Difference between revisions of "Part:BBa K1998001"

Latest revision as of 02:32, 21 October 2016

YCF54 - ChlM

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 912
Illegal BamHI site found at 316
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 478

Overview

This composite part consists of the genes yCF54 and chlM. They come from the chlorophyll biosynthetic pathway of Chlamydomonas reinhardtii. The genes encode the enzymes Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase (yCF54) and Magnesium-protoporphyrin O-methyltransferase (chlM). When combined with the Mg-chelatase plasmid BBa_K1998000, it can convert protoporphyrin IX to protochlorophyllide.

Biology & Literature

These two genes within this part are used within an operon in the biosynthesis pathway. cF54 works with the cTH1 gene and Plastocyanin within the oxidative cyclase pathway. It's interaction with other genes results in the catalysis of the biosynthesis pathway from Mg-protoporphyrin IX to Protochlorophyllide [1]. Deletion studies have detected the importance of yCF54 in maintaining levels of Mg-protoporphyrin IX methyl ester indicating that the yCF54 gene is critical to both the assembly and function of the cyclase complex [1].

The second gene in this part is chlM. chlM encodes for a magnesium protoporhyrin IX methytransferase. chlM's role in the pathway is to methylate Mg-protoporphyrin IX which then aids the catalysis of Mg-cheltase [2]. This then facilitates the formation of the final protochlorophyllide. Involved in the second step of the pathway, the chlM gene is involved in the transfer of a methyl group onto one of the rings of magnesium protoporphyrin which forms the magnesium prootoporhyrin IX monomethylester [3, 4].

Part Verification

Fig 1.Gel electrophoresis (1% agarose) provides evidence of successful assembly of pLac-ycf54-chlM (1.5 kbp) plasmid (Lane 3). The plasmid was assembled via 3A assembly and double digested to reveal the biobrick backbone (2000 bp) and the correct insert size.

Fig 2.PCR confirmation of the pLac-ycf54-chlM plasmid. Forward and reverse chlM primers were used to confirm the presence of the chlM gene. The presence of yCF54 gene was also confirmed using the pLac forward primer with the reverse yCF54 primer. This also confirmed the presence of the lac promoter followed by yCF54 (700 bp in total).

Protein information

yCF54
mass: 17.08kDa
sequence: MAPAAASADKATAAEYYALVCNAEWFFMDPQNESVAEQLREKVRFFKEQN KERDFFIVPNPKWLDAKFPEQAKQVKRPCVALVSTDKMWITFMKLRLDRV LKIDLKSMPASEVLAAGEALPDFKPDGKWTAPYARYTPGWWNVFLPNH

chlM
mass: 30.45kDa sequence: MASEIAQTADVGSLTFAVGGVGAVVGLGALLVATDHQKRRSEQMKSFDGD EKEAVKDYFNTAGFERWRKIYGETDEVNKVQLDIRTGHAQTVDKVLRWVD EEGSVQGITVADCGCGTGSLAIQLALRGAAVSASDISAAMASEAEQRYQQ AVAAGQGKAPKVAPKFEALDLESVKGKYDTVTCLDVMIHYPQDKVDAMIT HLAGLSDRRLIISFAPKTLSYSILKRIGELFPGPSKATRAYLHREEDVEAALK RAGFKVTKREMTATSFYFSRLLEAIRE

References

[1] Hollingshead S, Kopečná J, Jackson PJ, Canniffe DP, Davison PA, Dickman MJ, Sobotka R, Hunter CN. Conserved chloroplast open-reading frame ycf54 is required for activity of the magnesium protoporphyrin monomethylester oxidative cyclase in Synechocystis PCC 6803. Journal of Biological Chemistry. 2012 Aug 10;287(33):27823-33.

[2] Alawady A, Reski R, Yaronskaya E, Grimm B. Cloning and expression of the tobacco CHLM sequence encoding Mg protoporphyrin IX methyltransferase and its interaction with Mg chelatase. Plant molecular biology. 2005 Mar 1;57(5):679-91.

[3] Shepherd M, McLean S, Hunter CN. Kinetic basis for linking the first two enzymes of chlorophyll biosynthesis. FEBS Journal. 2005 Sep 1;272(17):4532-9.

[4] Meinecke L, Alawady A, Schroda M, Willows R, Kobayashi MC, Niyogi KK, Grimm B, Beck CF. Chlorophyll-deficient mutants of Chlamydomonas reinhardtii that accumulate magnesium protoporphyrin IX. Plant molecular biology. 2010 Apr 1;72(6):643-58.

