Difference between revisions of "Team:RHIT/Results"
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The team successfully produced a yeast expression plasmid, pSB416-GPD, by replacing the multiple cloning site of p416-GPD with the BioBrick prefix and suffix. This vector allows for easy overlap assembly and expression of standard BioBrick translational units in yeast. However, we have yet to remove an illegal <i>Pst</i>I site near the <i>URA3</i> gene, which currently prevents use of the vector for standard cloning of BioBrick parts by ligation. Once this restriction site is removed, the vector will be submitted to the Registry. | The team successfully produced a yeast expression plasmid, pSB416-GPD, by replacing the multiple cloning site of p416-GPD with the BioBrick prefix and suffix. This vector allows for easy overlap assembly and expression of standard BioBrick translational units in yeast. However, we have yet to remove an illegal <i>Pst</i>I site near the <i>URA3</i> gene, which currently prevents use of the vector for standard cloning of BioBrick parts by ligation. Once this restriction site is removed, the vector will be submitted to the Registry. | ||
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The team also successfully constructed a translational unit for the yeast mitochondrial ribosomal protein MRPS12, which includes the wild-type Kozak sequence and a yeast-optimized coding sequence. The full translational unit (BBa_K1729001) and a translational start sequence, which includes the wild-type Kozak sequence and the yeast-optimized mitochondrial localization signal of MRPS12 (BBa_K1729002), were each cloned into pSB1C3, sequence verified, and submitted to the Registry. | The team also successfully constructed a translational unit for the yeast mitochondrial ribosomal protein MRPS12, which includes the wild-type Kozak sequence and a yeast-optimized coding sequence. The full translational unit (BBa_K1729001) and a translational start sequence, which includes the wild-type Kozak sequence and the yeast-optimized mitochondrial localization signal of MRPS12 (BBa_K1729002), were each cloned into pSB1C3, sequence verified, and submitted to the Registry. | ||
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| + | In addition, we tested parent (P) and <i>MRPS12</i>-disrupted (D) haploid strains for growth on fermentable carbon (FC) and non-fermentable carbon (NFC) sources to show that inactivation of the <i>MRPS12</i> gene indeed prohibits growth on non-fermentable carbon sources (data not shown). We also cloned the newly-made MRPS12 translational unit (BBa_K1729001) into the pSB416-GPD vector we prepared. However, time constraints did not allow us to demonstrate that aerobic respiration could be restored in <i>MRPS12</i>-disrupted haploids transformed with the pSB416-GPD plasmid with (+) or without (-) the <i>MRPS12</i> gene (as diagrammed below).<br> | ||
| + | <img style="padding-right:15%;padding-left:15%;width:70%" src="https://static.igem.org/mediawiki/2015/d/d6/RHIT_plates.png"><br><br> | ||
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<b>Future Plans</b> | <b>Future Plans</b> | ||
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| − | Once we verify that introducing plasmid-borne <i>MRPS12</i> into disrupted strains will restore aerobic respiration, we plan to assemble a repressible promoter system to regulate the gene’s expression. We then hope to determine how regulation of aerobic respiration might affect the production of fermentation products and secondary metabolites in yeast cultures. | + | Once we verify that introducing plasmid-borne <i>MRPS12</i> into disrupted strains will restore aerobic respiration, we plan to assemble a repressible promoter system to regulate the gene’s expression. We then hope to determine how regulation of aerobic respiration might affect the production of fermentation products and secondary metabolites in yeast cultures.<br> |
| + | <img style="padding-left:5%;width:90%" src="https://static.igem.org/mediawiki/2015/a/af/RHIT_system.jpg"><br><br> | ||
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Latest revision as of 22:42, 18 September 2015
Project Results
Accomplishments
The team successfully produced a yeast expression plasmid, pSB416-GPD, by replacing the multiple cloning site of p416-GPD with the BioBrick prefix and suffix. This vector allows for easy overlap assembly and expression of standard BioBrick translational units in yeast. However, we have yet to remove an illegal PstI site near the URA3 gene, which currently prevents use of the vector for standard cloning of BioBrick parts by ligation. Once this restriction site is removed, the vector will be submitted to the Registry.
The team also successfully constructed a translational unit for the yeast mitochondrial ribosomal protein MRPS12, which includes the wild-type Kozak sequence and a yeast-optimized coding sequence. The full translational unit (BBa_K1729001) and a translational start sequence, which includes the wild-type Kozak sequence and the yeast-optimized mitochondrial localization signal of MRPS12 (BBa_K1729002), were each cloned into pSB1C3, sequence verified, and submitted to the Registry.
In addition, we tested parent (P) and MRPS12-disrupted (D) haploid strains for growth on fermentable carbon (FC) and non-fermentable carbon (NFC) sources to show that inactivation of the MRPS12 gene indeed prohibits growth on non-fermentable carbon sources (data not shown). We also cloned the newly-made MRPS12 translational unit (BBa_K1729001) into the pSB416-GPD vector we prepared. However, time constraints did not allow us to demonstrate that aerobic respiration could be restored in MRPS12-disrupted haploids transformed with the pSB416-GPD plasmid with (+) or without (-) the MRPS12 gene (as diagrammed below).
Future Plans
Once we verify that introducing plasmid-borne MRPS12 into disrupted strains will restore aerobic respiration, we plan to assemble a repressible promoter system to regulate the gene’s expression. We then hope to determine how regulation of aerobic respiration might affect the production of fermentation products and secondary metabolites in yeast cultures.
