Difference between revisions of "Part:BBa K3740044"

 
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<partinfo>BBa_K3740044 short</partinfo>
 
<partinfo>BBa_K3740044 short</partinfo>
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===Description===
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This composite part is a generator consisting of the pDawn promoter and X174 E.
  
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Lysis the engineered bacteria
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===Sequence and Features===
  
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<!-- Add more about the biology of this part here
 
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===Usage and Biology===
 
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<span class='h3bb'>Sequence and Features</span>
 
 
<partinfo>BBa_K3740044 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3740044 SequenceAndFeatures</partinfo>
  
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<partinfo>BBa_K3740044 parameters</partinfo>
 
<partinfo>BBa_K3740044 parameters</partinfo>
 
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=2021 SZPT-China=
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<h3>Biology</h3>
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<p>This composite part is a generator consisting of the pDawn (<partinfo>BBa_K1075044</partinfo>) promoter and X174 E (<partinfo>BBa_K1835500</partinfo>).</p>
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<h3>Usage</h3>
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<p>The target genes of pDawn blue light response system (<partinfo>BBa_K1075044</partinfo>) and X174 E (<partinfo>BBa_K1835500</partinfo>) were inserted 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 X174 E. Finally, the pDawn-RBSNNN-X174 E-rrnB T1 (<partinfo>BBa_K3740044</partinfo>) plasmid was constructed and introduced into <i>E. coli</i>. <i>E. coli</i> isolates that grow normally in the dark and cannot grow under blue light were screened out. Finally, the 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:szpt43.png|600px|thumb|center|Figure 1. Gene circuit of pDawn-RBSNNN-X174 E-rrnB T1.]]
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<h3>Characterization</h3>
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<h4>1. Batch screening of pSEVA331-pDawn-RBSNNN-X174 E-rrnB T1- pSEVA331in response to blue light lysis in <i>E. coli</i>.</h4>
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<p>Method: We use the random primer method to modulate the strength of the RBS (<partinfo>BBa_B0034</partinfo>) located upstream of X174 E. After introducing into <i>E. coli</i> DH5α, a drop plate assay was performed to screen the bacterial isolate that can grow normally in the dark but cannot under the blue light irradiation.</p>
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<br>
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<p>As shown in Figure 11, the 4th isolate that grew normally in the dark but did not under blue light, indicating that we successfully expressed the cleavage protein X174 E in E. coli.</p>

Revision as of 10:45, 19 October 2021


pDawn-B0034-X174 E-rrnB T1

Description

This composite part is a generator consisting of the pDawn promoter and X174 E.

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 X174 E (BBa_K1835500).

Usage

The target genes of pDawn blue light response system (BBa_K1075044) and X174 E (BBa_K1835500) were inserted into the pSEVA331 expression vector. Then, random primer guided mutagenesis method to modulate the strength of the RBS (BBa_B0034) located upstream of X174 E. Finally, the pDawn-RBSNNN-X174 E-rrnB T1 (BBa_K3740044) plasmid was constructed and introduced into E. coli. E. coli isolates that grow normally in the dark and cannot grow under blue light were screened out. Finally, the 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 pDawn-RBSNNN-X174 E-rrnB T1.

Characterization

1. Batch screening of pSEVA331-pDawn-RBSNNN-X174 E-rrnB T1- pSEVA331in response to blue light lysis in E. coli.

Method: We use the random primer method to modulate the strength of the RBS (BBa_B0034) located upstream of X174 E. After introducing into E. coli DH5α, a drop plate assay was performed to screen the bacterial isolate that can grow normally in the dark but cannot under the blue light irradiation.


As shown in Figure 11, the 4th isolate that grew normally in the dark but did not under blue light, indicating that we successfully expressed the cleavage protein X174 E in E. coli.