Difference between revisions of "Part:BBa K4583005"

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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).
 
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===
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We found that the expression of Pfic is not very stable（Fig.1）. In L31, Pfic was expressed at almost the same time as PesaRwt.
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  <img src="https://static.igem.wiki/teams/4583/wiki/pfic.png"width="540" height="210">
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  <figcaption><b>Fig. 1 </b>. Characterization results of Pfic-RBS(B0034)-GFP in L19 and L31</figcaption>
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==Reference==
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===Functional Parameters===
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[1] Jaishankar J, Srivastava P. Strong synthetic stationary phase promoter-based gene expression system for Escherichia coli. Plasmid. 2020 May;109:102491. doi: 10.1016/j.plasmid.2020.102491. Epub 2020 Feb 5. PMID: 32035079.
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Latest revision as of 11:48, 12 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

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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

We found that the expression of Pfic is not very stable（Fig.1）. In L31, Pfic was expressed at almost the same time as PesaRwt.

Fig. 1 . Characterization results of Pfic-RBS(B0034)-GFP in L19 and L31

Reference

[1] Jaishankar J, Srivastava P. Strong synthetic stationary phase promoter-based gene expression system for Escherichia coli. Plasmid. 2020 May;109:102491. doi: 10.1016/j.plasmid.2020.102491. Epub 2020 Feb 5. PMID: 32035079.