Part:BBa_K2587009

ptxD_opt

Most of the microorganisms, especially common used model organsims like Escherichia coli or Saccharomyces cerevisiae grow only very slow on phosphite as phosphorus source. Moreover in industry is not widely accepted to use antibiotic resistances as markers. Therefore a system is required, which allows avoidance of contamination by other microorganisms and at the same time represents a reliable selection marker. We present the use of the ptxD gene from Pseudomonas stutzeri together with a phosphite media (reduces growth of contaminants), which could abolish the use of antibiotics in the future and the more efficient use of a phosphorus source in the medium. In this case ptxD is codon optimised for S.cerevisiae.

Usage and Biology

• ptxD codes for phosphonate dehydrogenase
• oxidation of phosphite (phosphonate) using NAD+ and H₂0 to phosphate and NADH
• Selection marker for budding and fission yeast
• phosphite-oxidizing ability
• environmentally safe culture
• antibiotic free system
• pH optimum: 7.25- 7.75

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 93
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 6
  Illegal BamHI site found at 1044
  Illegal XhoI site found at 216
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 714
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 22
  Illegal BsaI.rc site found at 1051
  

Results Phosphite Measurement

Experimental Design

In our three-way co-culture we want to use phosphite as a non-metabolizable phosphorus source. Only our engineered S.  cerevisiae strain is able to convert it to phosphate for itself, as well as providing it to the other organisms.

To test if our construct with the codon optimized ptxD gene (ptxD_opt)14 works, we performed a plate reader experiment over 52 hours with different M2 media characteristics. S. cerevisiae and E. coli , both used as negative control, were cultivated in standard M2 medium15 with 1.5% glucose, 0.5% ammonium sulfate, histidine (1.56 mg/l), leucine (380 mg/l), lysine (1.52 mg/l) and uracil (18 mg/l). For some experiments, M2 medium was modified to contain phosphite (also known as phosphonic acid) instead of the originally used phosphoric acid. The same supplements were used to make the media only differ in the phosphorus source. Medium lacking uracil was used in the samples containing our construct, to maintain selection pressure. Five different constitutive promoters were tested. All samples were measured every 30 minutes in replicates of five. As sample size 200 µl were chosen. Every time, OD₆₀₀ and temperature were measured. The experiment was performed at room temperature, while the plates were shaken vigorously.

Table 2: Loading scheme of the 96 well plate for the OD measurement of different cultures, different colors represent different media compositions:

Data

First it had to be tested whether other organisms in the co-culture are able to use phosphite as a phosphorus source. To test this, we compared growth of E. coli and S. cerevisiae in normal M2 with M2 where phosphite is the only phosphorus source.

Figure 1: Growth of E. coli in different medium over 52 h in M2 medium with 1.5% glucose, 0.5% ammonium sulfate, histidine (1.56 mg/l), leucine (380 mg/l), lysine (1.52 mg/l) and uracil (18 mg/l) (blue) and in M2 with the same composition but only with phosphite instead of phosphate (yellow). The cell density was measured using OD₆₀₀.

As shown in Figure 1 in standard M2 medium E.  coli shows the common growth curve with a lag phase of 10 hours and a log phase over 10 hours. After 20 hours E. coli reaches the stationary phase with an OD₆₀₀ of nearly 0.4. In M2 medium with phosphite the bacteria stay in the lag phase and only reach an OD₆₀₀ of less than 0.1. At the end of the measurement a slow decrease of the population is visible.

Figure 2: Growth of S. cerevisiae over 52 h in M2 medium with 1.5% glucose, 0.5% ammonium sulfate, histidine (1.56 mg/l), leucine (380 mg/l), lysine (1.52 mg/l) and uracil (18 mg/l) (blue) and in M2 medium with the same composition but containing phosphite instead of phosphate (yellow), measured with OD₆₀₀.

S. cerevisiae shows the common growth curve similar to E. coli, with a lag phase until 10 hours and a stationary phase after 20 hours (Figure 2). Cells incubated in M2 medium, supplemented with phosphite as sole phosphorus source, no growth is detectable.

To figure out the strongest one, different promoters from the YTK toolbox6 were tested: TDH3 (BBa_K124002, Link: https://parts.igem.org/Part:BBa_K124002), CCW12, PGK1 (BBa_K122000), HHF2, TEF1 controlling the ptxD_opt gene in S. cerevisiae. All previously described experiments were also performed using M2 medium with phosphite as phosphorus source.

References

Kanda, Keisuke, et al. "Application of a phosphite dehydrogenase gene as a novel dominant selection marker for yeasts." Journal of biotechnology 182 (2014): 68-73.:
https://www.sciencedirect.com/science/article/pii/S016816561400193X