Team:Heidelberg/Selective Advantage
Selective Advantage
β-Agarase Assay
Background
For our project, it is important that our plasmid is not only taken up by the intestinal bacteria, but also permanently maintained in the population, if possible. For bacteria to maintain a plasmid, one must provide them with a selective advantage. For that purpose, plasmids containing antibiotic resistances are often used in the laboratory. Since our plasmid is to be introduced into the intestine,we aim to overcome this established antibiotic selection, also known as negative selection. Therefore, we aim to establish a selective advantage system with the means of dietary supplements. In short, the idea is that additional nutritional supplements can only be metabolized by the bacteria that have taken up the plasmid with the genes required for this metabolization, thus giving them a selective advantage.
In particular, we have looked at the sugar metabolism, since a specific sugar diet is favorable and easy to administer, thus making it comparably more tolerable for patients of different ages. However, simple sugar sources such as lactose are out of the question, since there are too many bacteria in the gut that can already metabolize lactose. An additional lactose supply would therefore also help these bacteria and would not bring any specific advantage for the bacteria we transformed. We hence looked for other sources of galactose, which are less accessible for bacteria. During our research we came across agar as a promising candidate for our purpose.
Agar is often used in the food industry as a thickening agent or as a vegan alternative to gelatin. It is a complex polysaccharide consisting of alternating 3-O-linked β-D-galactopyranose and 4-O-linked α-L-galactopyranose.
Agar cannot be degraded by most microorganisms, but there are some bacteria that metabolize agar as a carbon and energy source. These are mainly found in marine environments, where food resources are limited and agar is abundant in the form of the cell walls of some algae [1], [2].
This gave us the idea to use the ability of agar degradation as a selective advantage for our bacteria.
One of the enzymes present in agarolytic bacteria is the β-Agarase that hydrolyzes the β-(1,4) glycosidic bonds (see Fig. 1). We found a β-Agarase on the iGEM registry Part:BBa_K2094002 and decided to use this part for our project. We cloned this gene into a suitable plasmid and transformed this plasmid into E. coli to further characterize it.
Experiments and Results
The DNA was synthesized using the sequence from part BBa_K2094002. Amplification was performed via PCR. The DNA was digested with BamHI and NdeI restriction enzymes and after that ligated with a T4 ligase into a pET15b backbone. This construct includes a T7 promoter, lac operator and an ampicillin resistance. The construct was transformed into competent E. coli BL21 via heat shock.
Transformed E. coli BL21 were cultured on LB agar plates with carbenicillin for antibiotic selection and isopropyl β-D-1-thiogalactopyranoside (IPTG) to induce the expression of β-agarase. Agarolytic activity was confirmed by pit formation on the agar plates.
Assay of enzyme activity
A solution containing 4% agarose was melted and then solidified in 50 mL Erlenmeyer Flasks.
To the flasks with agar either the supernatant or cell suspension of E. coli BL21 with β-agarase or E. coli BL21 with mCherry was added (see Fig. 3).
Overnight cultures of E. coli BL21 with pET15b-β-agarase and E. coli BL21 with pET15b-mcherry were grown at 37 °C in LB medium. To the overnight cultures as well as to the in vivo experiments, carbenicillin was added for selection and IPTG was added to induce expression. Samples were incubated for 12 h at 37 °C with 70 rpm shaking. E. coli BL21 with pET15b-mcherry were used as a negative control to confirm that the occurrence of reducing sugars is due to the β-agarases and not to other metabolic pathways. By using bacteria having the same plasmid but with another insert, a possible influence of the pET15b vector can also be ruled out.
Briefly, 1.5 mL of sample solution was mixed with 0.5 mL of DNS reagent, the reaction was heated in boiling water for 5 min and then placed on ice for 5 min. Absorbance was measured at a wavelength of 540 nm, a standard curve of D-galactose dissolved in LB medium was used to determine the total amount of reducing sugars.
Samples were measured as described in table 1. We tested both the supernatant (hereafter in vitro) and the cell culture (hereafter in vivo) to find out whether or not β-agarase is secreted in the medium.
