Team:SZPT-CHINA/Parts/ImprovedParts
Improve
In 2015, TJU-China team submitted P170 Promoter (BBa_K1696005) which is regulated by the pH. When pH is between 6.0 and 6.5, it will be turned on and have the highest transcription efficiency. Conversely, when pH is neutral or alkaline, the expression efficiency will be lower.
But it’s a pity that there was no validation data in the project of TJU-China team. Otherwise, we want to use pH-regulated promoter in our project this year. However, in our project, the promoter regulated by lower pH than 6.0 is necessary. So, we used the modified promoter PrcfB(BBa_K3142000) in our project. Fig.1 are the DNA sequence of P170 (418bp) and PrcfB(112bp). Meanwhile，we perform fluorescence quantitative verification and protein expression.
A.P170
B.PrcfB
Fig.1 The DNA sequence of P170 and PrcfB
VERIFICATION BY FLUORESCENCE ASSAY
First, we inserted P170-sfGFP and PrcfB-sfGFP into the E. coli expression vector pET-28a(+)
respectively. Fig.2 shown that the PrcfB-sfGFP with size 1251bp and PrcfB-sfGFP with size 925bp was cloned to
pET-28a(+) vector successfully.
Fig2. The confirmation of recombinant expression
vector
A: Recombinant expression vector pET-28a(+)-P170-sfGFP; M:DNA Marker;
Lane 1: pET-28a(+)-P170-sfGFP/Hind III and Sal I ; Lane 2: PCR of pET-28a(+)-P170-sfGFP.
B: Recombinant expression vector pET-28a(+)-PrcfB-sfGFP; M:DNA Marker;
Lane 1: pET-28a(+)-PrcfB-sfGFP/ Hind III and Sal I; Lane 2:PCR of pET-28a(+)-PrcfB-sfGFP
Then the recombinant vector transformed into E. coli BL21. BL21，BL21 transferred with
pET-28a(+)-P170-sfGFP and BL21 transferred with pET-28a(+)-PrcfB-sfGFP were inoculated in LB medium with pH 5
respectively. After incubating for 18 h at 37 ° C. The bacterium was placed under UV light to excite GFP. The
results are shown in the Fig.3. It can be seen that in pH =5, the fluorescence using PrcfB is stronger than
P170.
Fig. 3 The regulation result of P170 promotor and
PrcfB promoter in pH 5
1: pET-28a(+)-PrcfB-sfGFP; 2: pET-28a(+)-P170-sfGFP;
3: pET-28a(+)
For a better comparison the regulation between PrcfB and P170, BL21，BL21 transferred with
pET-28a(+)-P170-sfGFP and BL21 transferred with pET-28a(+)-PrcfB-sfGFP were inoculated in LB medium with pH 5 and
pH 7 respectively. We measured the fluorescence intensity using fluorescence detection microplate reader. The
result is shown in Fig.4.The result shows that in pH 5, the fluorescence intensity using improved prompter PrcfB
is 239.78 and using unmodified promoter P170 is 123.693. While in pH7, the fluorescence intensity using PrcfB is
46.637 and using P170 is 101.407. This indicated that our improved PrcfB can be regulated by lower pH.
Fig.4 The Fluorescence Using Different pH Regulated
Promoter
VERIFICATION BY A11SG-AHPM PROTEIN EXPRESSION
First, we constructed a vector that regulates the expression of a protein of interest through the
PrcfB promoter. The vector has the characteristic that when the pH is less than 5.5, the PrcfB promoter strongly
expresses the LacR, and the LacR binds to the O1/O2 site to repress the production of the target protein. When the
pH is greater than 5.5, the PrcfB promoter is weakly expressed, and the protein of interest can be expressed
without being repressed. Fig.5 shows the regulation mechanism.
Fig.5 Diagram of regulation mechanism
MODEL
In order to know if PrcfB promotor can work Lactococcus lactis MG1363, We established a model of
pH-induced promoter regulation on the target protein of Lactococcus lactis MG1363. Then, we performed numerical
simulations on the model and sensitivity analysis on some key parameters. The results of this model are shown in
Fig.6
Fig. 6 The prediction result by established model
According to the model, when the pH was less than 5.5, the expression protein of the Lactococcus lactis MG1363 was
inhibited after 4 hours. It means that during the fermentation of lactic acid bacteria, when the pH of the
external environment drops below 5.5, the expression of the target protein will be inhibited after 4 hours. It is
beneficial to reduce the energy consumption of the bacteria, and is beneficial to increase the expression of our
target protein in the intestinal tract. When the pH of the external environment rises above 5.5, the expression of
the target protein can be observed after about 2 hours, and the expression rate is relatively stable. It indicates
that the corresponding protein of interest will be expressed by lactic acid bacteria two hours after it is
colonized into the intestine.
The regulation of PrcfB in A11sg-AHPM protein
From our model, we predicted that the vector containing the PrcfB promoter could express our
target protein at pH above 5.5, and could not express our target protein at pH less than 5.5. According to our
established model, we inoculated Lactic acid bacteria containing the vector into GM17 medium at pH 5, pH 5.5, PH 6,
and pH 7 for 30 hours, then collected the cells and verified by SDS-PAGE. Fig.7 is the expression result.
Fig.7 The expression of A11Sg-AHPM in different pH
M: DNA Marker; Lane 1: LAB MG1363(pMG36e-A11Sg-AHPM-P-L) cultured in pH5;
Lane 2: LAB MG1363(pMG36e-A11Sg-AHPM-AHPM-P-L) cultured in pH5.5; Lane 3: LAB MG1363(pMG36e-A11Sg-AHPM-P-L )cultured in pH6;
Lane4: LAB MG1363(pMG36e-A11Sg-AHPM-P-L) cultured in pH7
The results show when the pH is less than 5.5, PrcfB initiate the expression of LacR, the which
bind to O1/O2, then depressed the expression of A11Sg-AHPM. When pH≥5.5, the PrcfB is turned off and the
expression of LacR decrease, the expression of A11Sg-AHPM is initiated and the expression level increases along
with the increase of pH.
All the experimental results above show that the codons are successfully optimized, the length of
the sequence is reduced, and the function of the components is improved. This will facilitate the team that will
use this component to regulate at low pH in the future.
REFERENCE：
[1] Madsen S M, et al. Molecular characterization of the pH-inducible and growth phase-dependent
promoter P170 of Lactic acid bacteria. Molecular Microbiology, 1999, 32(1):75-87.
[2] Søren M. Madsen, et al. Two acid-inducible promoters from Lactic acid bacteria require the
cis-acting ACiD-box and the transcription regulator RcfB. Molecular Microbiology, 2005, 56(3):735-746.
[3] Ismail AKYOL, et al. Regulation of the acid inducible rcfB promoter in Lactic acid bacteria
subsp. Lactis. Annals of Microbiology, 2008, 58 (2) 269-273.