h1

TY  - THES
AU  - Müller, Carolin
TI  - Investigation of protein secretion in microscale cultivation systems with novel tools
PB  - RWTH Aachen University
VL  - Dissertation
CY  - Aachen
M1  - RWTH-2023-04029
SP  - Online-Ressource : Illustrationen, Diagramme
PY  - 2023
N1  - Veröffentlicht auf dem Publikationsserver der RWTH Aachen University
N1  - Dissertation, RWTH Aachen University, 2023
AB  - Until today, it is impossible to predict a suitable signal peptide for Sec-type secretion of heterologous proteins in Gram-positive bacteria. Instead, signal peptides have to be tested for each host and target protein under process conditions. In addition, the ribosome binding site and in particular the spacer between the Shine-Dalgarno sequence and the start codon of the signal peptide can also affect protein secretion. To accelerate the identification of suitable combinations of signal peptides and target proteins, automated workflows for targeted strain construction and high-throughput screening for heterologous protein secretion in Corynebacterium glutamicum were established, which can be easily adapted to different target proteins. A plasmid library with different Bacillus subtilis signal peptides was constructed in the newly designed pPBEx2-based plasmid pCMEx12, which allows the exchange of the ribosome binding site including the spacer sequence as well as the signal peptide sequence by cassette mutagenesis. In this method, the inserts are provided as hybridized oligonucleotides that are not fully complementary, but have overhangs that can be ligated to the restriction digested backbone. For target protein secretion with pCMEx-based plasmids, a reporter gene coding for a blue chromoprotein under the control of a constitutive promoter can be exchanged with the gene of interest by Golden Gate assembly, combining restriction and ligation in a one-pot setup. Since the chromoprotein leads to blue colonies after transformation, successful cloning can be detected by a change in colony color from blue to white, in addition to a restriction enzyme digest in which the number and size of DNA fragments depend on the insert. The gene of interest is then expressed in frame with an amino-terminal signal peptide and carboxyl-terminally linked to the 11th β-sheet of the green fluorescent protein (GFP, GFP11-tag) under the control of the inducible tac promoter. The molecular cloning steps of the Golden Gate assembly, the Escherichia coli heatshock transformation, the plasmid purification and the restriction digest were automated using the Opentrons OT-2 liquid handling robot with integrated Temperature or Magnetic Module. This reduced the process time for molecular cloning to about 58
LB  - PUB:(DE-HGF)11
DO  - DOI:10.18154/RWTH-2023-04029
UR  - https://publications.rwth-aachen.de/record/956251
ER  -