Team:JiangnanU China/Demonstrate
Project Demonstrate
Throughout the project, we screened two inducible promoters, mutated to obtain six phage resistance
parts, and found a phage resistance part from the literature, all of which were derived from E.
coli
BL21. We used reporter genes to detect the effectiveness of inducible promoters, and
overexpression
of resistance proteins to verify the effect of proteins on cell growth. We assembled the resistance
parts and promoters to obtain a strong strain that is immune to T4 phage.
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Inducible Promoters PputA and PglcF Can Respond toT4 Phage
Infection
We obtained phage-inducible promoters PputA and PglcF by
transcriptomics data, ligated
these two
different time-responsive inducible promoters to the fluorescent genes gfp and mcherry,
respectively,
and transformed them into E. coli BL21. The two strains BL21-PputA-gfp and
BL21-PglcF-mcherry
were
cultured overnight at 37 ° C for about 12-16 hours, then transferred to fresh LB medium at 1%, cultured
for about 2 hours. After that added 1% freshly centrifuged T4 phage solution, and shaken at 37 ° C for
30 min. Then take the 10 μL to make tablets, and fluorescence observation was performed by a
laser
confocal microscope. At the same time, we set up a mutant strain without phage as a control.
From the results, we can find that the strains E. coli BL21-PputA-gfp and
E. coli BL21-PglcF-mcherry
without phage showed
no signs of fluorescence. The two bacteria infected with phage showed obvious fluorescence. But overall,
the number of bacteria that show fluorescence was not that much, because not every bacterium was
infected with T4 phage. This indicated that the promoters we selected is silent in the absence of phage
infestation and does not affect the expression of subsequent proteins.
2.The Effectiveness of T4 Phage Resistance Proteins
We found a resistance protein, AbpAB, from the literature, and found that the protein only reduced
phage sensitivity. To obtain a strain that is fully immunized against the T4 phage, we mutated and
sequenced E. coli BL21 to obtain four key anti-phage-related genes gntR, rzpD, yhjH,
nuoE.
According to the advice of corporate stakeholders, if the Genetic Modified (GM) strain is to be applied in industry, our components cannot have a great influence on bacterial growth. Since it is not possible to directly see from the figure which element has the least influence on the growth of the bacteria, we use the Grey Relation Analysis（GRA） method to analyze the growth curve of the bacteria connecting the various components. We used the Entropy Weight Method (EWM) method to determine the weight of each growth point to select the most similarly modified strain (the highest correlation), which is the component that has the least impact on bacterial growth. At the same time, after consulting the industry experts, we revised the weights according to the experts' recommendations to evaluate the components again, making them more in line with the real situation of production, that is, the most suitable components for industrial production. Using GRA's two evaluations of the four components at different weights, we selected the component gntR to cascade abpAB to construct a stronger resistant strain for better industrial performance.
According to the advice of corporate stakeholders, if the Genetic Modified (GM) strain is to be applied in industry, our components cannot have a great influence on bacterial growth. Since it is not possible to directly see from the figure which element has the least influence on the growth of the bacteria, we use the Grey Relation Analysis（GRA） method to analyze the growth curve of the bacteria connecting the various components. We used the Entropy Weight Method (EWM) method to determine the weight of each growth point to select the most similarly modified strain (the highest correlation), which is the component that has the least impact on bacterial growth. At the same time, after consulting the industry experts, we revised the weights according to the experts' recommendations to evaluate the components again, making them more in line with the real situation of production, that is, the most suitable components for industrial production. Using GRA's two evaluations of the four components at different weights, we selected the component gntR to cascade abpAB to construct a stronger resistant strain for better industrial performance.
Overexpression of these four genes, further experimental results show that gntR has the best
anti-phage
effect. The experimental results are also consistent with the model prediction, and gntR has
better
anti-phage effect. Gene gntR is related to GntR family transcriptional regulator and galactonate
operon
transcriptional repressor. The specific mechanism is still unclear, and this remains to be explored. But
according to the literature, AbpAB can impair the synthesis of late gene transcripts, which resulted in
poor expression of late proteins and consequently no phage propagation, and impair the synthesis of the
phage DNA. Also, AbpAB have no effect on the bacterial growth. As for the GntR, we believe that it
affects the normal infection of phage by regulating cellular metabolism.
