Composite

Part:BBa_K5117014

Designed by: Jenny Sauermann, Lilli Kratzer, Katrin Lehmann   Group: iGEM24_TU-Dresden   (2024-08-31)


BsRBS-AtCelG

This part serves as translational unit composed of the ribosome binding site of Bacillus subtilis (BBa_K5117000) and the celG gene of Acetivibrio thermocellus (BBa_K5117004) encoding an endoglucanase (EC 3.2.1.4).


Biosafety level: S1

Target organism: Bacillus subtilis

Main purpose of use: Testing enzyme functionality in the host B. subtilis

Potential application: Degradation of cellulose


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1554
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 91
  • 1000
    COMPATIBLE WITH RFC[1000]


Enzyme characterization according to literature

The characterization of the enzyme included in this composite part can be found on the basic part page (BBa_K5117004) of the enzyme.


Construct Design

For compatibility with the BioBrick RFC[10] standard, the restriction sitesEcoRI, XbaI, SpeI, PstI and NotI were removed from the coding sequence (CDS). To make the part compatible with the Type IIS standard, BsaI and SapI sites were removed as well. This was achieved by codon exchange using the codon usage table of Bacillus subtilis (Codon Usage Database Kazusa). Upstream from the CDS, there is the ribosome binding site (RBS) for the host Bacillus subtilis followed by a 7 bp spacer. This composite part composed of RBS and CDS was flanked by the BioBrick prefix and suffix sequences and ordered via gene synthesis from IDT.


Construction of a template plasmid

In order to create a template from which this part could be amplified, the part was subcloned into a small vector pSB1C3 (Part:pSB1C3). For that purpose, the plasmid was isolated from E. coli DH10β, yielding a DNA concentration of 431.0 ng/µl. Afterwards, a Backbone PCR with pSB1C3 (Fig. 1) was performed, followed by a restriction digest of the amplified vector backbone and the part with EcoRI and PstI, which were purified via the HiYield® PCR Clean-up/Gel Extraction Kit (SLG, Germany), resulting in DNA concentrations of 32.6 ng/µl (digested pSB1C3) and 12.8 ng/µl.


Fig. 1: DNA Ladder (A) and agarose gel electrophoresis of pSB1C3 Backbone PCR (B). A: 1 kb Plus DNA Ladder from New England Biolabs (NEB). B: Backbone PCR of pSB1C3. Oligonucleotides for amplification can be found on the Experiments page. The correct PCR product has a size of 2043 bp. DNA fragments of other sizes represent unspecific bands. 1 kb Plus DNA Ladder (NEB) served as marker (M). The pSB1C3 backbone was purified by gel extraction resulting in a DNA concentration of 190.2 ng/µl.


After ligation, the plasmid was transformed into chemically competent E. coli DH10β cells. Transformants were selected by chloramphenicol resistance (35 µg/ml chloramphenicol) encoded on the pSB1C3 backbone. For the negative control, no DNA was added during the transformation procedure leading to no colony growth on selection plates. For the positive control, cells were transformed with the vector pSB1C3 resulting in a pink bacterial lawn due to the original RFP insert. On the selection plates of the target construct, white colonies were tested for the presence of the correct insert by Colony PCR and agarose gel electrophoresis (Fig. 2).


Fig. 2: Agarose gel electrophoresis: Insert amplification of pSB1C3-AtCelG by Colony PCR of transformed E. coli DH10β cells. Oligonucleotides for amplification can be found on the Experiments page. Numbers 1-4 correspond to chosen colonies. The correct PCR product has a size of 2030 bp. The negative control displayed no band. 1 kb Plus DNA Ladder (NEB) served as marker (M). Colony 1 resulted in the highest plasmid concentration and was subsequently verified by sequencing revealing the correct insert sequence.


Colonies with a band at the correct size of the insert were chosen for plasmid isolation according to the HiYield® Plasmid Mini DNA Kit (SLG, Germany). Finally, the plasmid was verified via sequencing by Microsynth Seqlab GmbH (DNA concentration: 250 ng/µl).


Cloning into inducible expression vectors

For testing the functionality and activity of this enzyme, the part was cloned into xylose-inducible expression vectors in order to overexpress the gene of interest. Replicative (pBS0E-xylR-PxylA) and integrative (pBS2E-xylR-PxylA) vectors were used, both with a xylose-inducible promoter for induced expression and a xylose repressor to decrease basal promoter activity (Popp et al. 2017). Whereas replicative plasmids provide a high copy number and result in high concentrations of target proteins, genomic integration (in this case into the lacA locus) ensures high stability but results in lower protein concentrations. The vectors were isolated from E. coli DH10β, resulting in DNA concentrations of 151.9 ng/µl pBS0E-xylR-PxylA and 127.6 ng/µl pBS2E-xylR-PxylA. These vectors were digested with EcoRI and PstI (Fig. 3) and purified via gel extraction using the HiYield® PCR Clean-up/Gel Extraction Kit (SLG, Germany).


