Regulatory

Part:BBa_K4583004

Designed by: Quanfeng Liang   Group: iGEM23_SDU-CHINA   (2023-10-04)
Revision as of 09:29, 5 October 2023 by Liang QF (Talk | contribs) (→‎Characterization using BFP in 3-plasmids bacteria)

PYU92

PYU92 is the promoter of the gene orf-O169, and it comes from Escherichia coli. It will reach its maximum expression at the late stationary phase and it is self-inducible.

Usage and Biology

When the bacteria enter the stationary phase, the physiological state of the bacteria changes significantly. During this phase, many genes will respond to make timely adjustments. This part BBa_K4583004(PYU92) is the promoter of the gene orf-O169. Its most notable feature is that it will be expressed in the late stationary phase. Moreover, it is a self-inducible promoter, which means that no additional inducers need to be added for expression. Exogenous inducers are expensive and need to be added artificially, whereas self-induced promoters are cost-effective and relatively stable. This part is also very safe because it comes from E. coli MG1655, a commonly engineered bacterium.

  • Late stationary phase promoter
  • Self-inducible promoter without additional inducers
  • Biosafety

There are 3 parts that have similar features characcterized by our team: BBa_K4583000(PYU3) , BBa_K4583001(PYU7), and BBa_K4583003(PYU16)

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.) and Molecular Devices SpectraMax i3x.

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).

Characterization using GFP in 2-plasmids bacteria

In this section we used the PACYC plasmid with BBa_K4583004(PYU92) upstream of GFP gene(Fig. 1). We transformed it into L19 and L31 with BBa_K4583009(PesaRwt), BBa_K4583010(PesaRc), BBa_K4583011(PesaRp) plasmids respectively (6 combinations in total) and characterized them using 24-well plates. The characterization results are shown in Fig. 2

Fig. 1 . Genetic Circuit when characterizing PYU92 using GFP

As can be seen from the characterization results, with the exception of two combinations (L31-PesaRwt-PYU7 and L31-PesaRc-PYU7) the expression time of this part is significantly different from that of other parts in terms of time and intensity.

In the case of the combination L19-PesaRwt-PYU7, for example, the expression of the other part peaked at about 14 h, whereas the expression of this part peaked at about 36 h.

Fig. 2 . Characterization results of PYU92 in the 2-plasmid bacteria

Characterization using BFP in 3-plasmids bacteria

In this section, we used the PACYC plasmid with PYU7 upstream of the BFP gene (Fig. 3). Based on the results of the last Characterization, we transformed it into L19 and L31 with BBa_K4583009(PesaRwt), BBa_K4583010(PesaRc), BBa_K4583011(PesaRp) and BBa_K4583012(PesaS) plasmid respectively (4 combinations in total) and characterized them using 24-well plates. The characterization results are shown in Fig. 4.

Fig. 3 . Genetic Circuit when characterizing PYU7 using BFP
From the characterization results, we can see that there is a significant delay in the expression of this part from the other promoters. PYU92 is expressed at the stationary phase and peaks at the late stationary phase.

For this characterization, we used a Molecular Devices SpectraMax i3x, which has a much higher precision. In addition, the difference between the 2-plasmids system and the 3-plasmids system may also account for the difference.

Fig. 4 . Characterization results of PYU92 in the 3-plasmids bacteria

Reference

[1] Talukder, A A et al. “RpoS-dependent regulation of genes expressed at late stationary phase in Escherichia coli.” FEBS letters vol. 386,2-3 (1996): 177-80. doi:10.1016/0014-5793(96)00426-7

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