@@ Line 14: / Line 14: @@
 ===Overview===
-This composite part is made up of the genes YCF54 and ChlM. They come from the chlorophyll biosynthetic pathway of Chlamydomonas reinhardtii. The two genes Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase (YCF54) and Magnesium-protoporphyrin O-methyltransferase (ChlM). When combined in Operon 3 of our chlorophyll biosynthetic pathway they are involved in converting protoporphyrin IX to protoin the presence of NADPH and O2.
+This composite part consists of the genes <i>yCF54</i> and <i>chlM</i>. They come from the chlorophyll biosynthetic pathway of <i>Chlamydomonas reinhardtii</i>. The genes encode the enzymes Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase (<i>yCF54</i>) and Magnesium-protoporphyrin O-methyltransferase (<i>chlM</i>). When combined with the Mg-chelatase plasmid BBa_K1998000, it can convert protoporphyrin IX to protochlorophyllide.
 <br><br>
 <html><center><img src="https://static.igem.org/mediawiki/2016/0/0f/T--Macquarie_Australia--ChlorophyllBiosynthesisDiagram.png" alt="ChlorophyllBiosynthesisDiagram" height="50%"width="75%"></center></html>
 ===Biology & Literature===
-These two genes within this part are used within an operon in the biosynthesis pathway. YCF54 works with the CTH1 gene and Plastocyanin within the oxidative cyclase pathway. It's interaction with other genes results in the catalysis of the biosynthesis pathway from Mg-protoporphyrin IX to Protochlorophyllide [1]. Deletion studies have detected the importance of YCF54 in maintaining levels of Mg-protoporphyrin IX methyl ester indicating that the YCF54 gene is critical to both the assembly and function of the cyclase complex [1].
+These two genes within this part are used within an operon in the biosynthesis pathway. <i>cF54</i> works with the <i>cTH1</i> gene and Plastocyanin within the oxidative cyclase pathway. It's interaction with other genes results in the catalysis of the biosynthesis pathway from Mg-protoporphyrin IX to Protochlorophyllide [1]. Deletion studies have detected the importance of <i>yCF54</i> in maintaining levels of Mg-protoporphyrin IX methyl ester indicating that the <i>yCF54</i> gene is critical to both the assembly and function of the cyclase complex [1].
-ChlM methylates Mg-protoporphyrin IX, facilitating the highly-regulated catalysis of Mg-chelatase and creating the final protochlorophyllide.
-ChlM methylates Mg-protoporphyrin IX, facilitating the highly-regulated catalysis of Mg-chelatase.
-The insertion of magnesium is the key component of the chlorophyll biosynthesis pathway.
-The plasmid is under the control of the lac promoter.
+The second gene in this part is <i>chlM</i>. <i>chlM</i> encodes for a magnesium protoporhyrin IX methytransferase. <i>chlM's</i> role in the pathway is to methylate Mg-protoporphyrin IX which then aids the catalysis of Mg-cheltase [2]. This then facilitates the formation of the final protochlorophyllide. Involved in the second step of the pathway, the <i>chlM</i> gene is involved in the transfer of a methyl group onto one of the rings of magnesium protoporphyrin which forms the magnesium prootoporhyrin IX monomethylester [3, 4].
+===Part Verification===
+<html><center><img src="https://static.igem.org/mediawiki/2016/b/bd/T--Macquarie_Australia--Importantresults.jpg" " width="50%" height="35%"></center> </html>
+<b>Fig 1.</b>Gel electrophoresis (1% agarose) provides evidence of successful assembly of <i>pLac-ycf54-chlM</i> (1.5 kbp) plasmid (Lane 3). The plasmid was assembled via 3A assembly and double digested to reveal the biobrick backbone (2000 bp) and the correct insert size.<br>
+<br>
+<br>
+<html><center> <img src="https://static.igem.org/mediawiki/2016/f/f2/T--Macquarie_Australia--CBPRes2.png" " width="35%" height="70%"></center> </html>
+<b>Fig 2.</b>PCR confirmation of the <i>pLac-ycf54-chlM</i> plasmid. Forward and reverse <i>chlM</i> primers were used to confirm the presence of the <i>chlM</i> gene. The presence of <i>yCF54</i> gene was also confirmed using the <i>pLac</i> forward primer with the reverse <i>yCF54</i> primer. This also confirmed the presence of the lac promoter followed by <i>yCF54</i> (700 bp in total).<br>
 ===Protein information===
-YCF54<br>
+<i>yCF54</i><br>
 mass: 17.08kDa <br>
 sequence: MAPAAASADKATAAEYYALVCNAEWFFMDPQNESVAEQLREKVRFFKEQN KERDFFIVPNPKWLDAKFPEQAKQVKRPCVALVSTDKMWITFMKLRLDRV LKIDLKSMPASEVLAAGEALPDFKPDGKWTAPYARYTPGWWNVFLPNH <br>
 <br><br>
-ChlM<br>
+<i>chlM</i><br>
 mass: 30.45kDa
 sequence: MASEIAQTADVGSLTFAVGGVGAVVGLGALLVATDHQKRRSEQMKSFDGD EKEAVKDYFNTAGFERWRKIYGETDEVNKVQLDIRTGHAQTVDKVLRWVD EEGSVQGITVADCGCGTGSLAIQLALRGAAVSASDISAAMASEAEQRYQQ AVAAGQGKAPKVAPKFEALDLESVKGKYDTVTCLDVMIHYPQDKVDAMIT HLAGLSDRRLIISFAPKTLSYSILKRIGELFPGPSKATRAYLHREEDVEAALK
@@ Line 42: / Line 43: @@
 ===References===
+[1] Hollingshead S, Kopečná J, Jackson PJ, Canniffe DP, Davison PA, Dickman MJ, Sobotka R, Hunter CN. Conserved chloroplast open-reading frame <i>ycf54</i> is required for activity of the magnesium protoporphyrin monomethylester oxidative cyclase in Synechocystis PCC 6803. Journal of Biological Chemistry. 2012 Aug 10;287(33):27823-33.
+<br><br>
+[2] Alawady A, Reski R, Yaronskaya E, Grimm B. Cloning and expression of the tobacco CHLM sequence encoding Mg protoporphyrin IX methyltransferase and its interaction with Mg chelatase. Plant molecular biology. 2005 Mar 1;57(5):679-91.
+<br><br>
+[3] Shepherd M, McLean S, Hunter CN. Kinetic basis for linking the first two enzymes of chlorophyll biosynthesis. FEBS Journal. 2005 Sep 1;272(17):4532-9.
+<br><br>
+[4] Meinecke L, Alawady A, Schroda M, Willows R, Kobayashi MC, Niyogi KK, Grimm B, Beck CF. Chlorophyll-deficient mutants of Chlamydomonas reinhardtii that accumulate magnesium protoporphyrin IX. Plant molecular biology. 2010 Apr 1;72(6):643-58.