Difference between revisions of "Part:BBa K4743034"

Revision as of 02:21, 10 October 2023

Astaxanthin production set

The design of the astaxanthin biosynthesis pathway incorporates a range of crucial enzymes that facilitate the synthesis of astaxanthin starting from acetyl-CoA. This design adheres to the yeast's monocistronic system and employs the Adh1 constitutive promoter and Adh1 terminator to enable gene expression within yeast cells. Within this pathway, genes such as chyB, crtE, crtI, CrtYB, and bkt are responsible for encoding catalytic enzymes, including beta-carotene 3-hydroxylase, geranylgeranyl diphosphate synthase, phytoene dehydrogenase, Bifunctional lycopene cyclase/phytoene synthase, and β-carotene ketolase. These enzymes collectively drive the production of astaxanthin.

To aid in the selection process, a hygromycin-resistance gene, hph, has been incorporated into the genetic construct. This allows for the identification and maintenance of yeast cells that have successfully integrated the desired genetic material. It's worth noting that the gene tHMG-1 is naturally present in the Kluyveromyces Marxianus' genome.

The entire genetic construct is integrated into the pklac2 expression vector, which possesses the capacity for replication in E. coli and stable integration into the yeast genome, particularly in kluyveromyces lactis.

Overall metabolic pathway of Astaxanthin

Figure 1. The Matabolic pathway of Astaxanthin production.

Phenotyping

Figure 2. The phenotyping of our strain for the third generation.

Quantitative check

Figure 3. The HPLC test for the third generation.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 8176
Illegal PstI site found at 8266
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 8176
Illegal NheI site found at 627
Illegal NheI site found at 915
Illegal NheI site found at 1119
Illegal NheI site found at 2274
Illegal NheI site found at 9773
Illegal NheI site found at 10061
Illegal NheI site found at 10265
Illegal PstI site found at 8266
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 8176
Illegal BglII site found at 725
Illegal BglII site found at 5611
Illegal BglII site found at 9871
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 8176
Illegal PstI site found at 8266
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 8176
Illegal PstI site found at 8266
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 4377
Illegal BsaI.rc site found at 12941

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 <partinfo>BBa_K4743034 short</partinfo>
-The engineered Kluyveromyces marxianus exhibits a red coloration as a result of successful genetic modification, indicating the accumulation of astaxanthin within the cells. This color change serves as a visual confirmation of the genetic engineering success. Yeast cells reproduce through a process known as budding, wherein some newly formed buds may still be attached to the parent cell. While these buds may lack the desired genes, they can survive the antibiotic selection process due to the antibiotic resistance conferred by the engineered parent cell. It was observed that the cell sizes within the culture vary, and not all of them display the red coloration, suggesting that not all cells in the culture contain the desired genes.
+The design of the astaxanthin biosynthesis pathway incorporates a range of crucial enzymes that facilitate the synthesis of astaxanthin starting from acetyl-CoA. This design adheres to the yeast's monocistronic system and employs the Adh1 constitutive promoter and Adh1 terminator to enable gene expression within yeast cells. Within this pathway, genes such as chyB, crtE, crtI, CrtYB, and bkt are responsible for encoding catalytic enzymes, including beta-carotene 3-hydroxylase, geranylgeranyl diphosphate synthase, phytoene dehydrogenase, Bifunctional lycopene cyclase/phytoene synthase, and β-carotene ketolase. These enzymes collectively drive the production of astaxanthin.
-To address this variation and further refine the culture, a three-generation streak selection method was employed, taking advantage of the easily observable red phenotype in the engineered Kluyveromyces marxianus.
+To aid in the selection process, a hygromycin-resistance gene, hph, has been incorporated into the genetic construct. This allows for the identification and maintenance of yeast cells that have successfully integrated the desired genetic material. It's worth noting that the gene tHMG-1 is naturally present in the Kluyveromyces Marxianus' genome.
+The entire genetic construct is integrated into the pklac2 expression vector, which possesses the capacity for replication in E. coli and stable integration into the yeast genome, particularly in kluyveromyces lactis.
 <span class='h3bb'><big>'''Overall metabolic pathway of Astaxanthin'''</big></span>