Difference between revisions of "Part:BBa K3740033"
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<h3>Usage</h3>
 
<h3>Usage</h3>
 
<p>The target gene of pDawn blue light response system (<partinfo>BBa_K1075044</partinfo>) and Lysis Cassette LKD16 (<partinfo>BBa_K3740032</partinfo>) were insert into the pSEVA331 expression vector. Then, random primer guided mutagenesis method to modulate the strength of the RBS (<partinfo>BBa_B0034</partinfo>) located upstream of LKD16. After introducing into <i>E. coli</i> DH5α, <i>E. coli</i> isolates that normally grow in the dark and cannot under the light of 470nm blue light were screened. Finally, the pDawn-RBSNNN-LKD16-rrnB T1 plasmid was extracted, and further introduced into <i>G. hansenii</i> ATCC 53582, in which the responsiveness of pDawn to blue light was also verified.</p>
 
<p>The target gene of pDawn blue light response system (<partinfo>BBa_K1075044</partinfo>) and Lysis Cassette LKD16 (<partinfo>BBa_K3740032</partinfo>) were insert into the pSEVA331 expression vector. Then, random primer guided mutagenesis method to modulate the strength of the RBS (<partinfo>BBa_B0034</partinfo>) located upstream of LKD16. After introducing into <i>E. coli</i> DH5α, <i>E. coli</i> isolates that normally grow in the dark and cannot under the light of 470nm blue light were screened. Finally, the pDawn-RBSNNN-LKD16-rrnB T1 plasmid was extracted, and further introduced into <i>G. hansenii</i> ATCC 53582, in which the responsiveness of pDawn to blue light was also verified.</p>
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[[File:szpt39.png|600px|thumb|center|Figure 1. Gene circuit of LKD16(<partinfo>BBa_K3740032</partinfo>).]]
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[[File:szpt.90.jpg|600px|thumb|center|Figure 1. Gene circuit of LKD16(<partinfo>BBa_K3740032</partinfo>).]]
 
<h3>Characterization</h3>
 
<h3>Characterization</h3>
 
<h4>1.Batch screening of pDawn-RBSNNN-LKD16-rrnB T1-pSEVA331 in response to blue light lysis in <i>E. coli</i>.</h4>
 
<h4>1.Batch screening of pDawn-RBSNNN-LKD16-rrnB T1-pSEVA331 in response to blue light lysis in <i>E. coli</i>.</h4>

Latest revision as of 18:02, 21 October 2021


pDawn-B0034-LKD16-rrnB T1

Description

This composite part is a generator consisting of the pDawn promoter and LKD16.

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


2021 SZPT-China

Biology

This composite part is a generator consisting of the pDawn (BBa_K1075044) promoter and LKD16 (BBa_K3740032).

Usage

The target gene of pDawn blue light response system (BBa_K1075044) and Lysis Cassette LKD16 (BBa_K3740032) were insert into the pSEVA331 expression vector. Then, random primer guided mutagenesis method to modulate the strength of the RBS (BBa_B0034) located upstream of LKD16. After introducing into E. coli DH5α, E. coli isolates that normally grow in the dark and cannot under the light of 470nm blue light were screened. Finally, the pDawn-RBSNNN-LKD16-rrnB T1 plasmid was extracted, and further introduced into G. hansenii ATCC 53582, in which the responsiveness of pDawn to blue light was also verified.

Figure 1. Gene circuit of LKD16(BBa_K3740032).

Characterization

1.Batch screening of pDawn-RBSNNN-LKD16-rrnB T1-pSEVA331 in response to blue light lysis in E. coli.

As shown in Figure 2, the 4th, 9th, 10th and 12th of the plate can grow normally in the dark, but not grow under blue light, indicating that LKD16 can induce lysis in E. coli in response to blue light.

Figure 2. The growth status of pDawn-RBSNNN-rrnB T1-LKD16-pSEVA331-DH5α under blue light (a) and dark conditions (b).

2. pDawn-RBSNNN-LKD16-rrnB T1-pSEVA331-8# in response to blue photolysis in G. hansenii ATCC 53582.

As shown in Figure 3, although 7th, 8th, 9th, 13th and 17th on the plate can grow under the dark, not grow under blue light, the growth of the bacteria under dark conditions is abnormal. Therefore, although the LKD16 cleavage protein has a cleavage function, its function is not very stable.

Figure 3. pDawn-RBSNNN-LKD16-rrnB T1-pSEVA331-G.hansenii ATCC 53582-8# cultured under blue light (a) and dark conditions (b).

References

[1] Ceyssens P J, Lavigne R, Mattheus W, et al. Genomic Analysis of Pseudomonas aeruginosa Phages LKD16 and LKA1: Establishment of the KMV Subgroup within the T7 Supergroup[J]. Journal of Bacteriology, 2006.