Team:BOKU-Vienna/Parts
Novel parts for novel approaches
Basic parts
Promoters
Enzyme therapies in the past often lacked the needed enzyme quantity. Thus, we wanted to find the optimal expression cassette for our enzymes. We tested nine different promotors, all of which are designed to function in L.plantarum. A list is shown below. P6 and P7 are from the iGEM registry and originate from the L.lactis genome. P1 to P5 come from the same synthetic promotor library (1) but have different expression strengths (1000, 1600, 700, 50 and 350 nU GusA activity for P1-P5 respectively). P8 originates from L.diolivorans.(2) Finally, P9 is an inducible promotor.(3)
| Internal name | Registry number | Short description |
|---|---|---|
| P1 | BBa_K3855000 | Strong constitutive Promotor (1000 nU) for L.plantarum |
| P2 | BBA_K3855001 | Strong constitutive Promotor (1600 nU) for L.plantarum |
| P3 | BBa_K3855002 | Medium constitutive Promotor (700 nU) for L.plantarum |
| P4 | BBA_K3855003 | Weak constitutive Promotor (350 nU) for L.plantarum |
| P5 | BBA_K3855004 | Weak constitutive Promotor (50 nU) for L.plantarum |
| P6 | BBa_K2253000 | Strong constitutive Promotor from Team Austin Texas 2017 from L.lactis |
| P7 | BBa_K2253001 | Strong constitutive Promotor from Team Austin Texas 2017 from L.lactis |
| P8 pGAP | BBa_K3855005 | Strong constitutive Promotor for L.diolivorans |
| P9 ORF | BBa_K3855006 | Inducible Promoter for L.plantarum |
Enzymes and modifications
Fructans can be separated into three major groups: inulins, levans, and graminins. While a levan is characterized by β-2,6-linked fructosyl residues and inulin by β-2,1-linked fructosyl residues, a graminin contains both of these linkages.
The first enzyme we selected was levanase. It originates from B.subtilis SacL mutants and has the ability to hydrolyze terminal, non-reducing (2->1)- and (2->6)-linked beta-D-fructofuranose residues in fructans. This enzyme can cleave both linkages but the Michaelis Menten constant for the hydrolysis of inulin as well as sucrose is too high for efficient fructan degradation. As a result, we included a second enzyme to increase the efficiency of the inulin breakdown. The endo-inulinase was chosen due to its ability to cleave the (2->1)- beta-D-fructosidic linkages in inulin. Since no sequence from a bacterial host was known, a gene from Aspergillus ficuum and from Kluyveromyces marxianus was used. Two different origins for the enzyme should work as a safety net in case one of the sequences would require different post-translational modifications. The last enzyme added to our combination was an invertase. Its purpose was the hydrolysis of terminal non-reducing beta-D-fructofuranoside residues in beta-D-fructofuranosides. With these three enzymes, we hope to completely degrade the 3 major fructan groups currently known.
We added a C-terminal 6 x His tag to all our enzymes for quantification and qualification purposes. Furthermore, only the levanase features a native secretion signal that functions in Lactobacillus. Thus, we chose two secretion signals that function in Lactobacillus and created respective composite parts for each enzyme that has no native secretion signal for our host. While this nearly doubled the number of constructs we had to handle in the lab, it allowed for some failsafe in case that one of the secretion signals might not work or cause unforeseen side effects. The mentioned parts are shown in the table below.
| Secretion signal a (BBa_K3855301) | Extracellular endo-inulinase INU2 (BBA_K3855100) | BBa_K3855500 |
| Secretion signal b (BBa_K3855302) | BBa_K3855501 | |
| Secretion signal a (BBa_K3855301) | Inulinase INU1 (BBA_K3855101) | BBa_K3855502 |
| Secretion signal b (BBa_K3855302) | BBa_K3855504 | |
| Secretion signal a (BBa_K3855301) | Sucrose-6-phosphat hydrolase sacA (BBA_K3855102) | BBa_K3855505 |
| Secretion signal b (BBa_K3855302) | BBa_K3855507 | |
| Native secretion signal | Levanase sacC (BBA_K3855103) | |
Terminators
To avoid homologous recombination, we used two different terminators. ttCAT (BBa_K3855200) for the “outer” enzymes, those who are in first and third place in the final expression cassette and PepNTT (BBa_K3855201) for the enzyme located in the middle of the expression cassette.
ttCAT comes from the same toolbox as P8 and hence was developed for L.diolivorans, while PepNTT has been published by Sørvig et al., being specifically designed for L.plantarum.
