Difference between revisions of "Part:BBa K3257030"
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We use this part as a reporter protein to verify protein expression. To characterize EGFP as a fluorescence protein, we have applied some different approach to observe and measure its fluorescence intensity. | We use this part as a reporter protein to verify protein expression. To characterize EGFP as a fluorescence protein, we have applied some different approach to observe and measure its fluorescence intensity. | ||
| − | Firstly, Escherichia coli (BL21) transformed with plasmid containing EGFP CDS are cultured in liquid medium. After overnight 37℃ culture, we observed the test tube under a fluorescence microscope and the bacteria solution appeared to give out green fluorescence as we expected. (Figure 1). | + | Firstly, ''Escherichia coli'' (BL21) transformed with plasmid containing EGFP CDS are cultured in liquid medium. After overnight 37℃ culture, we observed the test tube under a fluorescence microscope and the bacteria solution appeared to give out green fluorescence as we expected. (Figure 1). |
| − | [[File:EGFP in liquid bacteria culture.jpeg|center|200px|thumb|'''Figure 1. Bacteria Liquid culture observed by a fluorescence microscope''' The left side of the photo is bacteria (containing EGFP coding plasmid) liquid culture which appears to be green under excitation while the right side of the photo is empty. From the fluorescence microscope we can see that, EGFP can be expressed in E.coli cells and function well as a fluorescence protein.]] | + | [[File:EGFP in liquid bacteria culture.jpeg|center|200px|thumb|'''Figure 1. Bacteria Liquid culture observed by a fluorescence microscope''' The left side of the photo is bacteria (containing EGFP coding plasmid) liquid culture which appears to be green under excitation while the right side of the photo is empty. From the fluorescence microscope we can see that, EGFP can be expressed in ''E.coli'' cells and function well as a fluorescence protein.]] |
Secondly, we measured the fluorescence and OD600 of the liquid bacteria culture by a 96-well microplate reader. When stimulated by 485nm light, we can detect 528nm emitted light from EGFP (Figure 2). | Secondly, we measured the fluorescence and OD600 of the liquid bacteria culture by a 96-well microplate reader. When stimulated by 485nm light, we can detect 528nm emitted light from EGFP (Figure 2). | ||
| − | [[File:Fluorescence of EGFP.jpeg|center|400px|thumb|'''Figure 2. The relative fluorescence intensity of EGFP measured by 96-well plate reader''' EGFP represents the relative fluorescence intensity of BL21(DE3) transformed with plasmid encoding EGFP protein. The Negative represents the relative fluorescence intensity of BL21(DE3) transformed with plasmid encoding nonsense mutated EGFP protein. Significant difference between them shows that EGFP can be expressed and function well inside E.coli cells.]] | + | [[File:Fluorescence of EGFP.jpeg|center|400px|thumb|'''Figure 2. The relative fluorescence intensity of EGFP measured by 96-well plate reader''' EGFP represents the relative fluorescence intensity of BL21(DE3) transformed with plasmid encoding EGFP protein. The Negative represents the relative fluorescence intensity of BL21(DE3) transformed with plasmid encoding nonsense mutated EGFP protein. Significant difference between them shows that EGFP can be expressed and function well inside ''E.coli'' cells.]] |
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Latest revision as of 21:00, 21 October 2019
EGFP
Enhanced Green Fluorescence Protein(EGFP)
This part is an enhanced green fluorescence protein that gives out green light when excited by 485nm wavelength of light. It can be used as a reporter downstream a promoter to test its promoter strength or linked with a protein to characterize its expression.
We use this part as a reporter protein to verify protein expression. To characterize EGFP as a fluorescence protein, we have applied some different approach to observe and measure its fluorescence intensity. Firstly, Escherichia coli (BL21) transformed with plasmid containing EGFP CDS are cultured in liquid medium. After overnight 37℃ culture, we observed the test tube under a fluorescence microscope and the bacteria solution appeared to give out green fluorescence as we expected. (Figure 1).
Figure 1. Bacteria Liquid culture observed by a fluorescence microscope The left side of the photo is bacteria (containing EGFP coding plasmid) liquid culture which appears to be green under excitation while the right side of the photo is empty. From the fluorescence microscope we can see that, EGFP can be expressed in E.coli cells and function well as a fluorescence protein.
Secondly, we measured the fluorescence and OD600 of the liquid bacteria culture by a 96-well microplate reader. When stimulated by 485nm light, we can detect 528nm emitted light from EGFP (Figure 2).
Figure 2. The relative fluorescence intensity of EGFP measured by 96-well plate reader EGFP represents the relative fluorescence intensity of BL21(DE3) transformed with plasmid encoding EGFP protein. The Negative represents the relative fluorescence intensity of BL21(DE3) transformed with plasmid encoding nonsense mutated EGFP protein. Significant difference between them shows that EGFP can be expressed and function well inside E.coli cells.
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]