Difference between revisions of "Part:BBa K2300003"

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(→‎Protein information)
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K2300003 short</partinfo>
 
<partinfo>BBa_K2300003 short</partinfo>
−
 
−
The original HydEF page (BBa_K1998012) had the wrong sequence so we have created an updated page. The HydEF protein contains two unique domains that are homologous to two distinct prokaryotic proteins, HydE and HydF, which are found exclusively in organisms containing [FeFe] hydrogenase (Mulder et al., 2010). This part is the first gene of the hydrogenase maturation enzyme complex.
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2300003 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2300003 SequenceAndFeatures</partinfo>
 +
 +
===Overview===
 +
 +
The original HydEF page (BBa_K1998012) (https://parts.igem.org/Part:BBa_K1998012) had the wrong sequence so we have created an updated page. The HydEF protein contains two unique domains that are homologous to two distinct prokaryotic proteins, HydE and HydF, which are found exclusively in organisms containing [FeFe] hydrogenase (Mulder et al., 2010). This part is the first gene of the [FeFe] hydrogenase maturation enzyme complex.
 +
 +
All genes within this plasmid are sequences obtained from <i>Chlamydomonas  reinhardtii</i> and codon optimised to be expressed in <i>Escherichia coli</i>.
  
 
===Biology & Literature===
 
===Biology & Literature===
  
−
HydEF in the majority of [FeFe] containing organisms exists as two separately transcribed proteins (HydE and HydF). However in several algal species it the two genes are fused (Bock et al., 2006).
+
HydEF in the majority of [FeFe] containing organisms exists as two separately transcribed proteins (HydE and HydF). However in several algal species the two genes are fused (Böck et al., 2006). HydE (along with HydG) is a radical SAM (S-adenosyl methionine) enzyme which binds [4Fe-4S] clusters in the biosynthesis of the H-cluster (Mulder et al., 2010). HydF contains an N-terminal GTPase domain which is responsible for the P loop motif and FeS binding (Mulder et al., 2010). Both these genes have been shown to be essential for the maturation of the HydA hydrogenase (King et al., 2006). This is due to their role in the creation of the H-cluster.
  
 
===Part Verification===
 
===Part Verification===
 +
The entire Hydrogen Gas Producing Gene Cluster (BBa_K2300001) was sequenced and confirmed once it had been ligated together. This included the HydEF part.
 +
 +
To confirm the efficacy of the ribosome binding sites in our parts the Macquarie iGEM team of 2017 used the Salis Lab Ribosome Binding Site calculator from Penn State University. The results from this were that our ribosome binding site had a translation initiation rate of 1324.3.
 +
 +
<html><center><img src="https://static.igem.org/mediawiki/2016/b/bd/T--Macquarie_Australia--HYDShowGel.png" " width="50%" height="35%"></center> </html>
 +
<center> <b>Fig 1.</b> A gel consisting of EcorI/PstI for the hydEF part. The expected band for the hydEF (~3600 bp) part was observed.</center>
  
 
===Protein information===
 
===Protein information===
  
−
<i>HydEF</i><br>
+
HydEF<br>
 
Mass: 121.95 kDa<br>
 
Mass: 121.95 kDa<br>
 
Sequence: <br>
 
Sequence: <br>
Line 39: Line 49:
 
===References===
 
===References===
  
−
Mulder, D.W., Shepard, E.M., Meuser, J.E., Joshi, N., King, P.W., Posewitz, M.C., Broderick, J.B. and Peters, J.W., 2011. Insights into [FeFe]-hydrogenase structure, mechanism, and maturation. Structure, 19(8), pp.1038-1052.
+
Böck, A., King, P.W., Blokesch, M. and Posewitz, M.C., 2006. Maturation of hydrogenases. <i>Advances in Microbial Physiology</i>, 51, pp.1-225.
 +
 
