Team:Thessaly/Improve
Overview
It is important, as an iGEM team, to share our work through the parts registry but also build upon and
improve other igem existing parts. This year we decided to improve BBa_K523002, by altering the native
signal peptide sequence of BglX, a periplasmic beta glucosidase, with a N20- signal peptide BBa_K3866032, which
translocates the protein extracellularly. With this improved part, we engineered bacteria that are able
to valorize cellobiose and use it as a carbon source. In this page, we present the course of our
experiments, the results and how this improved part is implemented to our project.
Ensuring cell viability
Although humans are not able to digest cellulose, its partial digestion occurs in the gut by beneficial
microflora. In a healthy gut about 50% of cellulose is degraded by natural fermentation in the colon and
significant amounts of Short Chain Fatty Acids(SCFAs) are produced feeding our natural gut flora.
(Mudgil, D., & Barak, S., 2013).
For the last part of our project, we designed a probiotic supplement that produces the quantities of SCFAs missing inside the gut. Through our conversations with experts, specifically Dr. Meziti, we received feedback related to the survival of our probiotic in the unfriendly and antagonistic environment of the gut . Her opinion was that our probiotic bacteria should be sustainable and colonize the gut to survive. Building on this feedback, we thought that our engineered probiotic could benefit from a metabolite that exists inside the gut and ensure it’s viability. Through bibliographic research, we found that cellulose may be a possible target. Cellulose is the most abundant polysaccharide found in nature. It consists of linear chains of ß (1–4) linked D-glucose. (Li, S. et al., 2014).
It is a compound, which constitutes the 25% of the human stool inside the gut (Rose, C. et al., 2015) and as a dietary fiber can worsen gut irritation to people with gastrointestinal disorders (El-Salhy, M. et al., 2017). Additionally, decreased consumption of dietary fibers, such as cellulose, has been proposed to promote the emergence of inflammatory bowel diseases (Nagy-Szakal, D. et al., 2013). Those contributing factors led us to choose cellulose as the substrate for our engineered bacteria.
For the last part of our project, we designed a probiotic supplement that produces the quantities of SCFAs missing inside the gut. Through our conversations with experts, specifically Dr. Meziti, we received feedback related to the survival of our probiotic in the unfriendly and antagonistic environment of the gut . Her opinion was that our probiotic bacteria should be sustainable and colonize the gut to survive. Building on this feedback, we thought that our engineered probiotic could benefit from a metabolite that exists inside the gut and ensure it’s viability. Through bibliographic research, we found that cellulose may be a possible target. Cellulose is the most abundant polysaccharide found in nature. It consists of linear chains of ß (1–4) linked D-glucose. (Li, S. et al., 2014).
It is a compound, which constitutes the 25% of the human stool inside the gut (Rose, C. et al., 2015) and as a dietary fiber can worsen gut irritation to people with gastrointestinal disorders (El-Salhy, M. et al., 2017). Additionally, decreased consumption of dietary fibers, such as cellulose, has been proposed to promote the emergence of inflammatory bowel diseases (Nagy-Szakal, D. et al., 2013). Those contributing factors led us to choose cellulose as the substrate for our engineered bacteria.
Why did we choose to improve BglX?
In order for the probiotic to utilize cellulose, it has to degrade it into glucose. The degradation is
catalyzed by a metabolic pathway which consists of three enzymes. Those are cenA and cex
, which degrade cellulose to cellobiose (Greenberg, Warren, Kilburn & Miller, 1987). Cellobiose then is
broken down by BglX, the third enzyme in the metabolic pathway of cellulose degradation, into
glucose molecules, the energy currency of our engineered E.coli cells.
We chose to characterize the final reaction of cellulose degradation, cellobiose degradation, because we wanted to prove that the system works at its final form. It was, also, an opportunity to improve this already existing part in order to degrade cellobiose and also it was closely implemented to our project.
