Difference between revisions of "Part:BBa K4325018"
| Line 20: | Line 20: | ||
<h4>1.Batch screening of pDawn(cI-LVA)-LKD-LVA-T1 in response to blue light lysis in <i>E. coli</i>.</h4> | <h4>1.Batch screening of pDawn(cI-LVA)-LKD-LVA-T1 in response to blue light lysis in <i>E. coli</i>.</h4> | ||
<p>As shown in Figure 1, We inserted pDawn(cI-LVA)-RBS070-LKD-LVA-T1 into <i>E. coli</i> TOP10 and performed the agarose gel electrophoresis. Then we cultured monocloning site in the dark and under the light. After 12 hours, we found that the 4<sup>th</sup>, 5<sup>th</sup>, 6<sup>th</sup>, 14<sup>th</sup> bacteria grew in the dark and did not grow under the light,(Figure 2) which indicates that pDawn(cI-LVA)-RBS070-LKD-LVA-T1 was successfully expressed in <i>E. coli</i> TOP10.</p> | <p>As shown in Figure 1, We inserted pDawn(cI-LVA)-RBS070-LKD-LVA-T1 into <i>E. coli</i> TOP10 and performed the agarose gel electrophoresis. Then we cultured monocloning site in the dark and under the light. After 12 hours, we found that the 4<sup>th</sup>, 5<sup>th</sup>, 6<sup>th</sup>, 14<sup>th</sup> bacteria grew in the dark and did not grow under the light,(Figure 2) which indicates that pDawn(cI-LVA)-RBS070-LKD-LVA-T1 was successfully expressed in <i>E. coli</i> TOP10.</p> | ||
| − | [[File:K18 1.png|600px|thumb|center|Figure 1: | + | [[File:K18 1.png|600px|thumb|center|Figure 1: (1) pDawn(cI)-X174E-T1; (2) pDawn(cI-LVA)-X174E-LVA-T1; (3) pDawn(cI)-LKD-T1; <b>(4) pDawn(cI-LVA)-RBS070-LKD-LVA-T1</b> were constructed successfully.]] |
[[File:K18 2.png|600px|thumb|center|Figure 2:Growth condition of pDawn(cI-LVA)-RBS070-LKD-LVA-T1-TOP10 in the dark and under the light]] | [[File:K18 2.png|600px|thumb|center|Figure 2:Growth condition of pDawn(cI-LVA)-RBS070-LKD-LVA-T1-TOP10 in the dark and under the light]] | ||
Revision as of 17:44, 11 October 2022
pDawn-RBS070-LKD-LVA-T1
Description
This composite part is a generator consisting of pDawn(cI-LVA) (BBa_K1075044) and LKD-LVA (BBa_K4325011).
Usage
We inserted the pDawn(cI-LVA) blue light response system (BBa_K1075044) and the lysis gene with hydrolysis tag LKD-LVA (BBa_K4325011) into the pSEVA331 expression vector, which was inserted into E. coli TOP10 and screened for bacterial colony that grew in the dark but did not grow under blue light. Finally, the pDawn (cI-LVA)-LKD-LVA-T1 plasmids were inserted into G. hansenii ATCC53582 by electroporation to verify the responsiveness of pDawn(cI-LVA) to blue light.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2171
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 63
Illegal NgoMIV site found at 195
Illegal NgoMIV site found at 289
Illegal NgoMIV site found at 582
Illegal NgoMIV site found at 1076
Illegal NgoMIV site found at 1094
Illegal NgoMIV site found at 1184
Illegal AgeI site found at 414
Illegal AgeI site found at 1542 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1643
Illegal BsaI.rc site found at 525
2022 SZPT-China
Characterization
1.Batch screening of pDawn(cI-LVA)-LKD-LVA-T1 in response to blue light lysis in E. coli.
As shown in Figure 1, We inserted pDawn(cI-LVA)-RBS070-LKD-LVA-T1 into E. coli TOP10 and performed the agarose gel electrophoresis. Then we cultured monocloning site in the dark and under the light. After 12 hours, we found that the 4th, 5th, 6th, 14th bacteria grew in the dark and did not grow under the light,(Figure 2) which indicates that pDawn(cI-LVA)-RBS070-LKD-LVA-T1 was successfully expressed in E. coli TOP10.
References
[1]Robert Ohlendorf, Roee R. Vidavski, Avigdor Eldar et.al.From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression.Journal of Molecular Biology, 08 Jan 2012, 416(4):534-542.