Team:SZPT-CHINA/Improvement
Improvement
Background
Goal
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.