
Length:222 aa
Molecular mass:25,035 Da
Cellular localization:cytoplasmic
Biological function:transcription regulator
The PmrA protein is a cognate response regulator of the histidine kinase PmrB. Upon phosphorylation by PmrB, PmrA undergoes dimerization which dramatically increases its affinity for promoter DNA. This allows it to regulate expression of a number of genes, usually coding for LPS- modifying enzymes.
In our project, we used the PmrA unchanged, for it to serve as an transcription inductor for our reporter - the GFP, expressed under the control of the PmrC promoter, i.e. the promoter of one of the genes involved in LPS modification. Notably, however, there is no PmrC gene under its promoter in our constructs, so that neither this LPS-modifyi ng enzyme, nor any other enzymes of this kind, are expressed.
If you wish to study PmrA-PmrB system more closely, we suggest reading following papers:
[1] H. Liang, X. Deng, M. Bosscher, Q. Ji, M. P. Jensen, C. He, Engineering Bacterial Two-Component System PmrA/PmrB to Sense Lanthanide Ions, J.Am.Chem.Soc. 2013, 135, 2037−2039
[2] M. Wonsten, L. Kox, S. Chamnogpol, F. Soncini, E. Groisman,A Signal Transduction System that Responds to Extracellular Iron,Cell, Vol. 103, 113–125, September 29, 2000
SENT TO REGISTRY



BBa_K1459002 - C-term of PmrB from Salmonella enterica


PmrB is a transmembrane kinase. After binding iron (III) ion by binding peptide on extracellular loop, it's intracellular domain gains kinase activity and phosphorylates PmrA (BBa_K1459000).
PmrB C-term is a part of two-component system. When fused with some binding tag, PmrB(N-term), PmrA and pmrC-reporter, it is a viable detecting system.
If you wish to study PmrA-PmrB system more closely, we suggest reading following papers:
[1] H. Liang, X. Deng, M. Bosscher, Q. Ji, M. P. Jensen, C. He, Engineering Bacterial Two-Component System PmrA/PmrB to Sense Lanthanide Ions, J.Am.Chem.Soc. 2013, 135, 2037−2039
[2] M. Wonsten, L. Kox, S. Chamnogpol, F. Soncini, E. Groisman,A Signal Transduction System that Responds to Extracellular Iron,Cell, Vol. 103, 113–125, September 29, 2000
SENT TO REGISTRY



BBa_K1459003 - PmrA-PmrB(LBT) two-component system


PmrA-PmrB two-component system is native to Salmonella enterica and in its native state the system is responsible for chemotaxis. PmrB is a transmembrane protein with iron binding peptide on its extracellular loop. When PmrB binds iron (III) iron, the intracellular domain gains kinase activity and phosphorylates PmrA, which subsequently binds to pmrC promoter and induces expression of chemotaxis CheZ protein. In this part iron binding tag on the extracellular loop was exchanged with a lanthanide binding tag (LBT), to allow PmrA-PmrB two-component system to respond to lanthanide ions.
If you wish to study PmrA-PmrB system more closely, we suggest reading following papers:
[1] H. Liang, X. Deng, M. Bosscher, Q. Ji, M. P. Jensen, C. He, Engineering Bacterial Two-Component System PmrA/PmrB to Sense Lanthanide Ions, J.Am.Chem.Soc. 2013, 135, 2037−2039
[2] M. Wonsten, L. Kox, S. Chamnogpol, F. Soncini, E. Groisman,A Signal Transduction System that Responds to Extracellular Iron,Cell, Vol. 103, 113–125, September 29, 2000



BBa_K1459004 - PmrA-PmrB(LBT) with terminator (BBa_B1006)


PmrA-PmrB two-component system is native to Salmonella enterica and in its native state it is responsible for chemotaxis. PmrB is a transmembrane protein with iron binding peptide on its extracellular loop. When PmrB binds iron (III) iron, its intracellular domain gains kinase activity and phosphorylates PmrA, which subsequently binds to pmrC promoter and induces expression of chemotaxis CheZ protein. In this part iron binding tag on the extracellular loop was exchanged with a lanthanide binding tag (LBT), to allow PmrA-PmrB two-component system to respond to lanthanide ions.
If you wish to study PmrA-PmrB system more closely, we suggest reading following papers:
[1] H. Liang, X. Deng, M. Bosscher, Q. Ji, M. P. Jensen, C. He, Engineering Bacterial Two-Component System PmrA/PmrB to Sense Lanthanide Ions, J.Am.Chem.Soc. 2013, 135, 2037−2039
[2] M. Wonsten, L. Kox, S. Chamnogpol, F. Soncini, E. Groisman,A Signal Transduction System that Responds to Extracellular Iron,Cell, Vol. 103, 113–125, September 29, 2000
SENT TO REGISTRY



BBa_K1459005 - PmrA-PmrB(N-term)


