Part:BBa_K5299010

BG42 synthetic promoter

BG42 synthetic promoter, Zobel et al.

Biology

Zobel et al. identified the BG synthetic promoters by systematically analyzing promoter activities in E. coli and Pseudomonas strains, particularly P. aeruginosa and P. putida. They found that the consensus sequences, especially the −10 and −35 regions, closely resembled those of sigma-70 promoters in E. coli, which suggested similar transcriptional mechanisms across these species. Using an initial plasmid-based selection in E. coli PIR2 cells, they efficiently screened for effective synthetic promoters, confirming their comparable activity in both E. coli and Pseudomonas [1]

Experimental design

Our team used this promoter as part of a DBTL cycle aimed at finding the most proper promoter for the T7 polymerase production. We selected to test the promoters BG17, BG42, and BG37 based on findings from Zobel et al., which demonstrated their significant activation during the exponential growth phase [1,2]. Our objective was to evaluate their temporal activation profiles and relative strength. We began by cloning the promoter ordered from IDT, which included Golden Braid overhangs, into the pUPD2 vectors. The promoter was synthesized with its respective RBS (BBa_B0034) already integrated. We used E. coli DH5α cells for the cloning procedure due to their high transformation efficiency. Next, we transferred our part from the pUPD2 vector into the pDGB3a1 by combining it with an sfGFP reporter gene and BBa_B0030 terminator to assemble our level alpha constructs. This step enabled us to generate complete, functional genetic modules for further experimentation and analysis. After constructing all of our plasmid constructs, we transformed them into E. coli BL21 DE3 cells. The following day, we prepared liquid cultures to promote the growth and acclimatization of the transformed bacteria, allowing them to incubate overnight. On the third day, we centrifuged the cultures and washed the resulting pellets twice with NaOH. Subsequently, we performed a 1:100 dilution to measure the optical density at 600 nm (OD600). To achieve an OD600 of 0.1 with a final volume of 2 mL of M9 culture, we applied the dilution equation Cinitial x Vinitial= C final x V final. M9 medium was selected for this experiment due to the fluorescence interference associated with LB medium. We then transferred 200 μL of each diluted culture into the wells of a 96-well plate with a clear bottom. Each construct was tested in five technical replicates. The plate was incubated in a plate reader for 15 hours at 37°C, which is optimal for E. coli growth, while shaking at 180 rpm. Measurements were automatically recorded every hour, monitoring OD at 600 nm and sfGFP fluorescence at 515 nm.

Results

Figure 1: Bacterial growth curve, with M9 medium with 0,2% glucose as a carbon source, based on Optical Density at 600 nm measurements taken every 1 hour. Data points represent the average of five technical replicates, with any values showing a standard deviation greater than 0.2 excluded for clarity. Error bars correspond to standard deviation of n=5 replicates. Blank was subtracted.

By observing Figure 1 and analyzing the OD600 measurements, we notice significant bacterial growth between the 1h and 8h time points for every promoter except BG37. Specifically, for the E. coli cells with the BG37 promoter, there is an increase up to the 12h mark, with the steady increase in OD600 between 6h and 12h suggesting that the cells are in the exponential phase.

Figure 2: Normalized fluorescence intensity for J23119-RBS2-sfGFP-ter, osmy-RBS2-sfGFP-ter, BG37-RBS2-sfGFP-ter, BG17-RBS2-sfGFP-ter and BG42--RBS2-sfGFP-ter constructs during the time points: 3h, 6h, 10h and 15h. The RFUs (sfGFP measurements at 515 nm) are divided by cell growth (Optical Density at 600nm), in order to normalize all values. E. coli BL21 (DE3) cells with pDGB3a1 were used as the (-) control. Error bars correspond to standard deviation of n=5 replicates. Blank was subtracted.

Figure 3: Normalized fluorescence intensity for J23119-RBS2-sfGFP-ter, osmy-RBS2-sfGFP-ter, BG37-RBS2-sfGFP-ter, BG17-RBS2-sfGFP-ter and BG42--RBS2-sfGFP-ter constructs during the time points: 3h, 6h, 10h and 15h. The RFUs (sfGFP measurements at 515 nm) are divided by cell growth (Optical Density at 600nm), in order to normalize all values. E. coli BL21 (DE3) cells with pDGB3a1 were used as the (-) control. Error bars correspond to standard deviation of n=5 replicates. Blank was subtracted.

Based on the graphs (Figure 2 and Figure 3) and OD600 data, which show the cells remaining in the exponential phase at 12 hours, BG37 demonstrates strong activity during this period. BG42 shows early activation at 6 hours, with a sharp increase that surpasses BG37 and other promoters at 15 hours (~275 A.U.). This suggests it may be highly efficient in later phases of growth.

References

[1] Zobel, S., Benedetti, I., Eisenbach, L., de Lorenzo, V., Wierckx, N., & Blank, L. M. (2015). Tn7-Based Device for Calibrated Heterologous Gene Expression in Pseudomonas putida. ACS synthetic biology, 4(12), 1341–1351. https://doi.org/10.1021/acssynbio.5b00058

[2] Taş, H. (2020). Upgrading Pseudomonas putida as a Synthetic Biology chassis through inter-operativity of genetic devices [Doctoral dissertation, Universidad Autónoma de Madrid].

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]