Difference between revisions of "Part:BBa K4377003"
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===Design=== | ===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. ( result in Figure 1.) 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'' . | 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. ( result in Figure 1.) 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=== | ===Build=== | ||
====Cloning result==== | ====Cloning result==== | ||
Revision as of 00:11, 12 October 2022
Y167 mRFP Y240
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 736
Illegal AgeI site found at 848 - 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(Part:K4377001 and Part:K4377002), in an attempt to verify the feasibility of self-assembling. ( result in Figure 1.) 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 K4377007.
SDS page result
After expression we conducted SDS PAGE to verify the protein was produced. ( result in Figure 3.)
Test
Functional test
We used EDTA-Fe and H2O2 to catalyze dityrosine bonding, as Figure 4. shows 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 (Blue area in Figure 5.) , we decided to analyze the region of C-H bond in FTIR data (Figure 6.)