Difference between revisions of "Part:BBa K4937023"
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<p>TEF1p-SPE1-PRM9t-TDH3p-AtACL5-DIT1t:</p>
 
<p>TEF1p-SPE1-PRM9t-TDH3p-AtACL5-DIT1t:</p>
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https://static.igem.wiki/teams/4937/wiki/part/021-1.png
 
<p>Using the OAZ1 knockout strain we previously constructed, we generated yeast expression plasmids to overexpress SPE1 and <i>At</i>ACL5, to achieve overexpression of putrescine (and subsequently spermidine) and thermospermine. We initially obtained the plasmid backbone through PCR (Figure 1: 7-1 to 7-4) and the insert fragments (Figure 1: 7-7 is TEF1p-SPE1-PRM9t-TDH3p-AtACL5-DIT1t). Subsequently, we used homologous recombination to construct the complete plasmids and validated their successful construction through sequencing (Figure 2).</p>
 
<p>Using the OAZ1 knockout strain we previously constructed, we generated yeast expression plasmids to overexpress SPE1 and <i>At</i>ACL5, to achieve overexpression of putrescine (and subsequently spermidine) and thermospermine. We initially obtained the plasmid backbone through PCR (Figure 1: 7-1 to 7-4) and the insert fragments (Figure 1: 7-7 is TEF1p-SPE1-PRM9t-TDH3p-AtACL5-DIT1t). Subsequently, we used homologous recombination to construct the complete plasmids and validated their successful construction through sequencing (Figure 2).</p>
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https://static.igem.wiki/teams/4937/wiki/part/021-2.png
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<p style=" text-align: center;">Figure 1</p>
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https://static.igem.wiki/teams/4937/wiki/part/021-3.png
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<p style=" text-align: center;">Figure 2</p>
 
<p>Then we tested the growth of these strains under 35°C conditions (Figure 3). The results indicate that the expression of the SPE1-AtACL5 gene did not have a significant impact on the yeast's thermo-tolerance capabilities.</p>
 
<p>Then we tested the growth of these strains under 35°C conditions (Figure 3). The results indicate that the expression of the SPE1-AtACL5 gene did not have a significant impact on the yeast's thermo-tolerance capabilities.</p>
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https://static.igem.wiki/teams/4937/wiki/part/021-4.png
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<p style=" text-align: center;">Figure 3</p>
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Revision as of 08:16, 28 September 2023


TEF1p-SPE1-PRM9t-TDH3p-AtACL5-DIT1t

TEF1p-SPE1-PRM9t-TDH3p-AtACL5-DIT1t:

021-1.png

Using the OAZ1 knockout strain we previously constructed, we generated yeast expression plasmids to overexpress SPE1 and AtACL5, to achieve overexpression of putrescine (and subsequently spermidine) and thermospermine. We initially obtained the plasmid backbone through PCR (Figure 1: 7-1 to 7-4) and the insert fragments (Figure 1: 7-7 is TEF1p-SPE1-PRM9t-TDH3p-AtACL5-DIT1t). Subsequently, we used homologous recombination to construct the complete plasmids and validated their successful construction through sequencing (Figure 2).

021-2.png

Figure 1

021-3.png

Figure 2

Then we tested the growth of these strains under 35°C conditions (Figure 3). The results indicate that the expression of the SPE1-AtACL5 gene did not have a significant impact on the yeast's thermo-tolerance capabilities.

021-4.png

Figure 3


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