Table 1: Samples of the β-agarase assay. A brief description of each sample's content and the abbreviated form used in figure 4 are written in the first two columns. The absorbance value is the measurement we got when measuring the absorption at 540 nm with the TECAN and the total amount of reducing sugars was calculated using the standard curve and absorption value.
| Sample | Sample name Figure 4 | Absorbance Value | total amount of reducing sugars (mg/mL) |
|---|---|---|---|
| ß-agarase supernatant without agar | agarase no agar | 0.209400 | 0.057727 |
| in vivo positive control | agarase in vivo | 0.963100 | 0.658203 |
| in vitro positive control | agarase in vitro | 0.609800 | 0.376728 |
| mCherry supernatant without agar | neg. control no agar | 0.200600 | 0.050716 |
| in vivo negative control | neg. control in vivo | 0.188600 | 0.041156 |
| in vitro negative control | neg. control in vitro | 0.210600 | 0.058683 |
(A) Samples after the DNS assay was performed from left to right: agarase no agar, agarase in vitro, agarase in vivo, neg. control no agar, neg. control in vitro, neg. control in vivo. The more reducing sugars were present before the experiment, the more red the final solution is. (B) Absorbance measurements of the samples with and without agarase in vitro and in vivo plotted on the D-galactose standard curve. Measurements without agar and without agarase are all around 0.2 absorption and therefore cover each other. The absorption was measured at 540 nm.
As it can be seen in the table the absorption and the corresponding concentration of reducing sugars is the highest in the in vivo measurement with 0.658 mg/mL. This suggests that most β-agarase activity is found in our in vivo experiment. The in vitro experiment also shows the presence of reducing sugars with a concentration of 0.376728 mg/mL suggesting that our enzyme is to be found in the supernatant and must therefore be secreted.
The very little concentrations of reducing sugars for the negative control and the ß-agarase supernatant without agar indicate that the presence of reducing sugars is not attributable to other enzymes produced by the bacteria itself but only to the combination of the ß-agarase and agar as its substrate.
Discussion
With our experimental setup we were able to prove that the ß-agarase expressed by E. coli is able to break down solid agar into reducing sugars. Furthermore the positive results of the in vitro experiment show that the enzymatic reaction takes place in the supernatant separated from bacteria. Therefore it can be assumed that ß-agarase is secreted by E. coli.
Pursuant to publications, agar degradation by ß-agarase produces neoagarooligosaccharides with different degrees of polymerization having galactose residues at their reducing ends. These include, for example, neoagarotetraose, neoagarohexaose and neoagarooctaose [1]. For a more detailed characterization of our ß-agarase and the resulting products, an analysis of the supernatant, for example by mass spectroscopy, would be necessary.
It would also be necessary to find out whether agar can be cleaved by E. coli to the monomeric α-galactose-6-sulfate and galactose in order to be used for metabolism, or whether further enzymes would be necessary for the use of agar as carbon source.
Co-culture selective advantage
Background
To see whether the degradation of certain sugars really provides the bacteria with an advantage in growth over other bacteria, we co-cultured bacteria with sugar-degrading abilities with bacteria that do not have this ability and added the “selective solution” meaning the corresponding sugar source. Since we cannot say for sure whether β-agarase is sufficient to make agar accessible as an energy source in the form of galactose for bacterial metabolism, we also looked at β-galactosidase. When X-Gal is used as a substrate for the β-galactosidase, galactose is produced as it would also be the case for a complete agar degradation. With this extension of the experiment we want to check, weather galactose in general leads to an increased growth.
Experiments and results
β-galactosidase and β-agarase were the two genes tested in this assay regarding their suitability as a selective advantage in co-culture.