Fig. 3: Agarose gel electrophoresis: Restriction (Res) of expression vectors pBS0E-xylR-PxylA (pBS0EX) and pBS2E-xylR-PxylA (pBS2EX) with EcoRI and PstI. The digested plasmid backbones have sizes of 8114 bp and 7758 bp, respectively. Strong bands at approximately 9000 bp could represent undigested plasmids as well as digested ones due to high size inaccuracy of large bands. Weak bands at 1102 bp represent the RFP insert being cut out of the vector. Large bands were purified by gel extraction and resulted in 17.9 ng/µl and 31.3 ng/µl DNA for the digested vectors pBS0EX and pBS2EX, respectively. 1 kb Plus DNA Ladder (NEB) served as marker (M).


The enzyme part was amplified via PCR (Fig. 4) using the plasmid template pSB1C3-AtCelG and subsequently digested and purified via PCR clean up.


Fig. 4: Agarose gel electrophoresis: PCR of part AtCelG. Oligonucleotides for amplification can be found on the Experiments page. The correct PCR product has a size of 1767 bp. 1 kb Plus DNA Ladder (NEB) served as marker (M). AtCelG was purified by gel extraction resulting in a DNA concentration of 87.5 ng/µl.


After ligation, the plasmids were transformed into chemically competent E. coli DH10β and transformants were selected by ampicillin resistance (100 µg/ml ampicillin) encoded on the vector backbone. White colonies transformed with the expression plasmids were analyzed by Colony PCR and agarose gel electrophoresis (Fig. 5). Colonies with a band at the correct size of the insert were chosen for plasmid isolation. Finally, the replicative expression plasmid pBS0E-xylR-PxylA-AtCelG and the integrative expression plasmid pBS2E-xylR-PxylA-AtCelG were verified by sequencing and successfully generated (DNA concentrations: 600.7 ng/µl, 140.6 ng/µl).



















Fig. 5: Agarose gel electrophoresis: Insert amplification of pBS0E-xylR-PxylA-AtCelG (pBS0EX-AtCelG) and pBS2E-xylR-PxylA-AtCelG (pBS2EX-AtCelG) by Colony PCR of transformed E. coli DH10β cells. Oligonucleotides for amplification can be found on the Experiments page. Numbers 1-4 correspond to chosen colonies. Correct PCR products have a size of 1918 bp for pBS0EX-AtCelG and 2119 bp for pBS2EX-AtCelG. Negative controls (NC) displayed no band. Whereas the NC for pBS2EX is shown here, the NC for pBS0EX was loaded onto another gel and is therefore not depicted. 1 kb Plus DNA Ladder (NEB) served as marker (M). Colony 3 of pBS0EX-AtCelG and colony 2 of pBS2EX-AtCelG were verified by sequencing and contained the correct insert sequence.


Ultimately, these expression plasmids were transformed into the target host B. subtilis. Since this part was included in the strategy focused on the secretory expression of target enzymes, WB800N was chosen as an expression strain. This genetically engineered variant of B. subtilis W168 features the disruption of all extracellular proteases. The eight-extracellular-protease-deficient mutant is widely used in industrial applications, as it increases the stability of secreted proteins (Jeong et al. 2018). The transformants were selected by MLS resistance (1 micro;g/ml erythromycin and 25 micro;g/ml lincomycin) encoded on the vector backbones. The Bacillus transformation was carried out with early addition of DNA to growing WB800N cells (at OD600 ≈ 0.7) to not miss the timepoint of competence. Afterwards, cells were grown until OD600 ≈ 1.1-1.3 and the same procedure was followed as in the initial protocol (see Experiments page). The expression plasmids could be transformed into WB800N and colonies were verified by Colony PCR. The presence of replicative plasmids was tested by two primer pairs (double check), whereas both upstream and downstream integration into the lacA locus was checked for integrative plasmids (Fig. 6). Two colonies each with the correct insert size were chosen for cryo-conservation, serving as biological duplicates.


