Difference between revisions of "Part:BBa K3784005"

Latest revision as of 01:09, 20 October 2021

bg2 (Encoding β-galactosidase)

Patients with CKD are often accompanied by intestinal flora imbalance, and the number, composition, distribution and structure of intestinal flora have changed, which is manifested by the decrease of beneficial bacteria and the abnormal proliferation of pathogenic bacteria and conditional pathogens.

Therefore, we hope that our engineered bacteria can regulate the colony structure of the intestines, promote the proliferation of lactobacilli, bifidobacteria and other probiotics, and inhibit the proliferation of pathogenic bacteria such as Clostridium sporogenes. And galactooligosaccharide(GOS) is a very good choice. Studies have shown that galactooligosaccharide has a very good ability to promote the proliferation of intestinal probiotics such as bifidobacteria and lactobacilli, and it can inhibit the proliferation of intestinal pathogenic bacteria such as Clostridium sporogenes and reduce the production of intestinal endotoxins. At the same time, galactooligosaccharides can alleviate the intestinal inflammation caused by LPS stimulation and enhance the human intestinal barrier function.

We have heterologously expressed the β-galactosidase bga2 of strain Klebsiella oxytoca ZJUH1705 in the engineered bacteria. It has been confirmed that this enzyme has up to 45% of the ability to catalyze lactose to form galacto-oligosaccharides. This is a very high catalytic ability.

Characterization

To test whether our engineering bacteria can transcribe and encode the gene of β-galactosidase, we transformed the plasmid into E.coli BL21(DE3) and induced its expression. After the total RNA was extracted, the cDNA was obtained by reverse transcription. We use cDNA as a template to amplify each fragment, then check whether there is our target band by agarose gel electrophoresis. We chose rsmA (16S rRNA m(6)2A1518, m(6)2A1519 dimethyltransferase) as the reference gene and set a negative control. GOS was well expressed and the corresponding mRNA was successfully transcribed. The expression levels of reference genes in the experimental group (rsmAE) and the control group (rsmAC) were consistent.

bg2 gene expression level verification

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal PstI site found at 1708
12
INCOMPATIBLE WITH RFC[12]
Illegal PstI site found at 1708
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 1708
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 1708
Illegal NgoMIV site found at 176
Illegal NgoMIV site found at 1323
1000
COMPATIBLE WITH RFC[1000]

@@ Line 4: / Line 4: @@
 Patients with CKD are often accompanied by intestinal flora imbalance, and the number, composition, distribution and structure of intestinal flora have changed, which is manifested by the decrease of beneficial bacteria and the abnormal proliferation of pathogenic bacteria and conditional pathogens.
 Therefore, we hope that our engineered bacteria can regulate the colony structure of the intestines, promote the proliferation of lactobacilli, bifidobacteria and other probiotics, and inhibit the proliferation of pathogenic bacteria such as Clostridium sporogenes. And galactooligosaccharide(GOS) is a very good choice. Studies have shown that galactooligosaccharide has a very good ability to promote the proliferation of intestinal probiotics such as bifidobacteria and lactobacilli, and it can inhibit the proliferation of intestinal pathogenic bacteria such as Clostridium sporogenes and reduce the production of intestinal endotoxins. At the same time, galactooligosaccharides can alleviate the intestinal inflammation caused by LPS stimulation and enhance the human intestinal barrier function.
 We have heterologously expressed the β-galactosidase bga2 of strain Klebsiella oxytoca ZJUH1705 in the engineered bacteria. It has been confirmed that this enzyme has up to 45% of the ability to catalyze lactose to form galacto-oligosaccharides. This is a very high catalytic ability.
 ===Characterization===
-To test whether our engineering bacteria can transcribe and encode normally the gene of β-galactosidase, we extracted the enzyme RNA for cDNA, and used it as template for PCR after agarose gel electrophoresis.
+To test whether our engineering bacteria can transcribe and encode  the gene of β-galactosidase, we transformed the plasmid into E.coli BL21(DE3) and induced its expression. After the total RNA was extracted, the cDNA was obtained by reverse transcription. We use cDNA as a template to amplify each fragment, then check whether there is our target band by agarose gel electrophoresis. We chose rsmA (16S rRNA m(6)2A1518, m(6)2A1519 dimethyltransferase) as the reference gene and set a negative control. GOS was well expressed and the corresponding mRNA was successfully transcribed. The expression levels of reference genes in the experimental group (rsmAE) and the control group (rsmAC) were consistent.
+[[File:BNUZ-gp2.png|600px|border|center|]]
+<div class = "center">bg2 gene expression level verification</div><br>
 <!-- Add more about the biology of this part here
 ===Usage and Biology===