E.coli has a cryptic operon, consisting of six genes (CelABCDEF), which is inactive and contributes to the degradation of cellobiose (Reizer, J., et al., 1990). We cloned our bacteria with the protein, bglX, restoring the ability of these genes to catalyze the cellobiose degradation into glucose (Gao, D. et al., 2016).
Naturally, BglX has a native signal peptide for its transportation to the periplasm. This was a problem for us because we wanted BglX to be transported outside of our engineered bacteria so that BglX would come in contact with the extracellular cellobiose and degrade it into glucose, which then would be utilized by the same engineered bacteria. Through our bibliography, we found a signal peptide called N20 (Gao, D. et al., 2015) which was used for the extracellular transportation of other cellulases, like BglX. Our research led us to iGEM Edinburgh 2011 who used the native BglX (BBa_K523002) and 2 other enzymes in order to degrade cellulose to useful byproducts, but they could not make it possible. As a result, we decided to use the former native BglX by replacing its native signal peptide with our N20 signal peptide, so that we could transport it extracellularly and provide a useful carbon source to our engineered E.coli.
We chose to characterize the final reaction of cellulose degradation, cellobiose degradation, because we wanted to prove that the system works at its final form. It was, also, an opportunity to improve this already existing part in order to degrade cellobiose and also it was closely implemented to our project.
E.coli has a cryptic operon, consisting of six genes (CelABCDEF), which is inactive and contributes to the degradation of cellobiose (Reizer, J., et al., 1990). We cloned our bacteria with the protein, bglX, restoring the ability of these genes to catalyze the cellobiose degradation into glucose (Gao, D. et al., 2016).
Naturally, BglX has a native signal peptide for its transportation to the periplasm. This was a problem for us because we wanted BglX to be transported outside of our engineered bacteria so that BglX would come in contact with the extracellular cellobiose and degrade it into glucose, which then would be utilized by the same engineered bacteria. Through our bibliography, we found a signal peptide called N20 (Gao, D. et al., 2015) which was used for the extracellular transportation of other cellulases, like BglX. Our research led us to iGEM Edinburgh 2011 who used the native BglX (BBa_K523002) and 2 other enzymes in order to degrade cellulose to useful byproducts, but they could not make it possible. As a result, we decided to use the former native BglX by replacing its native signal peptide with our N20 signal peptide, so that we could transport it extracellularly and provide a useful carbon source to our engineered E.coli.
Figure 1: Schematic representation of the native bglX and its signal peptide (on the left)
and the improved bglX with the N20 signal peptide (on the right).
Experimental setup
The Transcriptional Units were assembled using
GoldenBraid method , adapted for bacterial chassis.
The functionality of the system was tested by transforming MC1061 bacteria with the final construct promoterJ23115-N20-bglX-terminator (BBa_K3866037), promoterJ23115-bglX-terminator (BBa_K3866033). We wanted to test the growth difference between the bacteria carrying either the native bglX orour improved N20-bglX constructs, while we also added an empty vector control. To ensure growth was observed due to the converted cellobiose, we incubated the strains in 10 ml of M9 minimal medium. We made a total of four different M9 media. The difference between them was the carbon source. The carbon source components for each medium were:
The symbols used in the following diagrams are described :
CB= M9 medium with cellobiose 1%.
GL= M9 medium with glucose 2%.
2= M9 medium with glucose 2% and cellobiose 1%.
(-)= M9 medium without carbon source.
N20= E.coli MC1061 bacteria transformed with the improved part promoterJ23115-N20-bglX-terminator.
SP= E.coli MC1061 bacteria transformed with the native part promoterJ23115-bglX-terminator.
EMPTY= E.coli MC1061 bacteria transformed with an empty vector.
The time points that have been received were at 0h, 2h, 4h, 6h, 8h, 10h, 24h.
