Team:SCUT-FSE-CHINA/Results
Results
N&P Pathways
Figure 1. Effects of different concentrations of formamide (a) and the constructed plasmid (b) on the growth of E. coli strain BL21(DE3) in normal LB media.
To examine the effects of formamide itself on the growth of E. coli, the medium was added with different concentrations of formamide. Considering the possible effect of inserted vector, we compared the growth curve of three different strains, including the original BL21(DE3), BL21(DE3)(pGEX) (BL21(DE3) with pGEX vector) and BL21(DE3)(pGEX-for) (BL21(DE3) with formamidase gene in pGEX vector), in normal LB media.
From Figure 1a, the inhibitory effect of formamide on the growth of E. coli is so slight that we can ignore it if we properly control the concentration of formamide. The results provided us a reference on designing the concentration of formamide in the culture media. From Figure 1b, the insertion of formamidase gene had no effect on the growth of E. coli.
Figure 2. Growth curves of E. coli containing formamidase gene in a basal MOPS medium (a) in the presence of formamide as the nitrogen source comparing with negative controls and (b) containing different concentrations of formamide.
To verify that our recombinant E. coli containing formamidase gene could work efficiently to utilize formamide as its nitrogen sources, we cultivated the recombinant one and the negative controls in a basal MOPS medium.
From Figure 2(a), E. coli containing formamidase gene, reached maximum concentration after 48 hours in the basal MOPS medium in the presence of 200 mM formamide. While E. coli without formamidase gene basically did not grow. From Figure 2(b), Under the condition of low concentration of formamide(<200mM), the bacteria grew fast with the increase of formamide concentration. However, when the concentration of formamide was higher than 200mM, the concentration of E. coli did not increase with the increase of formamide concentration. According to this result and the mentioned inhibitory effect of formamide, we optimized our MOPS medium with 200mM as the final concentration of formamide.
Figure 3. Growth curve of E. coli BL21(DE3) and the engineering E. coli BL21(DE3)(pGEX-ptx) expressing phosphite dehydrogenase in a medium contains Na2HPO3•5H20 (1.32mM) and NH4Cl serving as the phosphorus and nitrogen sources, respectively.
To examine the function of our recombinant E. coli containing phosphite dehydrogenase gene, we cultivated the recombinant one (BL21(DE3)(pGEX-ptx) and the negative control (original BL21(DE3) ) in a medium with Na2HPO3•5H2O (1.32mM) and NH4Cl as the phosphorus and nitrogen sources, respectively.
The E. coli expressing phosphite dehydrogenase outcompeted a control strain in phosphite-containing media after 72 hours. However, the negative control group still grew in a certain degree. We looked up the researches and presumed that, alkaline phosphatase(BAP) found in E. coli, will be increasingly express when the amount of bio-available phosphorus is low. Through the hydrolysis, the BAP then turn the phosphorus compounds in the environment into bio-available phosphorus. With the help of the BAP, the microbes can utilize the phosphorus compounds more effectively, which also facilitates the modified pathways.
Figure 4. Growth curves of E. coli (with two ways of constructed plasmids possessing the formamidase gene and phosphite dehydrogenase gene) in a basal MOPS medium (Na2HPO3•5H2O (1.32mM) , NH3CO (200mM))
In order to verify that the our recombinant E. coli with the recombinant plasmids of the formamidase gene and the phosphite dehydrogenase gene could work efficiently on simultaneously utilizing formamide and phosphite, we cultivated and compared the growth of three strains, including the original BL21(DE3), BL21(DE3)(pGEX-for-ptx and BL21(DE3)(pETDuet-for-ptx), on MOPS medium with NH3CO (200mM) as the sole nitrogen source and Na2HPO3•5H2O (1.32mM) as the sole phosphorus source.
The above results showed that the growth of recombinant strains of both construction ways are encouraging, indicating the recombinant strains were able to utilize formamide and phosphite at the same time, while the negative control group was unable to grow normally due to the lack of nitrogen and phosphorus sources.
Figure 5. (a)Picture of GFP fluorescence obtained from BL21(DE3) with the pGEX-f-p+pET28a-GFP construct cultured in LB medium (10X40); (b) Picture of GFP fluorescence obtained from BL21(DE3) with the pETDuet-f-p+pET28a-GFP construct cultured in LB medium (10X40); (c) Picture of GFP fluorescence obtained from BL21(DE3) with the pETDuet-GST-f-p+pET28a-GFP construct cultured in LB medium (10X40).
With GFP as a report to verify that exogenous protein could be properly folded in the engineering bacteria, we transfected our three kinds of expression vectors (pGEX-f-p+pET28a-GFP, pETDuet-f-p+pET28a-GFP and pETDuet-GST-f-p+pET28a-GFP) into BL21(DE3) and estimated the GFP expression level under a Fluorescence Microscope. According to Figure 5(a)-(c), significant GFP fluorescence in Escherichia coli was detected in all three samples, confirming that the recombinant vectors have been constructed successfully and then the GFP could be properly folded and expressed. Additionally, the observation that GFP fluorescence showed no significant difference among three expression vectors suggested that these ways of construction have similar expression efficiency.
