Pbr322

One Shot TOP10 chemically competent E. coli (Invitrogen™, Thermo Fisher Scientific, Dublin, Ireland) was used as the host strain for transformation. Recombinant E. coli transformed using pBR322 (Invitrogen™, Thermo Fisher Scientific, Ireland) was cultured in LB medium at 37 °C with the addition of 100 µgmL⁻¹ of ampicillin (AMP) and 30 µgmL⁻¹ tetracycline (TET). The recombinant strain was used as the resistant test organism throughout. Yeast mannan-rich fraction (MRF) from the cell wall of S. cerevisiae was provided by Alltech (Alltech Inc., Nicholasville, KY, USA). To ensure homogenous mixing, this suspension was then sonicated using an HTU Soni 130 ultrasonic processor at a power setting of 130 Watts for 3 min on ice. Aliquots were stored at 4 °C prior to use or at −70 °C for long-term storage. All antibiotics were obtained from Merck, Germany.

One Shot TOP10 chemically competent E. coli (Invitrogen) was used as the host strain for transformation. Recombinant E. coli transformed using pBR322 (Invitrogen) was cultured in LB medium at 37 °C with addition of 100 µg mL⁻¹ of ampicillin (AMP) and 30 µg mL⁻¹ tetracycline (TET). The recombinant strain was used as the resistant test organism throughout. Yeast mannan-rich fraction (MRF) from the cell wall of S. cerevisiae was provided by Alltech Biotechnology (Alltech Biotechnology, Nicholasville, KY). For determination of bacterial kinetic growth and respiratory analysis, the yeast fraction (0.1–0.5%) was ground and added to either fresh MHB or M9 minimal media (supplemented with 0.2% casamino acids and 10 mM glucose^{8 (link)}), respectively. To ensure homogenous mixing, this suspension was then sonicated using an HTU Soni 130 ultrasonic processor at a power setting of 130 Watts for 3 min on ice. Aliquots were stored at 4 °C prior to use or at − 70 °C for long term storage. All antibiotics and purified glucose [0.1% (w/v)] were obtained from Sigma.

The tetracycline inducible promoter (PLtetO-1) and a strong ribosome binding site (RBS, BBa_B0030) were obtained from the plasmid of pZE11, which was kindly provided by Bujard [26 (link)]. The PLtetO-1 promoter has two TetO2 operator sites. This promoter is tightly repressed by the Tn10-encoded Tet repressor (TetR) and can be activated by a supply of anhydrotetracycline (aTc).
All strains were constructed using standard cloning techniques. The sequences of the PCR primers used in this study are shown in Additional file 1: Table S1. The fusion PCR was used to construct the TetR-Venus fusion protein. The protocol is based on two rounds of PCR: The first PCR uses TetR-F/Linker-R and Linker-F/Venus-R as primer pairs, whose templates are the plasmid pBR322 (Invitrogen™) and E. coli SX4 genomic DNA from Xie [23 (link)], respectively. In the second PCR, the fragments from the first round were amplified and fused using primers TetR-F/Venus-R, resulting in the TetR-Venus fragment. The TetR-Venus fusion protein was connected with the flexible linker, which provided the proper distance between TetR and Venus to avoid interference.
The entire TetR-Venus fragment was digested with KpnI and XbaI and ligated into the pZE11 vector backbone, which placed the TetR-Venus under the control of the PLtetO-1 promoter, yielding the plasmid pZE11-PLtetO-1-TetR-Venus.