Difference between revisions of "Part:BBa K4583000"

Revision as of 03:33, 5 October 2023

PYU3

PYU3 is the promoter of the gene orf-0464, and it comes from Escherichia coli. It will reach its maximum expression at the late stationary phase.

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_K4583000) is the promoter of the gene orf-0464. Its most notable feature is that it will be expressed in the late stationary phase. Sequence and Features

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 Figure 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 Figure 2. This plasmid was then transferred 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
• 30^oC, 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).

Characterization using GFP in 2-plasmids bacteria

In this section we used the PACYC plasmid with PYU3 upstream of GFP. We transfected them into L19 and L31 with PesaRwt, PesaRc, PesaRp plasmids respectively (6 combinations in total) and characterized them using 24-well plates. The characterization results are shown in Fig 2

Characterization using BFP in 3-plasmids bacteria

Reference