Team:Northwestern/Project/Results

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<h1>Results</h1>
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<h1>Phytase</h1>
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    <p class="arrow-header">Design
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<p>At the core of Northwestern’s Phytastic Probiotic project are two main subprojects.
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<p>The first is to clone different phytases from different organisms into E. coli and then to engineer E. coli to constitutively express the phytases at varying levels. We used a lab strain of E. coli as a model chassis for this project, but our technology could be adopted to function in probiotic strains as well. The four phytases we chose were phytases from E. coli, citrobacter braaki, aspergillus niger, and bacillus subtilus. These phytases were selected based upon varying specific activities, pH optima, and temperature optima that are expected to be most compatible with physiological conditions of the stomach, as compared to other phytases. Our general methods comprised PCR from genomic DNA, gibson assembly,  and standard RFC10 biobrick assembly methods.
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<!--Cartoon picture of constitutive promoter and phytase system
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strongCP+4 Different Phytases
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newstrongCP + 4 Different Phytases-->
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    <p class="arrow-header">Construct
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<p>Each of the four phytases was PCR-amplified out of its respective organism’s genome.  However, due to the introns present in the the Aspergillus niger phytase coding gene, we had an intron-free version of this gene synthesized by IDT. We assembled these parts together using Gibson assembly. After PCR cleanup, gel electrophoresis was performed to verify that the expected band patterns were observed.
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<!--[gel picture for each of the PCR’d phytases]-->
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<img src="https://static.igem.org/mediawiki/2012/d/d1/AN_phy.png"> expected: 1500bp
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<img src=”https://static.igem.org/mediawiki/2012/3/35/CB_phy.png>  expected: 1302bp
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<img src=”https://static.igem.org/mediawiki/2012/c/ce/EC_phy.png> expected: 1299bp
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<p>After gel verifying and sequencing each of the phytases, an unexpected restriction site was discovered in the middle of the Bacillus subtilus phytase gene that prohibited biobrick assembly. Thus, we decided not to pursue this phytase construct further at this time. The three remaining phytases were cloned into protein expression backbones from the parts registry with constitutive promoters and ribosome binding sites already cloned into the vector. Several promoters from the same promoter library were used as each has varying relative expression strengths. Gel electrophoresis and sequencing was again performed to verify that the parts were assembled as expected.
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<!--[gel picture for each of the remaining 3 phytases with the correct CP’s] -->
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<p>A Niger was never successfully ligated with CP
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<img src="https://static.igem.org/mediawiki/2012/2/25/August_31_Gel_1_nsCP%2BEC_Phy_digests.jpg"> nsCP + EC phy (expected 1334)
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<img src="https://static.igem.org/mediawiki/2012/6/62/NSCP_%2B_CB_.png"> NSCP + CBphy (expected 1337)
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Revision as of 03:36, 4 October 2012

Phytase

Design

At the core of Northwestern’s Phytastic Probiotic project are two main subprojects.

The first is to clone different phytases from different organisms into E. coli and then to engineer E. coli to constitutively express the phytases at varying levels. We used a lab strain of E. coli as a model chassis for this project, but our technology could be adopted to function in probiotic strains as well. The four phytases we chose were phytases from E. coli, citrobacter braaki, aspergillus niger, and bacillus subtilus. These phytases were selected based upon varying specific activities, pH optima, and temperature optima that are expected to be most compatible with physiological conditions of the stomach, as compared to other phytases. Our general methods comprised PCR from genomic DNA, gibson assembly, and standard RFC10 biobrick assembly methods.

Construct

Each of the four phytases was PCR-amplified out of its respective organism’s genome. However, due to the introns present in the the Aspergillus niger phytase coding gene, we had an intron-free version of this gene synthesized by IDT. We assembled these parts together using Gibson assembly. After PCR cleanup, gel electrophoresis was performed to verify that the expected band patterns were observed. expected: 1500bp expected: 1302bp expected: 1299bp

After gel verifying and sequencing each of the phytases, an unexpected restriction site was discovered in the middle of the Bacillus subtilus phytase gene that prohibited biobrick assembly. Thus, we decided not to pursue this phytase construct further at this time. The three remaining phytases were cloned into protein expression backbones from the parts registry with constitutive promoters and ribosome binding sites already cloned into the vector. Several promoters from the same promoter library were used as each has varying relative expression strengths. Gel electrophoresis and sequencing was again performed to verify that the parts were assembled as expected.

A Niger was never successfully ligated with CP nsCP + EC phy (expected 1334) NSCP + CBphy (expected 1337)

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