Difference between revisions of "Part:BBa K4325027"
(→‎2022 SZPT-China)
Line 22: Line 22:
  
 
<h4>1.Batch screening of pDawn(CI-LVA)-LKD-T1-pSEVA331 in response to blue light lysis in <i>E. coli</i> TOP10.</h4>
 
<h4>1.Batch screening of pDawn(CI-LVA)-LKD-T1-pSEVA331 in response to blue light lysis in <i>E. coli</i> TOP10.</h4>
−
<p>As shown in Figure 2, except for the 16<sup>th</sup> bacterial colony,almost all of the plaque did not grow under blue light To further show the lysis effect of pDawn (cI-LVA) -LKD-T1-pSEVA331-TOP10, we measured the OD <sub>600</sub> values of pSEVA331-pDawn (cI-LVA) -LKD-T1-TOP10 periodically and ploting the growth curve diagram . As shown in Figure 3, pSEVA331-pDawn (cI-LVA) -LKD-T1-TOP10 was not excellent.</p>
+
<p>As shown in Figure 2, except for the 16<sup>th</sup> bacterial colony,almost all of the plaque did not grow under blue light. To further show the lysis effect of pDawn (cI-LVA) -LKD-T1-pSEVA331-TOP10, we measured the OD <sub>600</sub> values of pSEVA331-pDawn (cI-LVA) -LKD-T1-TOP10 periodically and ploting the growth curve diagram . As shown in Figure 3, pSEVA331-pDawn (cI-LVA) -LKD-T1-TOP10 was not excellent.</p>
 
[[File:K27 2.png|600px|thumb|center|Figure 2: <p></p> Growth situation of pSEVA331-pDawn (cI-LVA) -LKD-T1-TOP10 in the dark and under the blue light.]]
 
[[File:K27 2.png|600px|thumb|center|Figure 2: <p></p> Growth situation of pSEVA331-pDawn (cI-LVA) -LKD-T1-TOP10 in the dark and under the blue light.]]
 
[[File:K27 4.png|600px|thumb|center|Figure 3: <p></p> Growth curve diagram of pSEVA331-pDawn (cI-LVA) -LKD-T1-TOP10 in different growth conditions.]]
 
[[File:K27 4.png|600px|thumb|center|Figure 3: <p></p> Growth curve diagram of pSEVA331-pDawn (cI-LVA) -LKD-T1-TOP10 in different growth conditions.]]
  
 
<h3>2.pSEVA331-pDawn(cI-LVA)-LKD-T1 in response to blue photolysis in <i>G. hansenii</i> ATCC53582.</h3>
 
<h3>2.pSEVA331-pDawn(cI-LVA)-LKD-T1 in response to blue photolysis in <i>G. hansenii</i> ATCC53582.</h3>
−
<p>As shown in Figure 3, pDawn(cI-LVA)-LKD-T1 plasmid was successfully identified in <i>G. hansenii</i> ATCC53582 by agarose gel electrophoresis after electroporation the plasmid into our chassis bacteria.(electro-transforming)</p>
+
<p>As shown in Figure 3, pDawn(cI-LVA)-LKD-T1 plasmid was successfully identified in <i>G. hansenii</i> ATCC53582 by agarose gel electrophoresis after transformed the plasmid into our chassis bacteria.(electro-transforming)</p>
 
[[File:K27 5.png|600px|thumb|center|Figure 4:Successfully identified by agarose gel electrophoresis of  <p></p>  
 
[[File:K27 5.png|600px|thumb|center|Figure 4:Successfully identified by agarose gel electrophoresis of  <p></p>  
 
(1)pSEVA331-pDawn(cI-LVA)-RBS070-LKD-T1-<i>G. hansenii</i> ATCC53582 <p></p>
 
(1)pSEVA331-pDawn(cI-LVA)-RBS070-LKD-T1-<i>G. hansenii</i> ATCC53582 <p></p>

Revision as of 12:55, 10 October 2022

pDawn-LKD-T1

Description

This composite part is a generator consisting of pDawn (CI-LVA) (BBa_K1075044) and LKD (BBa_K4325004).

Usage

The pDawn (CI-LVA) blue light corresponding system (BBa_K1075044) and the lysis gene LKD (BBa_K4325004) were inserted into the pSEVA331 expression vector, which was inserted into E. coli TOP10 and screened out bacterial colony that grew in the dark but did not grow under blue light. Finally, the pSEVA331-pDawn (CI-LVA) -LKD-T1 plasmid was extracted and inserted into G. hansenii ATCC53582, after which we verified the responsiveness of pDawn (CI-LVA) to blue light.

Figure 1:

Gene circuit of pDawn (CI-LVA)-LKD-T1.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2171
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE 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
  • 1000
    INCOMPATIBLE 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-T1-pSEVA331 in response to blue light lysis in E. coli TOP10.

As shown in Figure 2, except for the 16th bacterial colony,almost all of the plaque did not grow under blue light. To further show the lysis effect of pDawn (cI-LVA) -LKD-T1-pSEVA331-TOP10, we measured the OD 600 values of pSEVA331-pDawn (cI-LVA) -LKD-T1-TOP10 periodically and ploting the growth curve diagram . As shown in Figure 3, pSEVA331-pDawn (cI-LVA) -LKD-T1-TOP10 was not excellent.

Figure 2:

Growth situation of pSEVA331-pDawn (cI-LVA) -LKD-T1-TOP10 in the dark and under the blue light.
Figure 3:

Growth curve diagram of pSEVA331-pDawn (cI-LVA) -LKD-T1-TOP10 in different growth conditions.

2.pSEVA331-pDawn(cI-LVA)-LKD-T1 in response to blue photolysis in G. hansenii ATCC53582.

As shown in Figure 3, pDawn(cI-LVA)-LKD-T1 plasmid was successfully identified in G. hansenii ATCC53582 by agarose gel electrophoresis after transformed the plasmid into our chassis bacteria.(electro-transforming)

Figure 4:Successfully identified by agarose gel electrophoresis of

(1)pSEVA331-pDawn(cI-LVA)-RBS070-LKD-T1-G. hansenii ATCC53582

(2)pSEVA331-pDawn (cI-LVA)-LKD-T1-G. hansenii ATCC53582

(3)pSEVA331-pDawn(cI-LVA)-RBS070-X174E-T1-G. hansenii ATCC53582

(4)pSEVA331-pDawn(CI-LVA)-X174E-T1-G. hansenii ATCC53582

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.