Difference between revisions of "Part:BBa K4377009"

 
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<partinfo>BBa_K4377009 parameters</partinfo>
 
<partinfo>BBa_K4377009 parameters</partinfo>
 
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===Introduction===
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This composite part contains constitutive promoter, RBS, mRFP, and two functional domains of Ubx protein. More detail about its crosslinking function is shown below.
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===Design===
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We designed mRFP(Part:E1010) as the functional protein between the two peptide regions(Part:K4377001 and Part:K4377002),  in an attempt to verify the feasibility of self-assembling. We choose mRFP as functional protein for testing because mRFP is easy to observe and check protein’s function.In order to purify our protein after expression, we added 10x His tag at the N terminal of Ubx protein. The reason we add 10x His tag at N terminal but not C terminal is to amplify the production of Ubx protein. To improve performance, we performed codon harmonization. The optimized sequence of codons will be more suitable for expression by our engineered ''E.coli'' .
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[[Image:BBa K4377003-biobrick.png|700px]]
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===Build===
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====Cloning result====
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With the same process of building Composite part K4377006, we successfully built Composite part K4377007.
 +
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[[Image:BBa K4377003-digest check.jpg|400px]]
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====SDS page result====
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After expression, we conducted SDS PAGE to verify the protein was produced.
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[[Image:BBa K4377003-sds page.jpg|400px]]
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===Test===
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====Functional test====
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We used EDTA-Fe and H2O2 to catalyze dityrosine bonding, as figures show that our sample(Composite part K4377007 ) has significant aggregation compared to the control (mRFP), since the bonding between tyrosines occurs when the C-H bond on the benzene ring breaks and forms C-C bonding, we decided to analyze the region of C-H bond in FTIR data
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[[Image:BBa K4377003-func test 1.png|400px]]
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[[Image:BBa K4377003-func test 2.jpg|800px]]

Revision as of 14:36, 13 October 2022


Y167 mRFP Y240

This composite part contains constitutive promoter, RBS, mRFP, and two functional domains of Ubx protein. More detail about its crosslinking function is shown below.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 711
    Illegal AgeI site found at 823
  • 1000
    COMPATIBLE WITH RFC[1000]


Introduction

This composite part contains constitutive promoter, RBS, mRFP, and two functional domains of Ubx protein. More detail about its crosslinking function is shown below.

Design

We designed mRFP(Part:E1010) as the functional protein between the two peptide regions(Part:K4377001 and Part:K4377002), in an attempt to verify the feasibility of self-assembling. We choose mRFP as functional protein for testing because mRFP is easy to observe and check protein’s function.In order to purify our protein after expression, we added 10x His tag at the N terminal of Ubx protein. The reason we add 10x His tag at N terminal but not C terminal is to amplify the production of Ubx protein. To improve performance, we performed codon harmonization. The optimized sequence of codons will be more suitable for expression by our engineered E.coli .

BBa K4377003-biobrick.png

Build

Cloning result

With the same process of building Composite part K4377006, we successfully built Composite part K4377007.

BBa K4377003-digest check.jpg

SDS page result

After expression, we conducted SDS PAGE to verify the protein was produced.

BBa K4377003-sds page.jpg

Test

Functional test

We used EDTA-Fe and H2O2 to catalyze dityrosine bonding, as figures show that our sample(Composite part K4377007 ) has significant aggregation compared to the control (mRFP), since the bonding between tyrosines occurs when the C-H bond on the benzene ring breaks and forms C-C bonding, we decided to analyze the region of C-H bond in FTIR data

BBa K4377003-func test 1.png

BBa K4377003-func test 2.jpg