This is N-terminal part of PmrA-PmrB two-component system native to Salmonella enterica. PmrA-PmrB two-component system is native to Salmonella enterica and in its native state it is responsible for chemotaxis. PmrB is a transmembrane protein with iron binding peptide on its extracellular loop. When PmrB binds iron (III) iron, it's intracellular domain gains kinase activity and phosphorylates PmrA, which subsequently binds to pmrC promoter and induces expression of chemotaxis CheZ protein. In this part iron binding tag on the extracellular loop was exchanged with a lanthanide binding tag (LBT), to allow PmrA-PmrB two-component system to respond to lanthanide ions. In this part, PmrB protein is truncated just before iron binding tag, which enables one to put any desired tag between two parts of PmrB, to construct a detecting system based on PmrA-PmrB system.
If you wish to study PmrA-PmrB system more closely, we suggest reading following papers:
[1] H. Liang, X. Deng, M. Bosscher, Q. Ji, M. P. Jensen, C. He, Engineering Bacterial Two-Component System PmrA/PmrB to Sense Lanthanide Ions, J.Am.Chem.Soc. 2013, 135, 2037−2039
[2] M. Wonsten, L. Kox, S. Chamnogpol, F. Soncini, E. Groisman,A Signal Transduction System that Responds to Extracellular Iron,Cell, Vol. 103, 113–125, September 29, 2000
SENT TO REGISTRY



BBa_K1459006 - pmrC


This is pmrC promoter native to Salmonella enterica. PmrA-PmrB two-component system is native to Salmonella enterica and in its native state it is responsible for chemotaxis. PmrB is a transmembrane protein with iron binding peptide on its extracellular loop. When PmrB binds iron (III) iron, the intracellular domain gains kinase activity and phosphorylates PmrA, which subsequently binds to pmrC promoter and induces expression of chemotaxis CheZ protein. In this part iron binding tag on the extracellular loop was exchanged with a lanthanide binding tag (LBT), to allow PmrA-PmrB two-component system to respond to lanthanide ions.
If you wish to study PmrA-PmrB system more closely, we suggest reading following papers:
[1] H. Liang, X. Deng, M. Bosscher, Q. Ji, M. P. Jensen, C. He, Engineering Bacterial Two-Component System PmrA/PmrB to Sense Lanthanide Ions, J.Am.Chem.Soc. 2013, 135, 2037−2039
[2] M. Wonsten, L. Kox, S. Chamnogpol, F. Soncini, E. Groisman,A Signal Transduction System that Responds to Extracellular Iron,Cell, Vol. 103, 113–125, September 29, 2000



BBa_K1459008 - pmrC-GFP-terminator


This is pmrC promoter from Salmonella enterica, with subsequent GFP and BBa_B1006 terminator. This part is one part of PmrA-PmrB detecting system. Upon phosphorylation by PmrB, PmrA binds to pmrC and induces expression of GFP.
If you wish to study PmrA-PmrB system more closely, we suggest reading following papers:
[1] H. Liang, X. Deng, M. Bosscher, Q. Ji, M. P. Jensen, C. He, Engineering Bacterial Two-Component System PmrA/PmrB to Sense Lanthanide Ions, J.Am.Chem.Soc. 2013, 135, 2037−2039
[2] M. Wonsten, L. Kox, S. Chamnogpol, F. Soncini, E. Groisman,A Signal Transduction System that Responds to Extracellular Iron,Cell, Vol. 103, 113–125, September 29, 2000
SENT TO REGISTRY



BBa_K1459010 - PmrB(LBT)


Protein name:PmrB
Other names:basS, parB
Gene name:basS
Source organism for the data:Salmonella enterica subsp. enterica serovar Typhimurium str. strain LT2 / SGSC1412 / ATCC 700720
UniProtKB signature:P36557/br> Gene sequence RefSeq accession number:NC_003197.1
Protein sequence RefSeq accession number:NP_463157.1
Length:356 aa
Molecular mass:40,262 Da
Cellular localization:inner plasma membrane
Biological function:Signal transduction via kinase acivities
PmrB(LBT) is a engineered PmrB gene, where PmrB is a sensor histidine kinase present in the inner cell membrane of many species of bacteria, including E. coli and S. enterica. With a 30 amino acid periplasmic loop, it is capable of binding iron (III) and aluminium ions. The binding event induces a conformational change of the protein, which leads to ATP phosphate-derived autophosphorylation of the C-terminal cytoplasmic domain, followed by transfer of the phosphate group onto the transcriptional regulator PmrA. As part of our project, the periplasmic iron/alumin ium-binding loop of the PmrB was substituted with a synthetic sequence - a lanthanide-binding ta g, intended to bind lanthanide ions, with terbium in particular. Such a binding event would then induce the aforementioned conformation change and phosphorylation of the PmrA, leading it to bind to the PmrC promoter, to allow for expression of the Green Fluorescent Protein - our reporter gene.
If you wish to study PmrA-PmrB system more closely, we suggest reading following papers:
[1] H. Liang, X. Deng, M. Bosscher, Q. Ji, M. P. Jensen, C. He, Engineering Bacterial Two-Component System PmrA/PmrB to Sense Lanthanide Ions, J.Am.Chem.Soc. 2013, 135, 2037−2039
[2] M. Wonsten, L. Kox, S. Chamnogpol, F. Soncini, E. Groisman,A Signal Transduction System that Responds to Extracellular Iron,Cell, Vol. 103, 113–125, September 29, 2000
SENT TO REGISTRY