We connect gntR with abpAB on pET28a, and transformed them into E. coli BL21. We
co-expressed abpAB and
gntR, and subsequently obtained a recombinant strain that is immune to phage.
3.Constitutive Expression of Resistance Proteins
In the case of determining that the combination resistance protein is effective, we choose the
constitutive promoter PrsmH to constitutively express the resistance protein in an
attempt to verify
whether the resistance protein has an effect on cell growth.
First we linked this constitutive promoter to gfp, demonstrating the validity of this
constitutive
promoter. The effect of overexpression of the resistance protein AbpAB on growth was then verified.
The results showed that PrsmH had a good effect, and overexpression of AbpAB had
no significant
effect on the growth of E. coli BL21.
The Effectiveness of The Overall Gene Circuit
We ligated the combined resistance protein gene and the kill switch P-1 (P-1 part链接)after the latent
phage-inducible promoter and the burst-inducible promoter, and performed the phage infection assay
on
the LB agar plate, and got very good resistance.
In addition, we inoculated E. coli BL21and recombinant E. coli
BL21-pET28a-PputA-abpAB-gntR-PglcF-P-1 in
LB liquid medium to raise the logarithmic growth phase, i.e. OD 0.6-0.8 . Then the fresh phage
solution
was inoculated at the same time and culture was continued for 1-2 h. As a result, it was found that the
recombinant grew well.
5.Application of Phage Resistant Strain in Fermentation Growth
We cooperated with NINGXIA EPPEN BIOTECH CO.,LTD (http://www.eppen.com.cn)to carry out small-scale
and pilot test fermentation experiments of resistant strain in the special fermentation laboratory
of Jiangnan University. This ensures that our experiments are controllable without any phage and
engineered bacteria leaking.
We transferred the constructed plasmid into a production strain producing γ-aminobutyric acid (GABA).
The fermentation laboratory is subjected to UV irradiation and ozone fumigation prior to formal fermentation to remove phage that may be present. TheE. coli BL21-pET28a-PputA-abpAB-gntR-PglcF-P-1, E. coli BL21-pET28a-PputA-abpAB and the control (E. coli BL21) were added to 1% T4 phage about 6 hours after inoculation, and the fermentation was continued for 10 hours. During the fermentation, the OD600 was measured, and the effects of the phage on the three were observed.
Then we used whole-cell transformation with a combination of resistant strain E. coli BL21-pET28a-PputA-abpAB-gntR-PglcF-P-1 and we got a good whole-cell transformation ability of the resistant strain (Figure 12).
From the results, our resistant composite part has great advantages in the production of γ-aminobutyric acid and are not threatened by T4 phage. The productivity of γ-aminobutyric acid is 278.3 g/L, and the molar conversion rate is really high, reaching 98.4%, which means that the circuit we built can be used in production without any impact (Figure 13).
We transferred the constructed plasmid into a production strain producing γ-aminobutyric acid (GABA).
The fermentation laboratory is subjected to UV irradiation and ozone fumigation prior to formal fermentation to remove phage that may be present. TheE. coli BL21-pET28a-PputA-abpAB-gntR-PglcF-P-1, E. coli BL21-pET28a-PputA-abpAB and the control (E. coli BL21) were added to 1% T4 phage about 6 hours after inoculation, and the fermentation was continued for 10 hours. During the fermentation, the OD600 was measured, and the effects of the phage on the three were observed.
Then we used whole-cell transformation with a combination of resistant strain E. coli BL21-pET28a-PputA-abpAB-gntR-PglcF-P-1 and we got a good whole-cell transformation ability of the resistant strain (Figure 12).
From the results, our resistant composite part has great advantages in the production of γ-aminobutyric acid and are not threatened by T4 phage. The productivity of γ-aminobutyric acid is 278.3 g/L, and the molar conversion rate is really high, reaching 98.4%, which means that the circuit we built can be used in production without any impact (Figure 13).
6.Models for Future Work
The phage-induced promoters are vital in our genetic circuit, which would response the phage stimulation
and start transcription of ant-P and P-1 (antimicrobial peptide) against the phage infection. And the
phage-induced promoters we used were selected from E. coli BL21, so there might be some potential
problems such as leakage and inclusion body due to the inappropriate promoter strength. Thus we
developed a quantitative design method for phage -induced promotors based on strength prediction using
Artificial Neural Network (ANN). The model allows us to choose or design promotors with desired strength
without extra experiments.
