Difference between revisions of "Part:BBa K3179004"

Latest revision as of 18:48, 21 October 2019

2Kt-E1A

This part is constructed by adding a 2X Kturn sequence before the E1A gene of Ad5. Due to the existence of 2X Kturn, the translation of E1A is regulated by L7Ae. E1A is an early gene of adenovirus and is essential in the commence of viral replication. This part has an intron which also exists in E1A of the wild adenovirus. Using the splicing system in eukaryotes can make it function as normal. In our design, 2kt-E1A is used as a response part to initiate viral gene expression and lyse target cells.

We integrate 2kt-E1A with miR885-5p-target and miR663b-target so that while miRNA885-5p and miRNA663b are absent in the cell, E1A can be translated and initiate viral gene expression to lyse target cells.

Usage and Biology

Since the gene product of the E1 can initiate the gene replication of adenovirus and it’s not been reported that it has any other function in eukaryotic cells, SYSU-CHINA decided to transfection pTRE-E1A, which is a plasmid containing E1A gene regulated by pTRE, with replication-defective adenovirus, which lacks the E1 gene, into HeLa cell. The cell experiment showed that replication-defective adenovirus can’t replicate and lyse cells. However, if there is E1A existed, the virus can be autonomously replicable and lyse cells. As we see, higher concentration of pTRE-E1A can make better effect.

The cell viability assay is shown below. It can be easily seen that the cell viabilities in experimental group are lower than the control ones. Although the effect is not as good as we thought, it still means that it can function.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal XbaI site found at 859
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 63
23
INCOMPATIBLE WITH RFC[23]
Illegal XbaI site found at 859
25
INCOMPATIBLE WITH RFC[25]
Illegal XbaI site found at 859
Illegal NgoMIV site found at 333
Illegal AgeI site found at 69
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3179004 short</partinfo>
-This part is constructed by adding 2X Kturn sequence before Adenovirus E1A gene. Due to the existence of 2X Kturn, the translation of E1A is controlled by L7Ae. E1A is an early gene of adenovirus and is essential in the commence of viral replication.
+This part is constructed by adding a 2X Kturn sequence before the E1A gene of Ad5. Due to the existence of 2X Kturn, the translation of E1A is regulated by L7Ae. E1A is an early gene of adenovirus and is essential in the commence of viral replication.
-This part has an intron which also exists in E1A in the original adenoviral genome. Thus using the splicing system in eukaryotes can make it function as normal. In our design, 2kt-E1A is used as a response part to startup viral gene expression and lyse target cells.
+This part has an intron which also exists in E1A of the wild adenovirus. Using the splicing system in eukaryotes can make it function as normal. In our design, 2kt-E1A is used as a response part to initiate viral gene expression and lyse target cells.
-We integrate 2kt-E1 with miR885-5p-target and miR663b-target so that while miRNA885-5p and miR663b-target are absent in the cell, E1A can be translated to startup viral gene expression and lyse target cells.
+We integrate 2kt-E1A with miR885-5p-target and miR663b-target so that while miRNA885-5p and miRNA663b are absent in the cell, E1A can be translated and initiate viral gene expression to lyse target cells.
+<br style="clear: both" />
+==Usage and Biology==
+[[File:T-SYSU-CHINA-pTRE-E.jpg|500px|thumb|left]]
+<br style="clear: both" />
+Since the gene product of the E1 can initiate the gene replication of adenovirus and it’s not been reported that it has any other function in eukaryotic cells, SYSU-CHINA decided to transfection pTRE-E1A, which is a plasmid containing E1A gene regulated by pTRE, with replication-defective adenovirus, which lacks the E1 gene, into HeLa cell. The cell experiment showed that replication-defective adenovirus can’t replicate and lyse cells. However, if there is E1A existed, the virus can be autonomously replicable and lyse cells. As we see, higher concentration of pTRE-E1A can make better effect.
+The cell viability assay is shown below. It can be easily seen that the cell viabilities in experimental group are lower than the control ones. Although the effect is not as good as we thought, it still means that it can function.
+<br style="clear: both" />
+[[File:T-SYSU-CHINA-pTRE-E1A.png|500px|thumb|left]]
+<br style="clear: both" />