 +
 
 +
King, P.W., Posewitz, M.C., Ghirardi, M.L. and Seibert, M., 2006. Functional studies of [FeFe] hydrogenase maturation in an <i>Escherichia coli</i> biosynthetic system. <i>Journal of Bacteriology</i>, 188(6), pp.2163-2172.
 +
 
 +
 
 +
Mulder, D.W., Boyd, E.S., Sarma, R., Lange, R.K., Endrizzi, J.A., Broderick, J.B. and Peters, J.W., 2010. Stepwise [FeFe]-hydrogenase H-cluster assembly revealed in the structure of HydA [Dgr] EFG. Nature, 465(7295), pp.248-251.
 +
 
 +
 
 +
Mulder, D.W., Shepard, E.M., Meuser, J.E., Joshi, N., King, P.W., Posewitz, M.C., Broderick, J.B. and Peters, J.W., 2011. Insights into [FeFe]-hydrogenase structure, mechanism, and maturation. <i>Structure</i>, 19(8), pp.1038-1052.
 +
 
  
−
Posewitz, M.C., King, P.W., Smolinski, S.L., Zhang, L., Seibert, M. and Ghirardi, M.L., 2004. Discovery of two novel radical S-adenosylmethionine proteins required for the assembly of an active [Fe] hydrogenase. Journal of Biological Chemistry, 279(24), pp.25711-25720.
+
Posewitz, M.C., King, P.W., Smolinski, S.L., Zhang, L., Seibert, M. and Ghirardi, M.L., 2004. Discovery of two novel radical S-adenosylmethionine proteins required for the assembly of an active [Fe] hydrogenase. <i>Journal of Biological Chemistry</i>, 279(24), pp.25711-25720.
 
Vancouver
 
Vancouver
  

Latest revision as of 00:03, 2 November 2017


HydEF (Updated 2017)

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Unknown
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Unknown
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 901
    Illegal AgeI site found at 1816
    Illegal AgeI site found at 3459
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2684
    Illegal BsaI.rc site found at 304
    Illegal BsaI.rc site found at 416

Overview

The original HydEF page (BBa_K1998012) (https://parts.igem.org/Part:BBa_K1998012) had the wrong sequence so we have created an updated page. The HydEF protein contains two unique domains that are homologous to two distinct prokaryotic proteins, HydE and HydF, which are found exclusively in organisms containing [FeFe] hydrogenase (Mulder et al., 2010). This part is the first gene of the [FeFe] hydrogenase maturation enzyme complex.

All genes within this plasmid are sequences obtained from Chlamydomonas reinhardtii and codon optimised to be expressed in Escherichia coli.

Biology & Literature

HydEF in the majority of [FeFe] containing organisms exists as two separately transcribed proteins (HydE and HydF). However in several algal species the two genes are fused (Böck et al., 2006). HydE (along with HydG) is a radical SAM (S-adenosyl methionine) enzyme which binds [4Fe-4S] clusters in the biosynthesis of the H-cluster (Mulder et al., 2010). HydF contains an N-terminal GTPase domain which is responsible for the P loop motif and FeS binding (Mulder et al., 2010). Both these genes have been shown to be essential for the maturation of the HydA hydrogenase (King et al., 2006). This is due to their role in the creation of the H-cluster.

Part Verification

The entire Hydrogen Gas Producing Gene Cluster (BBa_K2300001) was sequenced and confirmed once it had been ligated together. This included the HydEF part.

To confirm the efficacy of the ribosome binding sites in our parts the Macquarie iGEM team of 2017 used the Salis Lab Ribosome Binding Site calculator from Penn State University. The results from this were that our ribosome binding site had a translation initiation rate of 1324.3.

Fig 1. A gel consisting of EcorI/PstI for the hydEF part. The expected band for the hydEF (~3600 bp) part was observed.

