Part:BBa_K4850000

sacB Gene

The sacB gene was first obtained from Bacillus subtilis, and it encodes levansucrase, an enzyme involved in both sucrose hydrolysis and levan biosynthesis. In gram-negative bacteria like Escherichia coli (E. coli), levansucrase is secreted into the periplasmic space, where levan is synthesized as well. Levan cannot be metabolized by most gram-negative bacteria, so it is considered toxic to this group of organisms.

When sucrose is present in high concentrations, the interaction between the enzyme and substrate leads to the accumulation of both the enzyme and its product within the gram-negative bacteria's periplasmic space. As levan cannot be metabolized, this lethal accumulation within the cells could eventually lead to cell lysis.

Figure 1: The sacB gene encodes levansucrase, an enzyme known to convert sucrose to levan. Levan is toxic to gram-negative bacteria such as E. coli because these bacteria cannot metabolize levan. As a result, the accumulation of levan in the periplasmic space of the cells eventually leads to cell death.

Usage and Biology

Some common usage of the sacB gene include:

• Serve as an effective negative selection marker, enabling the isolation of target cells by subjecting them to growth in sucrose-containing media.
• Incorporated into the cell suicide cassette, allowing suppression of the cell growth under certain conditions.

The NUS-Singapore 2023 iGEM team introduced a model “OTTER”capable of predicting the RNA-RNA binding interaction and On/Off ratio of RNA switches. Recognizing the potential of the sacB gene, the team integrated sacB into their new selection method, known as the SIGNAL Assessment Workflow, to generate datasets in a high-throughput and highly selective manner for model training, thereby enhancing prediction precision and accuracy. The usage of the sacB gene includes:

• Functions as a versatile kill switch: The sacB gene serves as the core of the SIGNAL Assessment Workflow, particularly in the newly established diametric selection method. It allows precise and controlled cell death within a specific cell population, facilitating the selection of cells with favourable phenotypes, including high on/low off, high off/low on, high on/high off, and low on/low off.
• Maintain the cell selection quality: The sacB gene can eliminate cells with leaky gene expression effectively. This ensures that only cells with the desired gene regulation patterns are chosen for further analysis. Such precision is particularly vital in genetic experiments where accurate gene expression control is essential for reliable outcomes.

sacB Characterization

To understand the killing efficiency of the sacB gene under different sucrose concentrations and various levels of induction, a plasmid incorporating a pLac promoter, a green fluorescence protein (GFP) (BBa_K4850013), a protein linker [A(EAAAK)₂A] (BBa_K4850006), and the sacB gene (BBa_K4850000) was constructed. The pLac promoter was selected due to its inherent leakiness. This enables us to characterize the sacB gene for controlled cell elimination. For instance, it doesn't result in the elimination of all cells but rather preserves a fraction at specific sucrose concentrations. Consequently, only those cells displaying leaky gene expression or those with gene expression consistently in the ON state are targeted for elimination.

Figure 2: Plasmid with pLac promoter, GFP reporter protein, protein linker [A(EAAAK)₂A], and the sacB gene was constructed to characterize the sacB gene.

To characterize the sacB gene, the plasmid above was first transformed into the E. coli and cell stock was made. Then, the sacB cells were cultured in the LB media overnight. Subsequently, the cells were split into multiple tubes and cultured in fresh M9 media with different IPTG levels for 2 hours. Next, the cells were dispensed into a 96-well plate with each well holding pre-determined sucrose concentrations. The plate was then placed into a plate reader with a temperature set at 37 °C for continuous measurements of the optical density of the cells at 600nm (OD600) and GFP fluorescence over a 6-hour period, at 30-minute intervals. After the 6-hour measurement, the results were collected, analyzed, and plotted as line graphs to illustrate the changes in trend.

Results

The sacB cells were tested in the conditions shown in Table 1 and the changes in the OD600 of the cells were recorded. The results are listed from Figure 3 to Figure 10.

Table 1: 10 characterization tests were conducted for the sacB cells with various sucrose concentrations and induction levels as shown in the table.

Figure 3: The result was obtained by testing the sacB cells with 0 µM IPTG and 0%, 0.5%, 1%, 2.5%, as well as 5% sucrose. Data are represented as mean ± S.D. (n=3).

Figure 4: The result was obtained by testing the sacB cells with 25 µM IPTG and 0%, 0.5%, 1%, 2.5%, as well as 5% sucrose. Data are represented as mean ± S.D. (n=3).