Composite parts
We already mentioned the joint secretion signal-enzyme parts. To make them fully functional we combined each of those with the corresponding terminator and varying promotors. Our goal was to test all promotor-enzyme combinations. While we were able to assemble them all via Golden Gate cloning, we failed with the transformation for several combinations. It turned out that our enzymes were aversive for E.coli, resulting in functional mutations in many positive clones. This is further discussed in the trouble section.
In short, all positive BB2 Promotor P1-P8 -Enzyme -Terminator clones had a mutation. Most often a frameshift mutation or a deletion of the first base in the secretion signal A/B. Although no hint of this is found in the literature, all enzymes seem to be very toxic to E.coli, as even the weakest promotors (P4, P5, and P8) yielded non-functional mutants. After switching to an inducible promoter, we finally obtained non-mutated BB2 and BB3 clones.
This aversion seems to be E.coli specific, as the induction of the promotor in confirmed Lactobacillus clones did not result in significant growth inhibition. The following table shows our composite parts which were successfully expressed in E.coli and Lactobacillus.
| Promotor | Enzyme | Terminator | Composite part number |
|---|---|---|---|
| P9 | ENDI AF A | ttcat | BBa_K3855601 |
| P9 | ENDI AF B | ttcat | BBa_K3855602 |
| P9 | ENDI KM A | ttcat | BBa_K3855603 |
| P9 | ENDI KM B | ttcat | BBa_K3855604 |
| P9 | Invi A | ttcat | BBa_K3855605 |
| P9 | Invi B | ttcat | BBa_K3855606 |
| P9 | Levi | pepntt | BBa_K3855607 |
Backbone 3 (BBa_K3855401)
As we planned to assemble our constructs in E.coli and then switch to L.plantarum we needed a vector that works in both host organisms and is fitting for the Type II-S assembly system. We started from pSIP403 (4), which contains several illegal sequences forbidden for the Type II-S system. We split it into 5 pieces which we amplified individually, while simultaniously removing the restricted sites. A detailed page about how we did this and the troubles we faced in doing so is in the "Engineering" section.
In the end we created a “raw” version of the backbone which can be adapted to incorporate up to five inserts. It contains an erythromycin resistance and allows for an easy switch between the host organism. You can find it on the registry.
References
(1) Rud, I., Jensen, P. R., Naterstad, K., & Axelsson, L. (2006). A synthetic promoter library for constitutive gene expression in Lactobacillus plantarum. Microbiology, 152, 1011–1019. https://doi.org/10.1099/mic.0.28599-0
(2) Pflügl, S., Marx, H., Mattanovich, D., & Sauer, M. (2013). Genetic engineering of Lactobacillus diolivorans. FEMS microbiology letters, 344, 152–158. https://doi.org/10.1111/1574-6968.12168
(3) Sørvig, E., Grönqvist, S., Naterstad, K., Mathiesen, G., Eijsink, V. G., & Axelsson, L. (2003). Construction of vectors for inducible gene expression in Lactobacillus sakei and L.plantarum. FEMS microbiology letters, 229, 119–126. https://doi.org/10.1016/S0378-1097(03)00798-5
(4) Sørvig, E., Mathiesen, G., Naterstad, K., Eijsink, V., & Axelsson, L. (2005). High-level, inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum using versatile expression vectors. Microbiology, 151, 2439–2449. https://doi.org/10.1099/mic.0.28084-0