Part:BBa_K4583076

P2.1-RBS(B0034)-GFP

P2.1-RBS(B0034)-GFP

These parts are generated by multiple rounds of random mutation by BBa_K4583005(Pfic). BBa_K4583006(P1.1) is generated by the first round of mutation; BBa_K4583007(P2.1) is generated by the second round of mutation; and BBa_K4583008(P3.1) P3.1 is generated by the third round. Their expression intensity was higher than that of the wild type.

Usage and Biology

Constitutive gene expression would affect 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

There are 3 similar promoters: BBa_K4583005(Pfic), BBa_K4583007(P2.1), and BBa_K4583008(P3.1)。

Characterization

This promoter was placed upstream of GFP gene. 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.

Protocols

Our experimental conditions for characterizing this part were as follows:

• E. coli MG1655
• 30^oC, 48h, under vigorous shaking
• Plasmid Backbone: PACYC
• Equipment: Multi-Detection Microplate Reader (Synergy HT, Biotek. US)

We used GFP (excitation at 485 nm and emission at 528 nm)and mkate (excitation at 585 nm and emission at 640 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).

Characterization of Pfic, P1.1, P2.1, and P3.1

We compared the expression strength of Pfic, P1.1, P2.1, and P3.1 in both L19 (Fig. 1)and L31 (Fig. 2). You can found that there are obvious difference in the expression strength. The Expression of this promoter is very rapid, with peak expression in less than 4 hours

Fig. 1 . Characterization results of Pfic, P1.1, P2.1, and P3.1-RBS(B0034)-GFP in L19

Fig. 2 . Characterization results of Pfic, P1.1, P2.1, and P3.1-RBS(B0034)-GFP in L31

Characterization 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

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

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

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

Reference

[1] Jaishankar, J., & Srivastava, P. (2020). Strong synthetic stationary phase promoter-based gene expression system for Escherichia coli. Plasmid, 109, 102491. 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
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 902