Difference between revisions of "Part:BBa K1998000"

Revision as of 01:36, 21 October 2016

Mg-Chelatase Plasmid

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 4753
Illegal NotI site found at 2666
Illegal NotI site found at 4213
21
INCOMPATIBLE WITH RFC[21]
Unknown
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 1946
Illegal NgoMIV site found at 2765
Illegal NgoMIV site found at 3307
Illegal NgoMIV site found at 7968
Illegal AgeI site found at 6853
Illegal AgeI site found at 6883
Illegal AgeI site found at 7777
Illegal AgeI site found at 7945
Illegal AgeI site found at 9463
Illegal AgeI site found at 9517
Illegal AgeI site found at 9736
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 5553
Illegal BsaI.rc site found at 5709
Illegal BsaI.rc site found at 6330
Illegal BsaI.rc site found at 9862
Illegal SapI.rc site found at 7219
Illegal SapI.rc site found at 9792

Overview

The fate of the protoporphyrin IX (PPIX) is dependent upon which enzyme is present to convert PPIX to the next product. For instance, ferrochelatase (encoded by hemH) converts PPIX to FePPIX (heme). Our magnesium chelatase plasmid is an assembly of six biobricks previously registered by a past Macquarie iGEM team. These genes form the first part of the chlorophyll biosynthesis pathway. The six genes are in the order of chlI1, chlD, gun4, chlI2, cTH1, followed by chlH. At the beginning of each gene, a ribosome binding site can be found. plac is also located at the front of chlH. Another plac is at the beginning of chlI1. The genes encode for Magnesium chelatase subunit H (chlH), Magnesium chelatase subunit I (chli1/chli2), Magnesium chelatase subunit D (chlD), Genomes uncoupled 4 (gUN4) and Copper target 1 protein (cth1). These genes are involved in converting protoporphyrin IX to divinyl protochlorophyllide in the presence of NADPH and O2.

Biology & Literature

Genes chlI1 and chlI2 form a Mg-cheltase complex (Magnesium cheltase subunit I). This complex catalyses the insertion of magnesium ion into protoporphyrin IX to yield Mg-protoporphyrin IX by forming an ATP dependent hexameric ring complex and a complex with the chlD subunit. This complex then acts on protoporphyrin. The gUN4 gene encodes genomes uncoupled 4 protein. This is a tetrapyrrole-binding protein which controls the production of Mg-protoporhyrin IX. The cTH1 gene encodes for a copper target protein. It forms when oxygen and copper are present catalysing Mg-protoporphyrin IX mono methyl, and subsequently converts to divinyl protochlorophyllide when NADPH and O2 are present. The last gene is chlH. This gene encodes for the magnesium shelters subunit H. This acts as a chloroplast precursor, catalysing the first step in the chlorophyl biosynthesis pathway by insertion of an Mg2+ ion into protoporphyrin IX to generate Mg-protoporphyrin IX.

The chlH and gUN4 proteins bind to protoporphyrin IX and form an activated substrate complex which behaves as a substrate for the motor complex (ChlID complex) to insert magnesium into the bound Protoporphyrin IX upon ATP hydrolysis. The assembly of this complex requires milliMolar concentrations of Mg2+ and ATP to assemble. While the substrate complex also requires microMolar concentrations of Protoporphyrin IX and chlH for optimal activity.

Assembly and Design

Each individual biobrick was assembled in the following order using 3A assembly: pLac-chlI1-chlD-gun4-chlI2-cTH1-pLac-chlH [BBa_R0010], [BBa_K1326008], [BBa_K1080011], [BBa_K1080005] and [BBa_K1640019]. Five of these genes code for different subunits of the enzyme Magnesium chelatase, with the cTH1 gene encoding a component of a later enzyme in the pathway, the oxidative cyclase.

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

Characterisation and Verification

The following information shows the characterisation process of this plasmid as well as it's verification.

1. The digests of Mg-chelatase plasmid reveal the correct band sizes of the insert (approx 11 kbp). ChlH protein (approx. 150kDa) is also highly expressed and visible on SDS-PAGE.

