Difference between revisions of "Part:BBa K3753001"

Revision as of 14:44, 16 October 2021

PAAS-Vanda

Vanda hybrid phenylacetaldehyde synthase (PAAS) is capable of transforming L-phenylalanine (L-phe) into phenylacetaldehyde by oxidative decarboxylation. Subsequently, phenylacetaldehyde can be reducted into phenylethanol, which possesses elegant, fascinating and long-lasting fragrance.

Characterization

Fig.1 The growth curve of recombinant strains.

The figure above shows the changes of biomass concentration in WT, BY4741-pRS426-Petunia, BY4741-pRS426-Vanda, BY4741-pRS426-Rosa yeast over time. The OD₆₀₀ of different strain cultures is measured at the designated time points (0h, 24h,48h,72h). The result shows that the growth trend of the recombinant strain is basically the same as the wild-type strain, indicating that the introduction of heterogeneous gene has no significant effect on the growth of yeast.

Fig.2 The 2-PE production of the recombinant strains.
After 72 hours of fermentation, 2-PE production of the wild-type of BY4741, pRS426-Petunia, pRS426-Vanda and pRS426-Rosa was tested by HPLC. Data represent means of triplicate culture±standard error.

Saccharomyces cerevisiae BY4741 contains Ehrlich pathway and other metabolic pathways to operate simultaneously to produce 2-PE, so the wild-type of BY4741 has a certain amount of 2-PE production (1.205g/L). After the introduction of heterogeneous paas gene, the 2-PE production has remarkablely increased. Among them, the production of 2-PE produced by the yeast strain which was introduced petunia-paas increased the most(1.570g/L), followed by the strain which was introduced vanda-paas (1.514g/L) and rosa-paas (1.341g/L).

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1033
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 ===Characterization===
-Numerous experiments have shown that the growth of the modified strain was not significantly different from that of the wild type. Import genes had no significant effect on yeast growth and metabolism (Fig. 1). Because yeast contains Ehrlich pathway and other metabolic pathways, they can work together to produce 2-PE, Saccharomyces cerevisiae BY4741 wild-type contains a certain amount of 2-PE production (1.205g/L). After the introduction of exogenous paas gene, the 2-PE production has increased. Among Petunia PAAS <partinfo>BBa_K3753000</partinfo>, Vanda PAAS and Rosa PAAS <partinfo>BBa_K3753002</partinfo>, Petunia PAAS has the most notable effect. The 2-PE production of yeast cells carrying pRS426 plasmid with Petunia PAAS reached 1.570g/L, with Vanda PAAS (1.514g/L) and with Rosa PAAS (1.341g/L) following closely (Fig. 2).
 <br>
 <p> </p>
-[[File:OD.png|width='100%' valign='top'| |center|thumb|550px|''<b>Fig.1</b> Changes in WT, BY4741-pRS426-Petunia, BY4741-pRS426-Vanda, BY4741-pRS426-Rosa yeast biomass concentration over time. The OD600 of different strains cultures used for analysis of 2-PE was measured at the indicated time points (Samples 0h, 24h,48h,72h). Data represent means of triplicate cultures ± standard error.]]
+[[File:OD.png|width='100%' valign='top'| |center|thumb|550px|''<b>Fig.1</b> The growth curve of recombinant strains.]]
 <br>
+The figure above shows the changes of biomass concentration in WT, BY4741-pRS426-Petunia, BY4741-pRS426-Vanda, BY4741-pRS426-Rosa yeast over time. The OD<sub>600</sub> of different strain cultures is measured at the designated time points (0h, 24h,48h,72h). The result shows that the growth trend of the recombinant strain is basically the same as the wild-type strain, indicating that the introduction of heterogeneous gene has no significant effect on the growth of yeast.
 <br>
 <p> </p>
-[[File:2-PE.png|width='100%' valign='top'| |center|thumb|550px|''<b>Fig.2</b> Introduced plasmids with target gene paas elevates 2-PE levels. Yeast cells carrying pRS426 plasmid with Petunia PAAS , Vanda PAAS, Rosa PAAS or, as controls, the unrelated Saccharomyces cerevisiae BY4741 wild-type, were grown in fermentation media. Cultures analyzed sampling for 72h, followed by HPLC to determine 2-PE levels. Data represent means of triplicate culture±standard error.]]
+[[File:2-PE.png|width='100%' valign='top'| |center|thumb|550px|''<b>Fig.2</b> The 2-PE production of the recombinant strains.
 <br>
+After 72 hours of fermentation, 2-PE production of the wild-type of BY4741, pRS426-Petunia, pRS426-Vanda and pRS426-Rosa was tested by HPLC. Data represent means of triplicate culture±standard error.]]
+<p> </p>
+<br>
+''Saccharomyces cerevisiae'' BY4741 contains Ehrlich pathway and other metabolic pathways to operate simultaneously to produce 2-PE, so the wild-type of BY4741 has a certain amount of 2-PE production (1.205g/L). After the introduction of heterogeneous ''paas'' gene, the 2-PE production has remarkablely increased. Among them, the production of 2-PE produced by the yeast strain which was introduced ''petunia-paas'' increased the most(1.570g/L), followed by the strain which was introduced ''vanda-paas'' (1.514g/L) and ''rosa-paas'' (1.341g/L).