Part:BBa_K4937021

TEF1p-SPE1-PRM9t

TEF1p-SPE1-PRM9t:

This composite part was created by fusion PCR and used to exogenously express SPE1 gene and results in the overexpression of putrescine. This part was used in the oaz1Δ strain, in which the polyamine synthesis flux is stronger. We investigate whether this part can confer thermo-tolerance to resulted strain.

Using the OAZ1 knockout strain we previously constructed, we generated yeast expression plasmids to overexpress SPE1, to achieve overexpression of thermospermine. We initially obtained the plasmid backbone through PCR (Figure 1: 7-1 to 7-4) and the insert fragments (Figure 1: 7-5 is TEF1p-SPE1-PRM9t). Subsequently, we used homologous recombination to construct the complete plasmids and validated their successful construction through sequencing (Figure 2).

Figure 1

Figure 2

We performed HPLC test to ensure that our engineered strains have improved production of putrescine, spermidine (Figure 3,4,5) and thermospermine. As shown in figure 6, the strain contains plasmid that overexpress SPE1 has a higher production of putrescine and spermidine, namely 6.95 mg/L and 8.1 mg/L, respectively, compared to 5.00 mg/L and 6.7 mg/L in strain contains plasmid vector. As we didn’t get the standard of thermospermine, we didn’t get a directly evidence of thermospermine prodution.

Figure 3

Figure 4

Figure 5

Figure 6

Then we tested the growth of these strains under 35°C conditions (Figure 7). The results indicate that the expression of the SPE1 gene did not have a significant impact on the yeast's thermo-tolerance capabilities.

Figure 7

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1871
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1607
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 167