Fig. 6: Agarose gel electrophoresis: Insert amplification of pBS0E-xylR-PxylA-AtCelG (pBS0EX-AtCelG) and pBS2E-xylR-PxylA-AtCelG (pBS2EX-AtCelG) by Colony PCR of transformed B. subtilis WB800N cells. Oligonucleotides for amplification can be found on the Experiments page. Numbers 1-4 correspond to chosen colonies. 1 kb Plus DNA Ladder (NEB) served as marker (M). pBS0EX: Primer pair 1 was used for the amplification of a small fragment including AtCelG only (1767 bp). Primer pair 2 was used for amplification of a large fragment including xylR-PxylA-AtCelG (3398 bp). Colonies 3 and 4 of pBS0EX-AtCelG were correct and chosen for cryo-conservation. pBS2EX: Primer pair 3 was used to check downstream integration by amplification of a fragment including AtCelG and ‘lacA (2305 bp). Primer pair 4 was used to check upstream integration by amplification of a fragment including lacA’ and the erythromycin resistance gene erm (1370 bp).Colonies 1 and 4 of pBS2EX-AtCelG were correct and chosen for cryo-conservation. Negative controls of all primer pairs (NC 1-4) displayed no bands.


Expression of endoglucanases

For the expression and testing of heterologously expressed enzymes in B. subtilis, we induced protein production in cultures by adding 0.5 % xylose after reaching an OD600 of 0.5 – 0.6. After 24 hours, the supernatants were collected to test secreted endoglucanases. The enzyme activity of the supernatants was tested, with additional samples analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to confirm protein expression. Detailed procedures are available on the Experiments page.

From now on, we will refer to B. subtilis WB800N transformed with the replicative vector pBS0E-xylR-PxylA containing one of our enzymes, such as AtCelG, as "pBS0EX-AtCelG." Similarly, B. subtilis WB800N transformed with the integrative plasmid pBS2E-xylR-PxylA containing the same enzyme will be referred to as "pBS2EX-AtCelG."


CMCase activity determination

To assess the CMCase activity of heterologously expressed endoglucanase AtCelG in B. subtilis, we initially performed a qualitative assay on 1%-Carboxymethyl cellulose (CMC)-Agar plate (see the Experiments page) (Sharma et al. 2017). We decided to test pBS0EX-AtCelG (replicative vector), assuming it would exhibit higher activity than pBS2EX-AtCelG (integrative vector).

Following the expression of AtCelG, induced with 0.5% xylose, we applied 15 µl of the supernatant to wells at the center of 1%-CMC-agar plates. The plates were incubated at 50 °C for 24 hours, after which we stained them using the congo red method to visualize enzyme activity, as shown in Fig. 7. The control strain WB800N and cultures without the addition of inducer were used as controls. Halo formation, indicating CMC degradation, was used to determine enzyme activity.


Negligible halos were observed on the plate of WB800N, suggesting only basal endoglucanase activity in the control strain, confirming the presence of the eglS gene in B. subtilis. Similarly, negligible halo formation was observed in the uninduced culture, indicating minimal endoglucanase expression without induction, with only basal enzyme activity from eglS being present.


For pBS0EX-AtCelG induced with xylose, no halo formation was observed, indicating either failed production of the enzyme in B. subtilis or very low endoglucanase activity under the conditions tested.


Fig. 7: Qualitative CMCase activity assay of supernatant from pBS0EX-AtCelG on 1%-CMC-Agar plates. To determine CMCase activity, 15 µL of the supernatant was applied to 1%-CMC-Agar plates. After incubation at 50 °C for 24 hours, the plates were stained with congo red and destained with 1 M NaCl to visualize enzyme activity. Clear halos around the wells indicate CMC degradation by the expressed endoglucanase. Uninduced culture of pBS0EX-AtCelG and the strain WB800N were used as controls (see Experiments page).

References

Jeong H., Jeong D. E., Park S. H., Kim S. J., Choi S. K. (2018): Complete Genome Sequence of Bacillus subtilis Strain WB800N, an Extracellular Protease-Deficient Derivative of Strain 168. Microbiol Resour Announc. 7(18), e01380-18. https://doi.org/10.1128/MRA.01380-18

Popp P. F., Dotzler M., Radeck J., Bartels J., Mascher T. (2017): The Bacillus BioBrick Box 2.0: expanding the genetic toolbox for the standardized work with Bacillus subtilis. Scientific reports 7(1), 15058. https://doi.org/10.1038/s41598-017-15107-z

Sharma, P., & Guptasarma, P. (2017). Endoglucanase activity at a second site in Pyrococcus furiosus triosephosphate isomerase – Promiscuity or compensation for a metabolic handicap? FEBS Open Bio, 7(8), 1126–1143. https://doi.org/10.1002/2211-5463.12249


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