Difference between revisions of "Part:BBa K4879009"
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The resulting gene fragment was then PCR amplified with the respective primers to attain a higher copy number of the fragment. The amplification of the fragment was verified by running a 0.8% agarose gel, with the fragments expected to show at around the 3000 bp region. | The resulting gene fragment was then PCR amplified with the respective primers to attain a higher copy number of the fragment. The amplification of the fragment was verified by running a 0.8% agarose gel, with the fragments expected to show at around the 3000 bp region. | ||
| − | <html><img src="https://static.igem.wiki/teams/4879/wiki/bba-k4879009-pcr-gel.jpg"width="720"height="720"></html> | + | <html><img src="https://static.igem.wiki/teams/4879/wiki/bba-k4879009-pcr-gel-2.jpg"width="720"height="720"></html> |
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| + | Fig: Lane 1 is the DNA ladder, and lanes 4-7 contain the successfully amplified fragments | ||
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The amplified fragment was then gel-extracted and subsequently transformed into our chassis. | The amplified fragment was then gel-extracted and subsequently transformed into our chassis. | ||
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The transformed yeast culture was then plated onto the selection plates containing about 200 µg/ml of hygromycin B and were incubated at 30°C. The growth of the yeast on the selection plates confirms the functional validity of the expression construct. | The transformed yeast culture was then plated onto the selection plates containing about 200 µg/ml of hygromycin B and were incubated at 30°C. The growth of the yeast on the selection plates confirms the functional validity of the expression construct. | ||
| − | <html><img src="https://static.igem.wiki/teams/4879/wiki/bba-k4879009-plate.jpg"></html> | + | <html><img src="https://static.igem.wiki/teams/4879/wiki/bba-k4879009-plate.jpg"></html> |
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| + | Fig 2: The growth of transformed <i>Y. lipolytica</i> on the hygromycin B selection plate, confirming part functionality. | ||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
Latest revision as of 16:16, 11 October 2023
Yarrowia lipolytica HygR construct
The transcriptional unit for HygR, designed for expression in Y. lipolytica in order to delete the faa1 gene from our chassis.
Characterization
Cloning: The construct, along with homologous flanking sequences corresponding to the faa1 gene locus, were synthesized by iGEM’s DNA synthesis partner IDT. The resulting gene fragment was then PCR amplified with the respective primers to attain a higher copy number of the fragment. The amplification of the fragment was verified by running a 0.8% agarose gel, with the fragments expected to show at around the 3000 bp region.
Fig: Lane 1 is the DNA ladder, and lanes 4-7 contain the successfully amplified fragments
The amplified fragment was then gel-extracted and subsequently transformed into our chassis.
Validation: The transformed yeast culture was then plated onto the selection plates containing about 200 µg/ml of hygromycin B and were incubated at 30°C. The growth of the yeast on the selection plates confirms the functional validity of the expression construct.
Fig 2: The growth of transformed Y. lipolytica on the hygromycin B selection plate, confirming part functionality.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 1561
Illegal suffix found in sequence at 1831
Illegal EcoRI site found at 684
Illegal EcoRI site found at 778
Illegal EcoRI site found at 1825 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 684
Illegal EcoRI site found at 778
Illegal EcoRI site found at 1561
Illegal EcoRI site found at 1825
Illegal SpeI site found at 1832
Illegal PstI site found at 1846
Illegal NotI site found at 1567
Illegal NotI site found at 1839 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 684
Illegal EcoRI site found at 778
Illegal EcoRI site found at 1561
Illegal EcoRI site found at 1825 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 1561
Illegal suffix found in sequence at 1832
Illegal EcoRI site found at 684
Illegal EcoRI site found at 778
Illegal EcoRI site found at 1825 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 1561
Illegal EcoRI site found at 684
Illegal EcoRI site found at 778
Illegal EcoRI site found at 1825
Illegal XbaI site found at 1576
Illegal SpeI site found at 1832
Illegal PstI site found at 1846 - 1000COMPATIBLE WITH RFC[1000]