Figure 5: The result was obtained by testing the sacB cells with 50 µM IPTG and 0%, 0.5%, 1%, 2.5%, as well as 5% sucrose. Data are represented as mean ± S.D. (n=3).

Figure 6: The result was obtained by testing the sacB cells with 100 µM IPTG and 0%, 0.5%, 1%, 2.5%, as well as 5% sucrose. Data are represented as mean ± S.D. (n=3).

Figure 7: The result was obtained by testing the sacB cells with 0 µM IPTG and 0%, 0.01%, 0.05%, 0.1%, 0.25%, 0.5% as well as 1% sucrose. Data are represented as mean ± S.D. (n=3).

Figure 8: The result was obtained by testing the sacB cells with 25 µM IPTG and 0%, 0.01%, 0.05%, 0.1%, 0.25%, 0.5% as well as 1% sucrose. Data are represented as mean ± S.D. (n=3).

Figure 9: The result was obtained by testing the sacB cells with 50 µM IPTG and 0%, 0.01%, 0.05%, 0.1%, 0.25%, 0.5% as well as 1% sucrose. Data are represented as mean ± S.D. (n=3).

Figure 10: The result was obtained by testing the sacB cells with 100 µM IPTG and 0%, 0.01%, 0.05%, 0.1%, 0.25%, 0.5% as well as 1% sucrose. Data are represented as mean ± S.D. (n=3).

GFP expression levels across different concentrations of IPTG without sucrose demonstrated that we would expect IPTG and marker expression to be positively correlated. GFP cannot be used as an accurate indicator in the presence of expressed markers as cell death will influence protein production.

As can be seen from Figure 3, even at 0 µM IPTG, the growth curve of the cell deteriorates with increasing concentrations of sucrose. This can indicate that:

• There is significant leakiness of the pLac promoter, expressing enough marker to cause cell death even in the absence of the inducer or
• Sucrose has a negative effect on cell growth independent of sacB.

In the presence of any concentration of IPTG, a concentration of sucrose 1% and above caused immediate stunting of growth. However, for 0.5% sucrose, all curves had a distinct peak shape, where cells grew for a period of time before entering a death phase with decreasing OD600. Importantly, the speed at which the population entered the death phase was linearly correlated with the IPTG concentration.

Conclusion of sacB Characterization

The negative selection marker component of our diametric marker is meant to screen against leaky variants by killing them in culture. We now have found the ideal concentration of sucrose to use, 0.5%, which would allow us to tune the level of 'acceptable' leakiness. This acceptable leakiness can be quantified using the IPTG concentrations as a proxy. For example, based on the graphs, allowing cells to grow in 0.5% sucrose for 2 hours would guarantee that surviving cells had constructs that had leakiness equivalent to or less than a pLac promoter's expression at 25 µM IPTG, while growing it for only 1 hour would loosen the upper limit of leakiness, allowing any cell that had leakiness up to the equivalent of a pLac promoter's expression at 100 µM IPTG to survive in the media.

Proof of Killing Mechanism and Killing Effect

The characterization results indicate that the sacB gene is effective in eliminating cells. However, based on the OD600 results alone, it is inconclusive whether the sacB gene only kills the cells that have the sacB gene being actively expressed, or if it might also affect neighbouring cells with the sacB gene switched off. To confirm that the sacB gene selectively eliminates only those cells containing the sacB gene with active expression, and not the neighbouring cells with the sacB gene in an inactive state, an RFP cells and sacB cells co-culture experiment was conducted.

This experiment involved the incubation of different ratios of non-sacB-containing RFP cells and sacB cells under four different conditions: "Cell Only," "100 µM IPTG," "1% Sucrose," and "100 µM IPTG and 1% Sucrose." The setup is illustrated in the plate map below. The plate was then incubated at 37 °C for 6 hours. Subsequently, the plate was placed into a plate reader to measure its OD600, GFP fluorescence, and RFP fluorescence.

Figure 11: This is the plate map of the 96-well plate for RFP cells and sacB cells co-culture test. The test involved 5 types of cells with different RFP cell and sacB cell ratios and 4 test conditions.

Results

Figure 12: (Top) The figure displays the various triplicates of the cultures after 10 hours. The vertical axis denotes the proportion of the culture(i.e 75RFP25sacB reflects a starting culture that was 75% RFP-producing cells, 25% sacB-producing cells), and the horizontal axis reflects the different conditions used. (Bottom) The figure shows the various triplicates of the cultures after 10 hours under blue light. The RFP and GFP expressed can be clearly seen.