In Figure 2 we have our negative control and we see that in absence of carbon source, the SP-bglX and EMPTY bacteria don’t grow, but the N20-bglX bacteria have a growth advantage compared to the other two categories of bacteria, which indicates that it could improve cell fitness in an environment poor in nutrients.
In Figure 3, we can see that the N20-bglX bacteria grow more and faster than the SP-bglX and EMPTY bacteria in a 24 hour time-frame, which indicates that the engineered enzyme could provide an advantage in situations where cellobiose is the only abundant carbon source.
In Figure 4, it is evident that N20-bglX bacteria reach an exponential-like phase much quicker than the native peptide or the empty vector control bacteria, indicating that the presence of the engineered enzyme provides quicker valorization cellobiose and update of glucose by the cell which could also provide a decisive advantage of our probiotic in competitive environments where more than one carbon sources utilizable by the cell are present.
Finally, in Figure 5 we observe our positive control and we see that all bacteria grow well in rich nutrient conditions with glucose, with the N20 and SP -bglX bacteria growing faster than the empty bacteria.
The functionality of the system was tested by transforming MC1061 bacteria with the final construct promoterJ23115-N20-bglX-terminator (BBa_K3866037), promoterJ23115-bglX-terminator (BBa_K3866033). We wanted to test the growth difference between the bacteria carrying either the native bglX orour improved N20-bglX constructs, while we also added an empty vector control. To ensure growth was observed due to the converted cellobiose, we incubated the strains in 10 ml of M9 minimal medium. We made a total of four different M9 media. The difference between them was the carbon source. The carbon source components for each medium were:
- Glucose 2%
- Cellobiose 1%
- Glucose 2% and cellobiose 1%
- No carbon source
The symbols used in the following diagrams are described :
CB= M9 medium with cellobiose 1%.
GL= M9 medium with glucose 2%.
2= M9 medium with glucose 2% and cellobiose 1%.
(-)= M9 medium without carbon source.
N20= E.coli MC1061 bacteria transformed with the improved part promoterJ23115-N20-bglX-terminator.
SP= E.coli MC1061 bacteria transformed with the native part promoterJ23115-bglX-terminator.
EMPTY= E.coli MC1061 bacteria transformed with an empty vector.
The time points that have been received were at 0h, 2h, 4h, 6h, 8h, 10h, 24h.
In Figure 2 we have our negative control and we see that in absence of carbon source, the SP-bglX and EMPTY bacteria don’t grow, but the N20-bglX bacteria have a growth advantage compared to the other two categories of bacteria, which indicates that it could improve cell fitness in an environment poor in nutrients.
Figure 2: Growth (OD600) of E. coli MC1061 cells transformed with N20-BglX (N20),
native-peptide BglX (SP) or an empty vector (EMPTY), in a minimal M9 medium without a carbon source
( - ).
In Figure 3, we can see that the N20-bglX bacteria grow more and faster than the SP-bglX and EMPTY bacteria in a 24 hour time-frame, which indicates that the engineered enzyme could provide an advantage in situations where cellobiose is the only abundant carbon source.
Figure 3: Growth (OD600) of E. coli MC1061 cells transformed with N20-BglX (N20),
native-peptide BglX (SP) or an empty vector (EMPTY), in a minimal M9 medium with Cellobiose as a
carbon source ( CB ).
In Figure 4, it is evident that N20-bglX bacteria reach an exponential-like phase much quicker than the native peptide or the empty vector control bacteria, indicating that the presence of the engineered enzyme provides quicker valorization cellobiose and update of glucose by the cell which could also provide a decisive advantage of our probiotic in competitive environments where more than one carbon sources utilizable by the cell are present.
Figure 4: Growth (OD600) of E. coli MC1061 cells transformed with N20-BglX (N20),
native-peptide BglX (SP) or an empty vector (EMPTY), in a minimal M9 medium with Cellobiose and Glucose as a
carbon source.
