Difference between revisions of "Part:BBa K4377009"
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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. | 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=== | ===Design=== | ||
| − | We designed mRFP(Part:E1010) as the functional protein between the two peptide regions | + | We designed mRFP(Part:E1010) as the functional protein between the two peptide regions, 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'' . |
[[Image:BBa K4377009-biobrick.png|700px]] | [[Image:BBa K4377009-biobrick.png|700px]] | ||
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===Build=== | ===Build=== | ||
====Cloning result==== | ====Cloning result==== | ||
| − | With the same process of building Composite part K4377006, we successfully built Composite part | + | With the same process of building Composite part K4377006, we successfully built Composite part K4377009. |
[[Image:BBa K4377003-digest check.jpg|400px]] | [[Image:BBa K4377003-digest check.jpg|400px]] | ||
Revision as of 02:02, 14 October 2022
Y167 mRFP Y240
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 711
Illegal AgeI site found at 823 - 1000COMPATIBLE 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, 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 .
Build
Cloning result
With the same process of building Composite part K4377006, we successfully built Composite part K4377009.
SDS page result
After expression, we conducted SDS PAGE to verify the protein was produced.
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