Figure 6. Growth curve of three E. coli strains containing different plasmid (pGEX-f-p+pET28a-GFP, pETDuet-f-p+pET28a-GFP and pETDuet-GST-f-p+pET28a-GFP) in specific MOPS medium with formamide and phosphite as the nitrogen and phosphorus source (Na2HPO3•5H2O (1.32mM), NH3CO (200mM).
As shown in Figure 6, similar growth trends of three strains have been reported in lag, logarithmic and stationary phases. However, after a 24-hour lag, OD value of samples with corresponding expression vectors showed slight differences. The one with expression vector of fusion protein showed the best growth level, followed by the co-expression vector with GST tag and the co-expression vector without GST tag, successively. （GST used for increasing the solubility of exogenous protein. All three kinds of strains reached the stationary phase after 48 hours when their OD value fell on about 2.1-2.4. The result indicated that the construction way of fusion protein helps to promote the metabolic functions of the recombinant bacteria, whereas GST label also facilitates the expression of genes of formamidase and phosphite dehydrogenase.
Figure 7. (a) Picture of GFP fluorescence obtained from BL21(DE3) with the pGEX-f-p+pET28a-EGFP construct cultured in MOPS medium (Na2HPO3•5H2O (1.32mM) , NH3CO (200mM)) (10X40); (b)Picture of GFP fluorescence obtained from BL21(DE3) with the pETDuet-f-p+pET28a-GFP construct cultured in MOPS medium (10X40); (c) Picture of GFP fluorescence obtained from BL21(DE3) with the pETDuet-GST-f-p+pET28a-EGFP construct cultured in MOPS medium (10X40).
To further test the function of metabolic pathways that we modified, we continually cultivated the engineering bacteria of three different expression vectors in the MOPS medium. According to Figure 6 and 7, after 72 hours of cultivation and induction, the observation of GFP fluorescence in all three samples indicated that the recombinant bacteria have the abilities to utilize formamide and phosphite for their own growth and multiplication, as well as express the GFP. The results revealed that the existing culture conditions, including medium composition, pH, osmotic pressure, redox potential, cofactors, and folding mechanisms, had satisfied the normal folding and synthesis of the GFP. Next, we will try to prove that our design could be a suitable system for production of target products. To complete this, we plan on replacing the GFP with other genes of interest in the future.
Figure 8. Comparison between the growth situation of (a) the BL21(pGEX-f-p+pET28a-GFP) and unexpected bacteria in unsterile MOPS medium; (b)the BL21(pGEX-f-p+pET28a-GFP) and yeast in MOPS medium (Na2HPO3•5H2O) (1.32mM), NH3CO (200mM)). ( Click to show supplemental materials.)
We examined the respective population of our engineering strains and other species under co-culture circumstances by Flow cytometry. The results are showed as followed, which contain scatter plots and histograms in channel FITC-A.
Part 1 Culturing BL21(pGEX-f-p+pET28a-GFP) in unsterile specific MOPS medium
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Part 2 Co-culturing BL21(pGEX-f-p+pET28a-GFP) and yeast in specific MOPS medium
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To prove the function of the recombinant E. coli that are expected to outcompete the undesired microorganisms under limited culture environment, we conducted two experiments. First, we incubated the BL21(pGEX-f-p+pET28a-GFP) into unsterile MOPS medium. Another one is the co-culture of recombinant E. coli and yeast in MOPS medium. The samples were taken every six hours and estimated under flow cytometry.
According to Figure 8(a), the population of BL21(pGEX-f-p+pET28a-GFP) accounted for over 80% during the cultivation period. However, the percentage of other microorganisms (under 20%) remained much less than that of BL21(pGEX-f-p+pET28a-GFP). According to Figure 8(b), there are different growing trends of both strains. The proportion of the recombinant E. coli experienced an increase reaching to around 50% gradually, which exceeded that of yeast after 46 hours. On the contrary, with an initial proportion of 70.39%, the amount of yeast eventually fell to 34.26% with fluctuation.
The amount variation between them indicated that BL21(pGEX-f-p+pET28a-GFP) outcompeted (or surpassed) the unexpected bacteria in selective medium (MOPS). These results show that the recombinant E. coli possessing the modified pathways preponderate other species in the competition, which provides a basis for practical application of our project.
CRISPR/Cas9
Figure 9. Comparison of the growth situation between the BL21(DE3)(control group) (left) and the BL21(DE3) (pCas+pTarget-N20) (right) after infected by T7 phages (a) in LB plate; (b) in LB broth.
To test the function of the two-plasmid system in the BL21(DE3) (pCas+pTarget-N20) strain, we cultivated both the BL21(DE3) (pCas+pTarget-N20) and negative control BL21(DE3), with T7 phage added by two layer plating method and in liquid media when the bacteria reached Logarithmic growth period.
