Difference between revisions of "Part:BBa K4621003"

Latest revision as of 16:59, 5 October 2023

A whole functional sequence in front of the chitinase Chi19C gene, including its promoter.

Usage, Biology and Characterization：

chi19C hypothetical promoter is a functional fragment in chitinase Chi19C gene, which was cloned from SCUT-3 gene. In our project, through transcriptome analysis, qPCR and other means, we found that the gene expression of chitinase Chi19C was up-regulated when SCUT-3 was in chitin medium, and we speculated that its promoter chi19C hypothetical promoter may have properties that can be induced by chitin.

Clonal electrophoresis images of the fragments are shown in Figure. 1, with lane 3 representing chi19C hypothetical promoter.

Fig.1 Gel electrophoresis results

chi19C hypothetical promoter and genes encoding a series of enzymes responsible for 5-Hydroxyectoine synthesis (ectoperon) were overexpressed using an integrated plasmid in Streptomyces SCUT-3. The modified strain was inoculated in 40 mL LB medium and fermented at 40℃ and 220 rpm for 120h. The production of Ectoine and 5-Hydroxyectoin in the presence or without chitin (the first day of fermentation) was detected to evaluate the chitin inducability of the promoter. The results are shown in Figure. 2.

Fig.2 Fermentation of products with or without the addition of an inducer（chitin) (a).Ectoine (b).5-Hydroxyectoin(120h)

By adding chitin at different days after fermentation, we also determined that the optimal time for chitin induction of chi19C hypothetical promoter was the second day of fermentation, and the specific experimental data are shown in Figure 3:

Fig.3 Fermentation of the product of SCUT-3-EctC at different induction times

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 93
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K4621003 short</partinfo>
-This part contains the spacer sequence used to target a specific DNA fragment of a homoserine dehydrogenase gene, GQS52_08040 in SCUT-3. It must be co-expressed with a sgRNA that has a 20-base pair complementary sequence to the target gene that dCas9 can effectively silence the gene of interest [1]. In our case, we used it to target the binding site of RNP to effectively repress the transcription of the target gene. As a result, the production of the target product is intricately regulated.
+Usage, Biology and Characterization：
+chi19C hypothetical promoter is a functional fragment in chitinase Chi19C gene, which was cloned from SCUT-3 gene. In our project, through transcriptome analysis, qPCR and other means, we found that the gene expression of chitinase Chi19C was up-regulated when SCUT-3 was in chitin medium, and we speculated that its promoter chi19C hypothetical promoter may have properties that can be induced by chitin.
+Clonal electrophoresis images of the fragments are shown in Figure. 1, with lane 3 representing chi19C hypothetical promoter.
+https://static.igem.wiki/teams/4621/wiki/parts/gel-electrophoresis-results.png
+Fig.1 Gel electrophoresis results
+chi19C hypothetical promoter and genes encoding a series of enzymes responsible for 5-Hydroxyectoine synthesis (ectoperon) were overexpressed using an integrated plasmid in Streptomyces SCUT-3. The modified strain was inoculated in 40 mL LB medium and fermented at 40℃ and 220 rpm for 120h. The production of Ectoine and 5-Hydroxyectoin in the presence or without chitin (the first day of fermentation) was detected to evaluate the chitin inducability of the promoter. The results are shown in Figure. 2.
+https://static.igem.wiki/teams/4621/wiki/parts/effect-of-different-promoters-on-yield.png
+Fig.2 Fermentation of products with or without the addition of an inducer（chitin) (a).Ectoine (b).5-Hydroxyectoin(120h)
+By adding chitin at different days after fermentation, we also determined that the optimal time for chitin induction of chi19C hypothetical promoter was the second day of fermentation, and the specific experimental data are shown in Figure 3:
+https://static.igem.wiki/teams/4621/wiki/parts/effect-of-different-induction-times-on-yield-c.png
+Fig.3 Fermentation of the product of SCUT-3-EctC at different induction times
 <!-- Add more about the biology of this part here