Team:IISER Kolkata/Part Collection
Parts Collection
We have designed a collection Nitric Oxide (NO) sensors. For making this collection we have used different Transcription Factors (TFs) and corresponding Promoters (and TF binding site), the activity of these TFs depends on the presence or the absence of NO. There are mainly two types of NO dependent TFs, the first type of TFs repress gene expression in absence of NO but in presence of NO it can’t repress the gene expression (e.g. NsrR) and the second type of TFs activates the gene expression in presence of NO but in absence of NO it does nothing and the gene which is under control of corresponding promoter is not expressed (e.g. NorR, SoxR). Combining these two types of TFs and their corresponding Promoters (and TF binding site) we have designed a new kind of NO sensor which activates gene expression only within a particular range of NO.
Type I
BBa_K3011007
Constitutive Promoter (BBa_J23100) – RBS (BBa_B0032) – NsrR (BBa_K1682011) – Terminator (BBa_B0010) – PyeaR promoter (BBa_K216005) – RBS (BBa_B0032) – mRFP (BBa_E1010) – Terminator (BBa_B0012)
NsrR (BBa_K1682011) is a Nitric Oxide (NO) sensitive transcription repressor. This construct produces NsrR constitutively; in absence of NO, NsrR binds to the NsrR binding site present on the PyeaR promoter (BBa_K216005) and repress the transcription of mRFP (BBa_E1010). In presence of NO, NsrR binds to NO due to which NsrR can’t bind to the DNA (thus can’t repress gene expression) and mRFP will produce.
NsrR contains a Helix Turn Helix (HTH) motif and Fe-S cluster ([2Fe-2S] or [4Fe-4S]). In absence of NO, NsrR binds to a particular DNA sequence by using HTH motif (as a homodimer) and repress gene expression. In presence of NO, Fe-S cluster reacts with NO to form dinitrosyl iron complex, due to the formation of this complex NsrR can’t bind to that particular sequence anymore. Depending upon the sequence of NsrR binding site, the number of NO molecules (per cluster) which is required for abolishing DNA binding vary (2-10 NO / cluster).
Type II
BBa_K3011008
Constitutive Promoter (BBa_J23100) – RBS (BBa_B0032) – NorR (BBa_K256004) – Terminator (BBa_B0010) – pNO promoter (BBa_K256000) – RBS (BBa_B0032) – mRFP (BBa_E1010) – Terminator (BBa_B0012)
NorR (BBa_K256004) is a Nitric Oxide (NO) dependent transcription activator. This construct produces NorR constitutively; in presence of NO, NorR can activate the transcription of mRFP (BBa_E1010) which is under control of pNO promoter (BBa_K256000).
NorR contains a Helix Turn Helix (HTH) motif by which it can bind to a particular DNA sequence (as a homodimer). In presence of NO it can activate transcription of genes which is under control of pNO promoter. The regulatory domain of NorR contains a mononuclear non-haem iron centre, which reversibly binds NO. Binding of NO stimulates the ATPase activity of NorR, enabling the activation of transcription.
BBa_K3011009
Constitutive Promoter (BBa_J23100) – RBS (BBa_B0032) – SoxR (BBa_K554003) – Terminator (BBa_B0010) – SoxS promoter (BBa_K554000) – RBS (BBa_B0032) – mRFP (BBa_E1010) – Terminator (BBa_B0012)
SoxR (BBa_K554003) is a Nitric Oxide (NO) dependent transcription activator. This construct produces SoxR constitutively; in presence of NO, SoxR can activate the transcription of mRFP (BBa_E1010) which is under control of SoxS promoter (BBa_K554000).
SoxR contains a Helix Turn Helix (HTH) motif by which it can bind to a particular DNA sequence (as a homodimer) and a [2Fe-2S] cluster. In presence of NO, [2Fe-2S] cluster reacts with NO to form dinitrosyl iron complex, due to the formation of this complex SoxR can activate transcription of genes which is under control of SoxS promoter.
Type III
BBa_K3011010
This genetic circuit will produce RFP at particular range of Nitric Oxide (NO).
NorR and NsrR produce constitutively. When no NO is absent NsrR binds to a particular DNA sequence (NsrR binding site) and repress the transcription of RFP. When NO present at lower concentration, NsrR binds to NO and can’t repress transcription anymore. When NO present at higher concentration, NorR binds to NO, which then activates the transcription of TetR and final TetR bind to particular DNA sequence to repress the transcription of RFP.
See Model page for more details of Type III NO sensor.