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Team:Caltech/Results
From 2012.igem.org
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<h2>Degradation</h2> | <h2>Degradation</h2> | ||
<p> First, vectors that can stably replicate in Z. mobilis were constructed. Using the techniques Gibson assembly and homologous recombination in yeast, new vectors called pMQ95+tet and pMQ97+tet were constructed from replacing the original gene resistances on pMQ95 and pMQ97 with tetracycline and transformed into E. coli colonies. The next step was to identify and obtain the genes that code for the degradation enzymes of allose. It was found that one of the standard laboratory strains of E. coli had the genes for allose degradation enzymes, which are collectively called alsBACEK. This collection of genes was amplified out of the E. coli genome and used to construct new expression plasmids by joining the vectors and alsBACEK. Finally, the plasmid was transferred into Z. mobilis using 4 parent conjugation.</p> | <p> First, vectors that can stably replicate in Z. mobilis were constructed. Using the techniques Gibson assembly and homologous recombination in yeast, new vectors called pMQ95+tet and pMQ97+tet were constructed from replacing the original gene resistances on pMQ95 and pMQ97 with tetracycline and transformed into E. coli colonies. The next step was to identify and obtain the genes that code for the degradation enzymes of allose. It was found that one of the standard laboratory strains of E. coli had the genes for allose degradation enzymes, which are collectively called alsBACEK. This collection of genes was amplified out of the E. coli genome and used to construct new expression plasmids by joining the vectors and alsBACEK. Finally, the plasmid was transferred into Z. mobilis using 4 parent conjugation.</p> | ||
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| + | <p>The liquid minimal media cultures were plated on LB after two weeks, and significant growth was observed, indicating that growth had taken place in the minimal media cultures. To further isolate these organisms, each culture was then plated on solid minimal media with the carbon source added, and growth was again observed. As a control, the cultures were also plated on solid minimal media without the carbon source and failed to grow, indicating that they are not using agar as a carbon source. | ||
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| + | The 16S sequences of these organisms were amplified via PCR and sequenced, and compared with NCBI’s nucleotide database. The lignin-degrading cultures were identified to be <i>Sphingobacterium multivorum</i>, and the polystyrene-degrading cultures were identified to be <i>Raoultella terrigena</i>.</p> | ||
<a name="Biofuel"></a> | <a name="Biofuel"></a> | ||
Revision as of 23:13, 2 October 2012
Results Page
Degradation
First, vectors that can stably replicate in Z. mobilis were constructed. Using the techniques Gibson assembly and homologous recombination in yeast, new vectors called pMQ95+tet and pMQ97+tet were constructed from replacing the original gene resistances on pMQ95 and pMQ97 with tetracycline and transformed into E. coli colonies. The next step was to identify and obtain the genes that code for the degradation enzymes of allose. It was found that one of the standard laboratory strains of E. coli had the genes for allose degradation enzymes, which are collectively called alsBACEK. This collection of genes was amplified out of the E. coli genome and used to construct new expression plasmids by joining the vectors and alsBACEK. Finally, the plasmid was transferred into Z. mobilis using 4 parent conjugation.
The liquid minimal media cultures were plated on LB after two weeks, and significant growth was observed, indicating that growth had taken place in the minimal media cultures. To further isolate these organisms, each culture was then plated on solid minimal media with the carbon source added, and growth was again observed. As a control, the cultures were also plated on solid minimal media without the carbon source and failed to grow, indicating that they are not using agar as a carbon source.
The 16S sequences of these organisms were amplified via PCR and sequenced, and compared with NCBI’s nucleotide database. The lignin-degrading cultures were identified to be Sphingobacterium multivorum, and the polystyrene-degrading cultures were identified to be Raoultella terrigena.
Biofuel
Proteorhodopsin
Bacterial Animation
For the results of the bacterial animation project, we have successfully created two constructs that we have submitted to the Parts Registry as BioBricks: mCherry-LVA in pSB1C3 and mCherry-AAV in pSB1C3. We ran a RFP assay on our two parts and found that _______. We are still in the progress of making the bacterial animation system. On the side, we also had an exhibit at Cal Arts on bacterial plate art and had students draw on and make their own plate art.
