Difference between revisions of "Part:BBa K4583005"
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==Characterization== | ==Characterization== | ||
| + | Our characterization of this part is divided into two main parts. | ||
| + | * First, this promoter was placed upstream of<em> GFP </em>gene, forming a genetic circuit as shown in Fig. 1. This plasmid was transformed into a bacterium containing another plasmid for characterization. Green and red fluorescence were measured at fixed intervals to compare the expression time and intensity of the two. | ||
| + | * Second, this promoter was placed upstream of the <em> BFP </em> gene, forming a genetic circuit as shown in Fig. 3. This plasmid was then transformed into bacteria containing two other plasmids. Green, red and blue fluorescence were measured at fixed time intervals to compare the difference in expression time and intensity between this part and the other two parts. | ||
| + | For plasmid construction methods and other experimental procedures, see the Design page. | ||
===Protocols=== | ===Protocols=== | ||
| + | Our experimental conditions for characterizing this part were as follows: | ||
| + | * <em>E. coli</em> MG1655 | ||
| + | * 30<sup>o</sup>C, 48h, under vigorous shaking | ||
| + | * Plasmid Backbone: PACYC | ||
| + | * Equipment: Multi-Detection Microplate Reader (Synergy HT, Biotek, U.S.) | ||
| + | We used GFP (excitation at 485 nm and emission at 528 nm)and BFP (excitation at 400 nm and emission at 450 nm) to characterize this part. As our focus was mainly on the expression time, we processed the obtained fluorescence data by means of the following equation: x'=(x-min)/(max-x). This treatment makes all data fall between 0 and 1, which is easier to use for comparisons between different fluorescence data (since our focus is on expression time). | ||
===Characterizing using GFP in 2-plasmids Bacteria=== | ===Characterizing using GFP in 2-plasmids Bacteria=== | ||
<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
Revision as of 18:55, 5 October 2023
Pfic
Pfic is a wild-type promoter isolated from Gordonia sp. IITR100.
Usage and Biology
Constitutive gene expression affects growth by imposing a metabolic burden on the cell. Inducible gene expression systems are usually controlled by specific inducers. Inducers are expensive and often lead to irregular protein expression. To overcome this limitation, self-inducible promoters have been developed. The stationary phase promoter isolated from Gordonia sp. IITR100 does not require additional chemical inducers.
- Stationary Phase Promoter
- self-inducible
- Biosafety
Characterization
Our characterization of this part is divided into two main parts.
- First, this promoter was placed upstream of GFP gene, forming a genetic circuit as shown in Fig. 1. This plasmid was transformed into a bacterium containing another plasmid for characterization. Green and red fluorescence were measured at fixed intervals to compare the expression time and intensity of the two.
- Second, this promoter was placed upstream of the BFP gene, forming a genetic circuit as shown in Fig. 3. This plasmid was then transformed into bacteria containing two other plasmids. Green, red and blue fluorescence were measured at fixed time intervals to compare the difference in expression time and intensity between this part and the other two parts.
For plasmid construction methods and other experimental procedures, see the Design page.
Protocols
Our experimental conditions for characterizing this part were as follows:
- E. coli MG1655
- 30oC, 48h, under vigorous shaking
- Plasmid Backbone: PACYC
- Equipment: Multi-Detection Microplate Reader (Synergy HT, Biotek, U.S.)
We used GFP (excitation at 485 nm and emission at 528 nm)and BFP (excitation at 400 nm and emission at 450 nm) to characterize this part. As our focus was mainly on the expression time, we processed the obtained fluorescence data by means of the following equation: x'=(x-min)/(max-x). This treatment makes all data fall between 0 and 1, which is easier to use for comparisons between different fluorescence data (since our focus is on expression time).
Characterizing using GFP in 2-plasmids Bacteria
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]