Part:BBa_K4937020
OAZ1_upstream-TEF1p-SPE1-PRM9t-TDH3p-AtACL5-DIT1t-OAZ1_downstream
OAZ1_upstream-TEF1p-SPE1-PRM9t-TDH3p-AtACL5-DIT1t-OAZ1_downstream:
This composite part was created by fusion PCR and used to construct the oaz1Δ::spe1-Atacl5 strain by CRISPR/Cas9. The results strain has a greater flux of polyamine synthesis, especially putrescine and thermospermine. We successfully constructed this part, however, we didn’t construct the genome editing strain completely. Thus, there is not the thermo-tolerance test.
Our experiment consisted of three main phases: fragment construction, yeast transformation, and validation.
We initially obtained the short fragment (Figure 1A and 1B) through PCR. Subsequently, we amplified the SPE1 fragment (Figure 1C) from the previously used repair template. Following that, we obtained the AtACL5 fragment (Figure 1D) through fusion PCR. Finally, we acquired the complete repair template (Figure 1E) through fusion PCR.
Figure 1
However, numerous small colonies grew on the Delft plates after yeast transformation, as shown in Figure 2. These colonies could not be expanded in Delft media, and as a result, we never obtained positive clones. We suspect that this may be due to low transformation efficiency leading to a low positivity rate. Additionally, the use of uracil-containing medium during the preparation of competent cells and post-transformation recovery may have caused some cells that were not successfully transformed to maintain uracil for a period, allowing them to grow and divide, thereby leading to false positives.
Figure 2
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2336
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2072
Illegal BglII site found at 5129 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 632
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