BBa_K1459011 - PmrB(N-term)


PmrA-PmrB two-component system is native to Salmonella enterica and in its native state it is responsible for chemotaxis. PmrB is a transmembrane protein with iron binding peptide on its extracellular loop. When PmrB binds iron (III) iron, the intracellular domain gains kinase activity and phosphorylates PmrA, which subsequently binds to pmrC promoter and induces expression of chemotaxis CheZ protein.
In this part iron binding tag on the extracellular loop was exchanged with a lanthanide binding tag (LBT), to allow PmrA-PmrB two-component system to respond to lanthanide ions. This part was truncated just before the iron binding tag, and PmrB(N-term) is functionally complementar to PmrB(C-term).
If you wish to study PmrA-PmrB system more closely, we suggest reading following papers:
[1] H. Liang, X. Deng, M. Bosscher, Q. Ji, M. P. Jensen, C. He, Engineering Bacterial Two-Component System PmrA/PmrB to Sense Lanthanide Ions, J.Am.Chem.Soc. 2013, 135, 2037−2039
[2] M. Wonsten, L. Kox, S. Chamnogpol, F. Soncini, E. Groisman,A Signal Transduction System that Responds to Extracellular Iron,Cell, Vol. 103, 113–125, September 29, 2000
SENT TO REGISTRY



BBa_K1459012 - SENG lanthanide binding tag


This is a DNA sequence coding lanthanide binding tag described in literature. Its literatural dissociation constants are as follows:
KTb3+=18 nM
This is the lowest known value of dissociation constant for a Tb3+, thus making the binding strenght highest amongst known LBTs.
[1] J. M. Langdon, Development of Lanthanide-Binding Tags (LBTs) as Powerful and Versatile PeptidesFor Use in Studies of Proteins and Protein Interactions, © 2008 Massachusetts Institute of Technology All rights reserved



BBa_K1459013 - wSE3 lanthanide binding tag


This is sequence of DNA coding wSE3 lanthanide binding tag. It's dissociation constants are as follows:
KTb3+=2000 nM
[1] J. M. Langdon, Development of Lanthanide-Binding Tags (LBTs) as Powerful and Versatile PeptidesFor Use in Studies of Proteins and Protein Interactions, © 2008 Massachusetts Institute of Technology All rights reserved



BBa_K1459014 - Lanthanide Binding Tag


This is DNA sequence coding a lanthanide binding tag. This one is one of the best described LBTs in literature, with dissociation constants following:
KLa3+= 3500 nM
KCe3+= 950 nM
KNd3+= 270 nM
KEu3+= 62 nM
KGd3+= 84 nM
KTb3+= 57 nM
KDy3+= 71 nM
KEr3+= 78 nM
KYb3+= 100 nM
KLu3+= 128 nM
[1] M. Nitz, M. Sherawat, K. J. Franz, E. Peisach, K. N. Allen, B. Imperiali, Structural Origin of the High Affinity of a Chemically Evolved Lanthanide-Binding Peptide , Angew.Chem.Int.Ed. 2004, 43, 3682–368



BBa_K1459015 - 1L2Y short peptide


This is DNA sequence coding short peptide (PDB 1L2Y) is highly structured in water and could provide a structural foundation for small binding tags, such as we were planning to use it.
[1] Neidigh, J.W., Fesinmeyer, R.M., Andersen, N.H., Designing a 20-residue protein, Nat.Struct.Biol., 2002 9: 425-430


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Results


In what we succeded

We did succed in constructing the lanthanide sensor in BioBrick standard and cloning its parts into pSB1C3 and sending seven of them to the Registry.
As for 17.10.2014, we are trying to measure pmrC not activated by PmrA and also we are trying to measure GFP expression both in presence and in absence of lanthanide ions in the environment.
We also measured the relative strenght of pmrC promoter in the absence of lanthanides.

What would we do (given more time)

Given more time, we would certainly try to test more lanthanide binding tags and to construct a system to effectively bind those ions, not only detect them.
We would also try to quantify better our existing system.

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Cooperation with other iGEM Teams


During this year’s iGEM we have exchanged with the following teams:

  • Paris_Bettencourt – we participated in the iGEM newsletter, sending them information about our team, our project and trying to answer other teams questions from the previous newsletter
  • Toulouse – we sent them 4 of our BioBricks (BBa_K780003, BBa_K780002, BBa_K780001, BBa_K780000)
  • Groningen – we exchanged our official iGEM abstracts, translated their abstract into Polish and got our abstract translated into Dutch
  • Paris Saclay - we exchanged our official iGEM abstracts and we translated their into Polish and got our abstract translated into French
  • ETH Zurich – we filled in a survey about complexity in everyday life
  • Warwick - we filled in a survey about policy and practices
  • Valencia Biocampus - we filled in a survey

    • Medal Criteria


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