Difference between revisions of "Part:BBa K2973002"

Revision as of 18:26, 20 October 2019

T7-RBS- enhanced green fluorescent protein (EGFP)

This composite part consists of T7 Promoter (BBa_J64997) and T7 Terminator(BBa_K731721), Ribosomal Binding Site(BBa_B0034) and EGFP CDS (BBa_K1094400). EGFP (Enhanced Green Fluorescent Protein) is a version of GFP(green fluorescent protein) that produces enhanced bioluminescence signal in the green zone of the noticeable spectrum and is being used widely as a protein reporter. This protein is produced by the jellyfish (Aequorea Victoria).

Usage and Biology

This part was used as a positive control for our in vitro protein synthesis experiments.

During our experiments, one of the reporter genes that we tested was EGFP (Enhanced Green Fluorescent Protein). To test its functionality, we added its sequence downstream of Pardee's Toehold Switch 32B. The in vitro transcription/ translation reactions were done using the PURExpress® In Vitro Protein Synthesis kit. To enable translation of the protein from the toehold regulated construct, different amounts of trigger sequence were added in the reaction. Also, a reaction without a trigger sequence was included, as a negative control and a leakage measure. To reduce the cost of the reaction, we lowered the reaction volume from 25 to 7 μL.

After a 3-hour incubation in a PCR machine at 37℃, protein expression was measured using a plate reader. To measure eGFP, an excitation step at 488nm was required before visualizing at 515nm, where the protein’s emission wavelength is. The results of the assay can be seen in the graph below.

As expected, the non-regulated eGFP emitted the highest fluorescence, providing a positive control and confirming the protocol we used was functional. The toehold-regulated construct produced robust signal in decreasing concentrations of trigger (75nM, 15nM, 7nM). It is interesting that the signal remained the same even with a 10-fold decrease in trigger concentration, while the no trigger control had an almost 2-fold lower fluorescence. HTML img Tag

Figure 1: eGFP Fluorescence after in vitro protein expression.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 776
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 During our experiments, one of the reporter genes that we tested was EGFP (Enhanced Green Fluorescent Protein). To test its functionality, we added its sequence downstream of Pardee's Toehold Switch 32B. The <i>in vitro</i> transcription/ translation reactions were done using the PURExpress® <i>In Vitro</i> Protein Synthesis kit. To enable translation of the protein from the toehold regulated construct, different amounts of trigger sequence were added in the reaction. Also, a reaction without a trigger sequence was included, as a negative control and a leakage measure. To reduce the cost of the reaction, we lowered the reaction volume from 25 to 7 μL.
-After a 3-hour incubation in a PCR machine at 37oC, protein expression was measured using a plate reader. To measure eGFP, an excitation step at 488nm was required before visualizing at 515nm, where the protein’s emission wavelength is. The results of the assay can be seen in the graph below.
+After a 3-hour incubation in a PCR machine at 37&#8451;, protein expression was measured using a plate reader. To measure eGFP, an excitation step at 488nm was required before visualizing at 515nm, where the protein’s emission wavelength is. The results of the assay can be seen in the graph below.
 As expected, the non-regulated eGFP emitted the highest fluorescence, providing a positive control and confirming the protocol we used was functional. The toehold-regulated construct produced robust signal in decreasing concentrations of trigger (75nM, 15nM, 7nM). It is interesting that the signal remained the same even with a 10-fold decrease in trigger concentration, while the no trigger control had an almost 2-fold lower fluorescence.