Difference between revisions of "Part:BBa K3598043"

 
 
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This part is for the expression of mTyr-CNK in our project.  PJExD promoter is regulated by EilR, and  several cationic dyes act as efficient low-cost inducers.
 
This part is for the expression of mTyr-CNK in our project.  PJExD promoter is regulated by EilR, and  several cationic dyes act as efficient low-cost inducers.
  
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===Usage and Biology===
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===Background and Description===
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<span class='h3bb'>To achieve co-expression of adhesive/cohesive protein and tyrosinase, we need a new inducible system different from T7-LacI inducible promoter to control the expression of tyrosinase. Thus, we constructed BBa_K3598043, which consists EilR repressor and PJExD promoter and uses cationic dye as inducer.</span>
  
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[[File:T--BEIJING_4ELEVEN--Figure_1_BBa_K3598043.PNG|300px|thumb|center|Figure 1. The overall design of BBa_K3598043]]
 
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<span class='h3bb'>Sequence and Features</span>
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<br>
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<span class='h3bb'>EilR promoter starts the expression of EilR, an repressor to PJExD promoter, so the expression of mTyr_CNK will not start spontaneously. Cationic dyes act as an inducer that blocks the repression of PJExD promoter, starting the expression of mTyr_CNK. For purification of mTyr_CNK, we add 7*His tag</span>
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<br>
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<br>
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===Experiment and Results===
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<span class='h3bb'>We used LB medium to cultivate the E.coli BL21 chasis that contains our inducible system, and use 1μM CV as inducer, inducing the production at 25℃ for 24 hours. 100mL culture was collected for purification. The purified mTyr_CNK was verificated by SDS-PAGE. W0 and W1 in Figure 2 are the unpurified liquid, which have notable strand of mTyr_CNK. E1, E2, and E3 are the liquid gained after first purification, second purification, and third purification respectively, in which the strands of E2 are comparatively intensive. We used BCA assay to measure the E2 concentration of mTyr_CNK, which is 1.44mg/mL.</span>
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[[File:T--BEIJING 4ELEVEN--Results_Fig.20 EilR promoter-CNK.png|500px|thumb|center|Figure 2. SDS-PAGE gel analysis of mTyr-CNK produced by our inducible system]]
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<partinfo>BBa_K3598043 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3598043 SequenceAndFeatures</partinfo>
  
  
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K3598043 parameters</partinfo>
 
<partinfo>BBa_K3598043 parameters</partinfo>
 
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Latest revision as of 03:28, 26 October 2020


EilR_EilR promoter_PJExD promoter_mTyr-CNK_7*His

This part is for the expression of mTyr-CNK in our project. PJExD promoter is regulated by EilR, and several cationic dyes act as efficient low-cost inducers.

Background and Description

To achieve co-expression of adhesive/cohesive protein and tyrosinase, we need a new inducible system different from T7-LacI inducible promoter to control the expression of tyrosinase. Thus, we constructed BBa_K3598043, which consists EilR repressor and PJExD promoter and uses cationic dye as inducer.

Figure 1. The overall design of BBa_K3598043


EilR promoter starts the expression of EilR, an repressor to PJExD promoter, so the expression of mTyr_CNK will not start spontaneously. Cationic dyes act as an inducer that blocks the repression of PJExD promoter, starting the expression of mTyr_CNK. For purification of mTyr_CNK, we add 7*His tag

Experiment and Results

We used LB medium to cultivate the E.coli BL21 chasis that contains our inducible system, and use 1μM CV as inducer, inducing the production at 25℃ for 24 hours. 100mL culture was collected for purification. The purified mTyr_CNK was verificated by SDS-PAGE. W0 and W1 in Figure 2 are the unpurified liquid, which have notable strand of mTyr_CNK. E1, E2, and E3 are the liquid gained after first purification, second purification, and third purification respectively, in which the strands of E2 are comparatively intensive. We used BCA assay to measure the E2 concentration of mTyr_CNK, which is 1.44mg/mL.

Figure 2. SDS-PAGE gel analysis of mTyr-CNK produced by our inducible system



Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 916
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 346
    Illegal AgeI site found at 496
  • 1000
    COMPATIBLE WITH RFC[1000]


Functional Parameters