2. Mass spectrometry confirms the presence of magnesium chelatase subunits. important 1st

Fig 1. Protein expression of the Mg-chelatase plasmid induced with IPTG. Lane 2 is the uninduced culture. Highly expressed band at approximately 144 kDa represents the ChlH protein. The MW of other proteins of interest include ChlI1 (40 kDa), ChlD (63 kDa), Gun4 (24 kDa), ChlI2 (40 kDa) and CTH1 (43 kDa).

@@ Line 37: / Line 37: @@
 <br>
 . Mass spectrometry confirms the presence of magnesium chelatase subunits.
-<img src=" https://static.igem.org/mediawiki/2016/b/bd/T--Macquarie_Australia--Importantresults.jpg", alt="important 1st " height="200%" width="400%"></center>
+<html><img src=" https://static.igem.org/mediawiki/2016/b/bd/T--Macquarie_Australia--Importantresults.jpg", alt="important 1st " height="200%" width="400%"></center>
 <figcaption><div class="ex2"  align="justify"> <font face="Corbel" font style="line-height:1.5" font size="2" color="#404040"><b>Fig 1.</b> Protein expression of the Mg-chelatase plasmid induced with IPTG. Lane 2 is the uninduced culture. Highly expressed band at approximately 144 kDa represents the <i>ChlH</i> protein. The MW of other proteins of interest include <i>ChlI1</i> (40 kDa), <i>ChlD</i> (63 kDa), Gun4 (24 kDa), <i>ChlI2</i> (40 kDa) and CTH1 (43 kDa).</center></figcaption></div></font><br>
 <br>
@@ Line 60: / Line 60: @@
 <figcaption><div class="ex2"  align="justify"> <font face="Corbel" font style="line-height:1.5" font size="2" color="#404040"><b>Fig 5. </b>Fluorescence excitation and emission spectra demonstrates the production of Mg-PPIX upon the addition of Mg-chelatase plasmid to ΔhemeH mutants. Mg-PPIX has an emission and excitation spectra of 418nm and 592nm respectively.</center></figcaption></div></font><br>
 <img src="https://static.igem.org/mediawiki/2016/9/96/T--Macquarie_australia--POC4_.jpeg", alt="hemH mutants 2" height="200%" width="400%"></center>
-<figcaption><div class="ex2"  align="justify"> <font face="Corbel" font style="line-height:1.5" font size="2" color="#404040"><b>Fig 6. </b>Emission and excitation spectra of the in vitro assay of Mg-chelatase from the Mg-P plasmid. Zero time control is the initial emission/excitation spectra when Mg-chelatase is added to 10mM PPIX, 10mM ATP and 15mM MgCl2. After incubating the assay under constant reagents as the control at room temperature, Mg-PPIX production increases. When additional 50mM MgCl<sub>2</sub> is added to the assay in comparison to the control, Mg-PPIX production increases further. However, upon addition of 50mM MgCl<sub>2</sub> including 20nM of ChlID complex, the production of Mg-PPIX remains the same. Since adding additional ChlID enzyme to the last assay does not change Mg-PPIX production, the ChlID complex is not limiting.</center></figcaption></div></font><br>
+<figcaption><div class="ex2"  align="justify"> <font face="Corbel" font style="line-height:1.5" font size="2" color="#404040"><b>Fig 6. </b>Emission and excitation spectra of the in vitro assay of Mg-chelatase from the Mg-P plasmid. Zero time control is the initial emission/excitation spectra when Mg-chelatase is added to 10mM PPIX, 10mM ATP and 15mM MgCl2. After incubating the assay under constant reagents as the control at room temperature, Mg-PPIX production increases. When additional 50mM MgCl<sub>2</sub> is added to the assay in comparison to the control, Mg-PPIX production increases further. However, upon addition of 50mM MgCl<sub>2</sub> including 20nM of ChlID complex, the production of Mg-PPIX remains the same. Since adding additional ChlID enzyme to the last assay does not change Mg-PPIX production, the ChlID complex is not limiting.</center></figcaption></div></font><br></html>
 Further information can be found here: http://2016.igem.org/Team:Macquarie_Australia/Proof