Difference between revisions of "Part:BBa K1640018"
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S-adenosyl-L-methionine + magnesium protoporphyrin IX ⇌ S-adenosyl-L-homocysteine + magnesium protoporphyrin IX 13-methyl ester.
 
S-adenosyl-L-methionine + magnesium protoporphyrin IX ⇌ S-adenosyl-L-homocysteine + magnesium protoporphyrin IX 13-methyl ester.
  
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This part allows E. coli to express the Chlamydomonas reinhardtii-derived ChlM gene involved in the chlorophyll biosynthesis pathway.
 
  
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This part allows ''E. coli'' to express the ''Chlamydomonas reinhardtii''-derived ChlM gene involved in the chlorophyll biosynthesis pathway.
  
 
===Part functionality===
 
===Part functionality===
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<figcaption><i><b>Figure 5.</b> Catalysis of Mg-protoporphyrin IX to Mg-protoporphyrin IX monomethylester by ChlM(4).</i></figcaption>
 
<figcaption><i><b>Figure 5.</b> Catalysis of Mg-protoporphyrin IX to Mg-protoporphyrin IX monomethylester by ChlM(4).</i></figcaption>
  
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<p>To demonstrate functionality of ChlM, we added a lac promoter creating a lac + ChlM composite part (Figure 4). This was transformed into E. coli strains DH5a, K12 and XL-blue. Protein was induced using auto-induction media ZYM5052 (5).</p>
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<p>To demonstrate functionality of ChlM, we added a lac promoter creating a lac + ChlM composite part (Figure 4). This was transformed into <i>E. coli</i> strains DH5a, K12 and XL-blue. Protein was induced using auto-induction media ZYM5052 (5).</p>
  
 
<h5>Functional assay of ChlM</h5>
 
<h5>Functional assay of ChlM</h5>
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<p>When compared to ChlM pET (positive control) expression, which successfully converted 12% of the precursor molecule, it is apparent that our expression of ChlM was very successful.</p>
 
<p>When compared to ChlM pET (positive control) expression, which successfully converted 12% of the precursor molecule, it is apparent that our expression of ChlM was very successful.</p>
  
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<b>Visualisation</b>
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<br>The following gel image shows the expected CHlM banding in lanes 6 and 7, comprising of a EcoRI digest and EcoRI + PstI double digest respectively. This part has been sequenced to confirm design with final biobrick.
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===<span class='h3bb'>Sequence and Features</span>===
 
===<span class='h3bb'>Sequence and Features</span>===
 
<partinfo>BBa_K1640018 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1640018 SequenceAndFeatures</partinfo>

Latest revision as of 08:33, 22 September 2015

CHlM

ChlM gene from Chlamydomonas reinhardtii, encodes magnesium protoporphyrin IX methyltransferase, a protein involved in chlorophyll biosynthesis.

Usage and Biology

The autotrophic gene ChlM is involved in the chlorophyll biosynthetic pathway in the formation of chlorophyll a (Meinecke et al. 2010). It encodes the enzyme magnesium protoporphyrin IX methyltransferase, involved in the second step of chlorophyll biosynthesis from protoporphyrin IX (Block et al, 2002). ChlM utilizes ubiquitous methylating agent S-Adenosyl-L-methionine as a cofactor in the catalysis of Mg-protoporphyrin IX through methyl group transfer, forming Mg-protoporyphyrin IX monomethyl ester (Gibson et al., 1963);


S-adenosyl-L-methionine + magnesium protoporphyrin IX ⇌ S-adenosyl-L-homocysteine + magnesium protoporphyrin IX 13-methyl ester.


This part allows E. coli to express the Chlamydomonas reinhardtii-derived ChlM gene involved in the chlorophyll biosynthesis pathway.

Part functionality

We have demonstrated the functionality of ChlM enzyme encoding magnesium protoporphyrin IX methyltransferase (lac+ChlM: BBa_K1640018), an enzyme within the third operon in the chlorophyll-a biosynthesis pathway. ChlM catalyses the methylation of a carboxyl group in magnesium-protoporphyrin IX using cofactor S-Adenosyl-L-methionine (SAM), yielding magnesium-protophorphyrin IX mono-methyl ester (3);

S-adenosyl-L-methionine + magnesium protoporphyrin IX ⇌ S-adenosyl-L-homocysteine + magnesium protoporphyrin IX 13-methyl ester.


Figure 5. Catalysis of Mg-protoporphyrin IX to Mg-protoporphyrin IX monomethylester by ChlM(4).

To demonstrate functionality of ChlM, we added a lac promoter creating a lac + ChlM composite part (Figure 4). This was transformed into E. coli strains DH5a, K12 and XL-blue. Protein was induced using auto-induction media ZYM5052 (5).

Functional assay of ChlM

Cell lysate containing over expressed ChlM was added with substrates and products were separated by FPLC (Figure 3).


Figure 6. FPLC chromatogram of the products of ChlM activity alongside positive and negative controls. The substrate MG-PPIX elutes at 3.04 mins, while the product Mg-PPIX mono-ester elutes at 4.22 mins.

The FPLC chromatogram data enabled determination of ChlM enzyme activity (Figure 3). This data enabled further determination of levels of Mg-Protoporphyrin IX converted to Mg-Protoporphyrin IX-ME by ChlM. In this instance, 26% of the precursor molecule Mg-Protoporphyrin IX had been catalysed by the enzyme ChlM into Mg-Protoporphyrin IX-ME.

When compared to ChlM pET (positive control) expression, which successfully converted 12% of the precursor molecule, it is apparent that our expression of ChlM was very successful.

Visualisation
The following gel image shows the expected CHlM banding in lanes 6 and 7, comprising of a EcoRI digest and EcoRI + PstI double digest respectively. This part has been sequenced to confirm design with final biobrick.



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 225
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


References

Block, M., Tewari, A., Albrieux, C., Maréchal, E. and Joyard, J. (2002). The plant S-adenosyl-l-methionine:Mg-protoporphyrin IX methyltransferase is located in both envelope and thylakoid chloroplast membranes. European Journal of Biochemistry, 269(1): 240-248.

Gibson, K.D., Neuberger, A. and Tait, G.H. (1963). Studies on the biosynthesis of porphyrin and bacteriochlorophyll by Rhodopseudomonas spheroides. 4. S-Adenosylmethionine–magnesium protoporphyrin methyltransferase. Biochemical Journal, 88(2): 325-334.

Meinecke, L., Alawady, A., Schroda, M., Willows, R., Kobayashi, M., Niyogi, K., Grimm, B. and Beck, C. (2010). Chlorophyll-deficient mutants of Chlamydomonas reinhardtii that accumulate magnesium protoporphyrin IX. Plant molecular biology, 72(6): 643-658.