According to Figure 9(a), obvious plaque can be seen on both plates after phage infection. However, the plaque amount on the negative control plate was much more than that on the plate of BL21(DE3) (pCas+pTarget-N20). The liquid media are supposed to be more transparent when the phage infection is more severe. According to Figure 9(b), the concentration of the experimental strain was significantly higher than that of the control group after introducing T7 phage, which confirmed our CRISPR/Cas9 system did work to resist this specific phage.
Figure 10. Growth curve of BL21(DE3) (pCas+pTarget-N20) with and without the T7 phage infection and E. coli BL21(DE3)(negative control) with the T7 phage infection in LB media.
To monitor the growth situation of the BL21(DE3) (pCas+pTarget-N20) strain, we cultivated both the BL21(DE3) (pCas+pTarget-N20) and negative control BL21(DE3), with T7 phage added in liquid LB medum when the bacteria reached logarithmic growth period. To estimate the resist efficiency of the CRISPR system, we also set a control group when BL21(DE3) (pCas+pTarget-N20) was cultivated without phage infection during the period. We sampled and detected both three groups every two hours.
As shown in Figure 10, obviously, the growth curves of BL21(DE3) (pCas+pTarget-N20) with and without the T7 phage infection were similar to each other, indicating that the growth of BL21(DE3) (pCas+pTarget-N20) did not be affected by T7 phage. In addition, the concentration of the negative control BL21(DE3) dropped sharply four hours after T7 phage added. The above results convinced that the recombinant strain functioned quite well to resist the phage T7 in LB medium, and the efficiency of resistance reached nearly 100%.
Robust System
Figure 11. Growth curve of BL21(DE3) (pCas+pTarget-N20+for+ptx) with and without the T7 phage infection and E. coli BL21(DE3)(for+ptx)(negative control) with the T7 phage infection in LB media.
In order to verify the function of BL21(DE3) (pCas+pTarget-N20+for+ptx) which processed the functional vectors pGEX-for-ptx from N&P Pathways as well as pCas and pTarget-N20 from CRISPR/Cas9 system, we cultivated and compared the growth of BL21(DE3) (pCas+pTarget-N20+for+ptx) and BL21(DE3) (for+ptx), with T7 phage added in liquid LB media when the bacteria reached logarithmic growth period. We also set a control group when BL21(DE3) (pCas+pTarget-N20+for+ptx) was cultivated without phage infection, to estimate the function efficiency.
According to Figure 11, the concentration of BL21(DE3) (pCas+pTarget-N20+for+ptx) and BL21(DE3) (for+ptx) both experienced dramatical decreases about six hours after T7 phage. BL21(DE3) (for+ptx) stood at a low concentration since then. However, after remaining stable for about 36 hours, BL21(DE3) (pCas+pTarget-N20+for+ptx) recovered from the invasion of T7 phage and eventually climbed to a satisfying concentration comparative to the one without phage infection. These results proved that the Robust system with three plasmids worked well in resisting phage infection, although a period of recovery was required.
Figure 12. Growth curve of BL21(DE3) (pCas+pTarget-N20+for+ptx) with and without the T7 phage infection and E. coli BL21(DE3)(for+ptx)(negative control) in specific MOPS medium.
Next, similar experiment was conducted to examine the entire function of our Robust system in specific MOPS medium. The figure showed that the concentration of BL21(DE3) (pCas+pTarget-N20+for+ptx) did not decrease after T7 phage infection. On the other hand, lower growth rate and relatively lower final concentration of the the group with T7 phage were observed compared to the one without phage infection during this period. The encouraging growth situation of BL21(DE3) (pCas+pTarget-N20+for+ptx) in MOPS medium without phage infection indicated that the CRISPR/Cas did not affect the function of N&P Pathways. Meanwhile, CRISPR/Cas system still worked to resist T7 phage and the efficiency of resistance can reached nearly 60%. The efficiency was slightly lower than that culture in LB medium. This may due to lack of enough nutrition in the specific MOPS medium, and we will continue to optimize the concentration of formamide and phosphite of the medium to achieve a satisfactory efficiency.
Discussion
In this project, we have successfully confirmed that our design of N&P pathways could be a suitable system for production of target products (here we use GFP as an example) in MOPS medium (with formamide as the sole nitrogen source and phosphite as the sole phosphorus source). Moreover, we have verified that the CRISPR/Cas functioned well in our Robust E. coli to resist the T7 phage in the specific medium. Therefore, it is indicated that our Robust system has been built up successfully. We devised to replace the GFP with other genes of interest. In this way, variable fermented products can be produced in specific MOPS medium.
With our Robust E. coli, open(unsterilized) fermentative process are expected to be operated in a more economic friendly way, as well as be free from the bacterial contamination and phage infection. To make our design become more applicable in practical production, we will try to figure out ways to improve the efficiency of the Robust system in the future.
Reference
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