E. coli DH5ɑ expressing the pUC19_LacZ plasmid with the β-galactosidase were cultured together in LB medium with E. coli DH5ɑ expressing pUC19_RFP (as a negative control) at 37 °C, 180 rpm. X-Gal was added as the “selective solution” Samples were taken at 0 h, 4 h, 6 or 7 h and 24 h, diluted and spread on LB agar plates with carbenicillin. The agar plates were incubated at 37 °C overnight and the colony forming units (CFU) counted. Colonies were distinguished by their colour: colonies with β-galactosidase appear white, colonies with RFP appear red (see Fig. 8).
When both E. coli strains are grown together without X-Gal, this type of E. coli seems to have a disadvantage against E. coli with RFP, as there were always more red colonies (with RFP) observed than white ones (with LacZ) (see Fig. 9 A+B). In the co-culture of these two strains with the addition of X-Gal (see Fig 9 C+D) it first seems that both grow equally well, but the last measurement at 24 h is surprisingly low. It seems like both strains died between 6 h and the final measurement. However in the ratio between E. coli with LacZ and E. coli with RFP, it can be seen, that there are always at least 1.5 times more E. coli with LacZ than with RFP.
The same assay was performed using E. coli BL21 expressing the pET15b-β-agarase plasmid and E. coli BL21 expressing the pET15b-β-mcherry plasmid. The co-culture was grown in LB medium containing a thin layer of solidified agar on the bottom as the “selective solution” (see Fig. 10)
In the co-culturing of E. coli with agarase and with mCherry it first seemed like E. coli mCherry grew faster than E. coli agarase, but somewhere between 6 and 24 hours E. coli agarase had overgrown the other strain. As this overall growth is the same, one might think that agarase does not provide a selective advantage to the bacteria expressing it. However, the total CFU for E. coli β-agarase with agar (see Fig. 10 D) is almost four times as high as in E. coli agarase without agar (see Fig. 10 B). This might be the result of a surplus of available nutrients from the agar degradation.
Discussion
In figure 9 it can be seen that without the addition of X-Gal, E. coli RFP grows faster/ higher than E. coli β-galactosidase. The cause for this observation remains unclear. The ratio calculations indicate as well that there is always more E. coli RFP present when no X-Gal is added.
For the positive control with the addition of X-Gal the opposite observation is the case. Even if after 7 hours both bacterial strains decreased in growth, E. coli with the β-galactosidase increased its ratio in the co-culture. Furthermore there were always more E. coli with β-galactosidase than with RFP present.
The results suggest the trend that the counted colonies of the E. coli strain with the pUC19 LacZ gene have a slight selective advantage before the E. coli strain with the RFP Plasmid.
Regarding figure 10, the β-Agarase did not show any selective advantage in comparison to the RFP plasmid. Here the solubility of agarose was a major problem.
Another parameter that could be checked for in further experiments would be to run the experiment in nutritient-poor conditions using minimal medium.
An assay with more frequent measurements would be necessary for stronger significance.
We had planned to detect this in Big Scale with the help of Beckmann Coulter but the company decided for another project. Therefore our first finding needs to be tested further.
References
[1] Chi, W. J., Chang, Y. K., & Hong, S. K. (2012). Agar degradation by microorganisms and agar-degrading enzymes. Applied microbiology and biotechnology, 94(4), 917–930. https://doi.org/10.1007/s00253-012-4023-2
[2] Su, Q., Jin, T., Yu, Y., Yang, M., Mou, H., & Li, L. (2017). Extracellular expression of a novel β-agarase from Microbulbifer sp. Q7, isolated from the gut of sea cucumber. AMB Express, 7(1), 220. https://doi.org/10.1186/s13568-017-0525-8
[3] G. L. Miller. (1959): Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Analytical Chemistry. Vol. 31(3):426-428. DOI: 10.1021/ac60147a030
[2] Su, Q., Jin, T., Yu, Y., Yang, M., Mou, H., & Li, L. (2017). Extracellular expression of a novel β-agarase from Microbulbifer sp. Q7, isolated from the gut of sea cucumber. AMB Express, 7(1), 220. https://doi.org/10.1186/s13568-017-0525-8
[3] G. L. Miller. (1959): Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Analytical Chemistry. Vol. 31(3):426-428. DOI: 10.1021/ac60147a030
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