Proof of expression

● Background
An inducible promoter is often used to control the expression of lysis protein in bacteria. However, a background level of lysis protein could often cause great pressure to the host bacteria. Since accumulation of lysis protein would induce autolysis, or drive the emergence of loss-of-function mutations in coding sequence (CDS) or ribosome-binding sequence (RBS) [1].
● Goal
Modulate the expression of lysis protein by random mutagenesis in the RBS region, which can enable our host bacteria to produce effective lysis proteins in a more steady fashion.
● Proof of expression
Screening the suitable RBS for our system by the auto-lysis action
Mutagenesis at specific position of RBS (BBa_B0034) was achieved using random primers and PCR, and the constructed plasmid was transformed into E. coli DH5α. 12 single colonies were selected to streak on two new LB plates to have two identical copies. Both plates were incubated at 37 ℃ for 12-16 hours, with one under blue light with a weavelenth of 470nm while the other under dark condition (Figure 1). Colonies that grew normally in the dark but not under the blue light irradiation met our needs, since the expression of lysis protein successfully expressed at the latter illumination condition could inhibit bacterial growth. The culture of eligible bacterial isolates were then sent for Sangon sequencing to obtain the corresponding RBS sequence ((Table 1&2).
Figure 1. Colony growth of E. coli DH5α-pDawn-RBSNNN-LKD-pSEVA331 under blue light (Left) or in the dark (Right).
Fluorescence quantitative analysis to screen the RBS
The RBS region of J23100-B0034-sfGFP -pSEVA331(BBa_K3740058) was replaced respectively by the abovemetioned RBS004 (BBa_K3740018), RBS009 (BBa_K3740008), and RBS010 (BBa_K3740010) to obtain an array of resultant plasmids as listed in Table 1. These plasmids were then transformed into E. coli competent cells.
Using micro-plate reader, we quantified the fluorescence intensity when the optical absorbance of bacterial culture at 600nm was around 0.2 [2], to determine the strength of the mutated RBS and B0034.
As shown in Figure 2 and Table 2, the reporter gene expression level in isolate 2, 3 and 4 was much lower than that of isolate 1. The average fluorescence intensity of sfGFP induced by RBS004 (BBa_K3740018), RBS009 (BBa_K3740008) and RBS010 (BBa_K3740010) was less than 3% of B0034 (BBa_B0034), which means reducing the pressure to the host bacteria.
Table 1. Information related to the plasmids derived from B0034
Number Full name of corresponding plasmid RBS ID
1 J23100-B0034-sfGFP-pSEVA331 B0034
2 J23100-RBS004-sfGFP-pSEVA331 B0034
3 J23100-RBS009-sfGFP-pSEVA331 RBS009
4 J23100-RBS010-sfGFP-pSEVA331 RBS009
Figure 2. The sfGFP inducible expression by different RBS. 1, isJ23100-B0034-sfGFP-pSEVA331-DH5α; 2, is J23100-RBS004-sfGFP-pSEVA331-DH5α; 3, is J23100-RBS009-sfGFP-pSEVA331-DH5α; 4, is J23100-RBS010-sfGFP-pSEVA331-DH5α
Table 2. Information related to RBS used in this study
number RBS BBa sequence average fluorescence intensity (a.u.) ratio
1 B0034 BBa_B0034 aaagaggagaaa 23498.40742 1
2 RBS004 BBa_K3740018 aaGgaTgTGaaa 420.0416198 0.017875323
3 RBS004 BBa_K3740008 aaGgaCgTGaaa 607.8410133 0.025867328
4 RBS010 BBa_K3740010 aaCgaAgCGaaa 255.1001708 0.010856062
● References
[1]Dai Z , Lee A J , Roberts S , et al. Versatile biomanufacturing through stimulus-responsive cell-material feedback [J]. Nature Chemical Biology, 2019, 15(10): 1017-1024. [2]Zhang H M , Chen S , Shi H , et al. Measurements of Gene Expression at Steady State Improve the Predictability of Part Assembly [J]. Acs Synthetic Biology, 2016, 5(3): 269.