Finally, in Figure 5 we observe our positive control and we see that all bacteria grow well in rich nutrient conditions with glucose, with the N20 and SP -bglX bacteria growing faster than the empty bacteria.
Figure 5: Growth (OD600) of E. coli MC1061 cells transformed with N20-BglX (N20),
native-peptide BglX (SP) or an empty vector (EMPTY), in a minimal M9 medium with Glucose as a
carbon source ( GL ).
Conclusion
Our live biotherapeutic solution includes a system that will provide the ability to the probiotic to
utilize cellulose and produce SCFAs with higher yield. That’s how the microbial imbalance that may
exist in the gut, will be restored
In conclusion, we found a way to make probiotic bacteria use cellulose, found in the gut, for their growth in a more beneficial way. We engineered E.coli to utilise cellobiose by expressing the protein N20-bglX, and proved that they have a higher growth rate than the bacteria with native signal peptide- bglX. This means that the first one can utilize both carbon sources, cellobiose and glucose, in a much more efficient way.
In conclusion, we found a way to make probiotic bacteria use cellulose, found in the gut, for their growth in a more beneficial way. We engineered E.coli to utilise cellobiose by expressing the protein N20-bglX, and proved that they have a higher growth rate than the bacteria with native signal peptide- bglX. This means that the first one can utilize both carbon sources, cellobiose and glucose, in a much more efficient way.
References
- Mudgil, D., & Barak, S. (2013). Composition, properties and health benefits of indigestible carbohydrate polymers as dietary fiber: a review. International journal of biological macromolecules, 61, 1–6. https://doi.org/10.1016/j.ijbiomac.2013.06.044
- Li, S., Bashline, L., Lei, L., & Gu, Y. (2014). Cellulose synthesis and its regulation. The arabidopsis book, 12, e0169. https://doi.org/10.1199/tab.0169
- Rose, C., Parker, A., Jefferson, B., & Cartmell, E. (2015). The Characterization of Feces and Urine: A Review of the Literature to Inform Advanced Treatment Technology. Critical reviews in environmental science and technology, 45(17), 1827–1879. https://doi.org/10.1080/10643389.2014.1000761
- El-Salhy, M., Ystad, S. O., Mazzawi, T., & Gundersen, D. (2017). Dietary fiber in irritable bowel syndrome (Review). International journal of molecular medicine, 40(3), 607–613.https://doi.org/10.3892/ijmm.2017.3072
- Nagy-Szakal, D., Hollister, E. B., Luna, R. A., Szigeti, R., Tatevian, N., Smith, C. W., Versalovic, J., & Kellermayer, R. (2013). Cellulose supplementation early in life ameliorates colitis in adult mice. PloS one, 8(2), e56685. https://doi.org/10.1371/journal.pone.0056685
- Greenberg, N., Warren, R., Kilburn, D., & Miller, R. (1987). Regulation, initiation, and termination of the cenA and cex transcripts of Cellulomonas fimi. Journal Of Bacteriology, 169(2), 646-653. doi: 10.1128/jb.169.2.646-653.1987
- Reizer, J., Reizer, A., & Saier, M. H., Jr (1990). The cellobiose permease of Escherichia coli consists of three proteins and is homologous to the lactose permease of Staphylococcus aureus. Research in microbiology, 141(9), 1061–1067.https://doi.org/10.1016/0923-2508(90)90079-6
- Gao, D., Wang, S., Li, H., Yu, H., & Qi, Q. (2015). Identification of a heterologous cellulase and its N-terminus that can guide recombinant proteins out of Escherichia coli. Microbial cell factories, 14, 49. https://doi.org/10.1186/s12934-015-0230-8
- Gao, D., Luan, Y., Liang, Q., & Qi, Q. (2016). Exploring the N-terminal role of a heterologous protein in secreting out of Escherichia coli. Biotechnology and bioengineering, 113(12), 2561–2567. https://doi.org/10.1002/bit.26028