Figure 13: (Left) RFP level of the cells against time in 100 µM IPTG. (Right) RFP Level of the cells against time in 1% sucrose solution. All data are represented as mean ± S.D. (n=3).

Figure 14: (Left) The RFP level of the cells against time in a mixed solution with 100 µM IPTG and 1% sucrose solution. (Right) The RFP level of the cells against time without any additive. All data are represented as mean ± S.D. (n=3).

Figure 13 and Figure 14 show the expression of RFP over time in each of the different conditions. As the OD600 cannot be used to directly differentiate the cells expressing RFP and the cells expressing GFP-sacB, RFP was assumed to be directly proportionate with the amount of RFP-expressing cells.

From the condition without any additives, we can see that the amount of RFP produced over time is correlated with the seed proportion of RFP-expressing cells. From the condition where 1% Sucrose and 100uM IPTG are added, it can be seen that there is significantly more RFP expression independent of the seed proportion.

Figure 15: (Left) The OD600 over time graphs of 100% RFP cells under different conditions. (Right) The OD600 over time graphs of 75% RFP cells co-cultured with 25% sacB cells under different conditions. All data are represented as mean ± S.D. (n=3).

Figure 16: (Left) The OD600 over time graphs of 50% RFP cells co-cultured with 50% sacB cells under different conditions. (Right) The OD600 over time graphs of 25% RFP cells co-cultured with 75% sacB cells under different conditions. All data are represented as mean ± S.D. (n=3).

Figure 17: The OD600 over time graphs of 100% sacB cells under different conditions. Data are represented as mean ± S.D. (n=3).

Figure 15 to Figure 17 show the overall OD600 of each coculture seed proportion in each condition over 6 hours.  Across all conditions the addition of sucrose caused the OD to drop slightly, even for cultures that did not have any GFP-sacB producing cells.

As expected, the addition of sucrose and IPTG to cultures that were fully GFP-sacB expressing cells led to a collapse in OD600, similar to what was observed in the GFP-sacB characterization. However, for cocultures, the addition of both IPTG and sucrose saw a restoration of normal growth compared to the addition of sucrose only, except for cultures that were fully RFP-expressing cells.

Conclusion

As RFP-expressing cells continue to grow in 1% sucrose and IPTG, conditions that, from our data, evidently produce sufficient levan to kill the GFP-sacB producing cells, we can conclude that levan only negatively affects cells that GFP-sacB was produced in.

In addition, it seems that sucrose has a negative effect on cell growth, independent of sacB expression. This effect is relieved in the presence of IPTG and GFP-sacB producing cells. Our hypothesis is that GFP-sacB producing cells metabolize the sucrose in the media into levan, which is evidently kept intracellular. Even though this kills the GFP-sacB producing cells, it removes the sucrose from the media, alleviating the negative effect on other cells.

Thus, not only does sacB not negatively affect the growth of surrounding cells, it provides a growth advantage to non-sacB producing cells, which assists in our use of the marker to distinguish leaky and non-leaky RNA switches.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

[1] Liu, Y., Miao, J., Traore, S. M., Kong, D., Lü, Y., Zhang, X., Nimchuk, Z. L., Liu, Z., & Zhao, B. (2016). SacB-SacR Gene Cassette As the Negative Selection Marker to Suppress Agrobacterium Overgrowth in Agrobacterium-Mediated Plant Transformation. Frontiers in Molecular Biosciences, 3. https://doi.org/10.3389/fmolb.2016.00070

[2] Lim, J. Y. (2010, January 1). A Brief Overview of Escherichia coli O157:H7 and Its Plasmid O157. PubMed Central (PMC). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3645889/#:~:text=Escherichia%20coli%20(E.,Most%20E.

[3] Miller, S. I., & Salama, N. R. (2018). The gram-negative bacterial periplasm: Size matters. PLOS Biology, 16(1), e2004935. https://doi.org/10.1371/journal.pbio.2004935

[4] Anguluri, K., La China, S., Brugnoli, M., De Vero, L., Pulvirenti, A., Cassanelli, S., & Gullo, M. (2022). Candidate acetic acid bacteria strains for Levan production. Polymers, 14(10), 2000. https://doi.org/10.3390/polym14102000

[5] Lee, J. C. (n.d.). Secretory Production of Rahnella aquatilis ATCC 33071 Levansucrase Expressed in Escherichia coli. https://www.jmb.or.kr/journal/view.html?spage=1232&volume=14&number=6