Proof of concept
Proof of concept
We were able to successfully express our fructan degrading enzymes in Lactobacillus and show their secretion (see Slot Blot in the Results). Additionally, we encapsulated bacteria within a biocontainer, demonstrated their retention and overall survival and thiolated the biocontainer in order to achieve mucoadhesion (see Scaffolds Section in the Results).
Furthermore, we provided a kinetic model that showed that it is possible to sufficiently degrade fructans in the small intestine and as a consequence, not exceed the clinical threshold of fructans therein (see Model).
We successfully engineered the bacteria L.plantarum through Golden Gate Assembly with a plasmid encoding the enzymes endo-inulinase, levanase and invertase with the inducible promotor ORF (BBa_K3855006) and could prove the transformation with a correctly cloned plasmid by Sanger-sequencing.
To prove that our His-tagged enzymes are actually expressed and secreted by L.plantarum, we induced the enzyme expression in liquid cultures of the positive clones with Sakacin A Inducing Peptide / Sakacin P Inducing Peptide (SapIP/SppIP).
Subsequently, we performed a Slot Blot and applied the supernatant of our liquid culture to a membrane. We stained the blot using an anti-His-tag antibody-HRP conjugate. This method provided a simple, straightforward, and simultaneously very sensitive detection of His-tagged proteins in the supernatants of our clones.
We encapsulated E.coli with a commercially available cell encapsulation kit (Cell-in-a-box by Austrianova), which contains two differently charged polysaccharide solutions. To form a scaffold, the anionic solution containing the cells is dripped into the cationic solution with a syringe.
In order to investigate whether cells survive the encapsulation and simultaneously do not escape the capsules, we plated out the supernatant and the capsule content. Our results showed that the E.coli cells survived the process and that they cannot escape the capsules. Thereby, we could prove that it is possible to contain the cells in the scaffolds.
It was proven that bovine serum albumin (BSA) could diffuse out of our scaffold, but results have shown that the diffusion rate is rather slow. We assume that since BSA is much bigger than our enzymes, the diffusion rate of our enzymes would be much higher, although this still needs to be investigated.
One novel trait of our scaffolds is the mucoadhesion, which can be achieved with thiolated chitosan. The added thiol groups form strong covalent bonds with the cysteines present in surface proteins of the mucosa through disulfide bridges. By adding the thiolated chitosan to the cationic solution of the kit, we could form stable thiolated scaffolds.
Due to the time constraints, further experiments like the determination of the ideal concentration of thiolated chitosan, the experimental pore size adjustment as well as the mucoadhesion testing of the scaffold with the added thiolated chitosan could not be performed.
With our kinetic model we could successfully show the degradation of fructans in the small intestine by Lactobacillus. We simulated that inulin and levan present in an average portion of a pizza Margherita can be broken down by our enzyme mix so that the clinical threshold is not exceeded and additionally, that no inulin or levan can reach the large intestine. The critical degradation of inulin and levan that leads to bloating and stomachaches by intestinal bacteria occurs in the large bowel. By the administration of our Friendzyme pill, these effects can therefore be prevented.
Induction of orfXP promotor (BBa_K3855006)
Inducer: Sakacin A Inducing Peptide / Sakacin P Inducing Peptide (SapIP/SppIP)
- Make o/n cultures in MRS-medium (De Man, Rogosa and Shape agar, selective culture medium to favour the luxuriant growth of lactobacilli) + antibiotics (Erythromycin), put on 30 °C and 180 rpm in an anaerobic jar.
- The next morning, dilute the cultures to an OD600 of 0.1 and incubate further until an OD600 of 0.3 is reached.
- Induce with stock solution (25 µg/mL of Inducer SapIP/SppIP) and add the appropriate amount to the respective culture so the final concentration of the inducer equals 25 ng/mL (= 1:1000).
- Incubate either until an OD600 of 1.8 or o/n and harvest the supernatant (13000 rpm, 3min)
His Link purification
According to protocol of HisLink™ Spin Protein Purification System V1320 by Promega
Deviations: Centrifugation Protocol 3.E. - 1.: instead of lysing 700 µL of bacterial culture by adding 70 µL of FastBreak reagent, we used 770 µL supernatant and adjusted the pH > 7 with NaOH
Sodium-dodecyl-sulfate-polyacrylamide gel (SDS-PAGE)
- 10.5 µL sample (not normalized)
- 3.75 µL loading buffer (NuPage® LDS sample buffer (4X))
- 0.75 µL 1M Dithiothreitol (DTT)
- 1x NuPAGE™ MES SDS Running Buffer
- PageRuler™ Prestained Protein Ladder by Thermo Scientific™
Polyacrylamide Gel (PAGE)
- NuPAGE® 10 % BT 1.0
- Put sample mix on 70 °C for 20 min to break disulfide bonds
- Load 5 µL ladder and 15 µL sample mix onto page, 200 V for ~1 h
- Wash with reverse osmosis water (R-O-H2O) to remove running buffer
- Stain with Coomassie brilliant blue R-250 solution for 1 h
- Wash with RO-H2O on rocker at ~15 rpm to remove stain (change RO-H2O every 30 minutes)
According to manual of PR 600 Slot Blot Filtration Manifolds version 80-6295-84/ Rev. C/07-00 by Amersham Biosciences
- supernatant was loaded as much as possible until the membrane clogged, other samples: 150 µL
- Trans-Blot® Turbo™ Mini 0.2 µm PVDF Transfer Pack by BioRad
- 6x-His Tag Monoclonal Antibody (HIS.H8)
- 1-Step™ Ultra TMB-Blotting Solution by Thermo Scientific™
- Incubate membrane from Slot Blot in PBS-T (= 1x PBS + 0,1% Tween20) + 2% BSA for at least 2 h or o/n at 4 °C (blocking)
- Continue at room temperature, wash 3x for 10 min with PBS-T
- Incubate 1 h in 12 mL diluted detection antibody in PBS-T + 2% BSA (1:2000 – 6 µL in 12 mL)
- Wash 3x for 10 min with PBS-T
- Develop Blot with 4 mL Blotting Solution until desired intensity of the chemiluminescent signal is reached
- Rinse with RO-H2O