Difference between revisions of "Part:BBa K4583008"

Latest revision as of 21:10, 30 September 2024

Variant of wild type promoter Pfic--P3.1

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

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.

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

B0030 vs B0034 in E.coli BL21 (DE3) - Thessaly 2024

Group: Thessaly 2024

Purpose
P3.1 (BBa_K4583008) was used in our promoter testing experiments. We wanted to test its strength and phase activation, as we have included it in our project design.

Methods
Measurements for OD 600nm and fluorescence (488nm,515nm) were taken over the course of 16 hours, in a 96-well microplate. Clonings were done according to the Golden Braid method, leaving us with level α constructs in the pDGB3a1 backbone BBa_K4213058.

Constructs and pDGB3a1 (empty vector) transformed into E.coli BL21 (DE3) chassis, incubated at 37oC, 180rpm for 16 hours.

P3.1 was inserted into the constructs BBa_K5299200,BBa_K5299201. For more information, head over to these pages.

Plated 200 ul 5 times, out of each single liquid bacterial culture, created from the same bacterial colony, in order to establish accuracy through technical repeats.

Medium used was M9 due to minimal interference, with D-glucose serving as the carbon source. Also, served as blank, plated 5 times.

Measurements were normalised as such: using the average price of fluorescence for the 5 technical repeats and dividing it by the average price of OD.
Standard deviation included in the graphs.

Results
Measurements were taken over the course of 16 hours. Here are the results (mentioned in the graphs as P3.1 B0030 and P3.1 B0034 respectively).

Figure 1: Constructs with the BBa_J23119 or the P3.1 (BBa_K4583008) or the BBa_J45992 promoter, the BBa_B0030 RBS, the BBa_I746916 sfGFP and the BBa_B0015 terminator. The pDGB3a1 served as a negative control. BBa_J23119 and BBa_J45992 serve as phase activation controls. BBa_J45992 also serves as strength control, due to the same phase activation.

Figure 2: Constructs with the BBa_J23119 or the P3.1 (BBa_K4583008) or the BBa_J45992 promoter, the BBa_B0034 RBS, the BBa_I746916 sfGFP and the BBa_B0015 terminator. The pDGB3a1 served as a negative control. BBa_J23119 and BBa_J45992 serve as phase activation controls. BBa_J45992 also serves as strength control, due to the same phase activation.