Protein information

HydEF
Mass: 121.95 kDa
Sequence:
MAHSLSAHSRQAGDRKLGAGAASSRPSCPSRRIVRVAAHASASKATPDVPVDDLPPAHARAAVAAANRRARAMASAEAAAETLGDFLGLGKGGLSP GATANLDREQVLGVLEAVWRRGDLNLERALYSHANAVTNKYCGGGVYYRGLVEFSNICQNDCSYCGIRNNQKEVWRYTMPVEEVVEVAKWALENGI RNIMLQGGELKTEQRLAYLEACVRAIREETTQLDLEMRARAASTTTAEAAASAQADAEAKRGEPELGVVVSLSVGELPMEQYERLFRAGARRYLIRIET SNPDLYAALHPEPMSWHARVECLRNLKKAGYMLGTGVMVGLPGQTLHDLAGDVMFFRDIKADMIGMGPFITQPGTPATDKWTALYPNANKNSHMK SMFDLTTAMNALVRITMGNVNISATTALQAIIPTGREIALERGANVVMPILTPTQYRESYQLYEGKPCITDTAVQCRRCLDMRLHSVGKTSAAGVWGDPA SFLHPIVGVPVPHDLSSPALAAAASADFHEVGAGPWNPIRLERLVEVPDRYPDPDNHGRKKAGAGKGGKAHDSHDDGDHDDHHHHHGAAPAGAAA GKGTGAAAIGGGAGASRQRVAGAAAASARLCAGARRAGRVVASPLRPAAACRGVAVKAAAAAAGEDAGAGTSGVGSNIVTSPGIASTTAHGVPRINI GVFGVMNAGKSTLVNALAQQEACIVDSTPGTTADVKTVLLELHALGPAKLLDTAGLDEVGGLGDKKRRKALNTLKECDVAVLVVDTDTAAAAIKSGRLA EALEWESKVMEQAHKYNVSPVLLLNVKSRGLPEAQAASMLEAVAGMLDPSKQIPRMSLDLASTPLHERSTITSAFVKEGAVRSSRYGAPLPGCLPRW SLGRNARLLMVIPMDAETPGGRLLRPQAQVMEEAIRHWATVLSVRLDLDAARGKLGPEACEMERQRFDGVIAMMERNDGPTLVVTDSQAIDVVHPW TLDRSSGRPLVPITTFSIAMAYQQNGGRLDPFVEGLEALETLQDGDRVLISEACNHNRITSACNDIGMVQIPNKLEAALGGKKLQIEHAFGREFPELESG GMDGLKLAIHCGGCMIDAQKMQQRMKDLHEAGVPVTNYGVFFSWAAWPDALRRALEPWGVEPPVGTPATPAAAPATAASGV

References

Böck, A., King, P.W., Blokesch, M. and Posewitz, M.C., 2006. Maturation of hydrogenases. Advances in Microbial Physiology, 51, pp.1-225.


King, P.W., Posewitz, M.C., Ghirardi, M.L. and Seibert, M., 2006. Functional studies of [FeFe] hydrogenase maturation in an Escherichia coli biosynthetic system. Journal of Bacteriology, 188(6), pp.2163-2172.


Mulder, D.W., Boyd, E.S., Sarma, R., Lange, R.K., Endrizzi, J.A., Broderick, J.B. and Peters, J.W., 2010. Stepwise [FeFe]-hydrogenase H-cluster assembly revealed in the structure of HydA [Dgr] EFG. Nature, 465(7295), pp.248-251.


Mulder, D.W., Shepard, E.M., Meuser, J.E., Joshi, N., King, P.W., Posewitz, M.C., Broderick, J.B. and Peters, J.W., 2011. Insights into [FeFe]-hydrogenase structure, mechanism, and maturation. Structure, 19(8), pp.1038-1052.


Posewitz, M.C., King, P.W., Smolinski, S.L., Zhang, L., Seibert, M. and Ghirardi, M.L., 2004. Discovery of two novel radical S-adenosylmethionine proteins required for the assembly of an active [Fe] hydrogenase. Journal of Biological Chemistry, 279(24), pp.25711-25720. Vancouver