Team:Harvard/Results/MAGE

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For a detailed explanation of how MAGE works, check out our [https://2011.igem.org/Team:Harvard/Technology/MAGE MAGE technology page].
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For a step-by-step procedure, see [https://2011.igem.org/Team:Harvard/Protocols Protocols].
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=Building the selection strain: MAGE=
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[[File: HARVMAGEresults2.png|right|410px|Figure 2: Sequence Alignment of Selection Strain Clone with HisB and PyrF Reference Sequences]]
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To test the zinc fingers constructed by chip synthesis, we used a one-hybrid system tying zinc finger binding to cell survival (Meng et al, 2005).  Under the control of a zinc finger binding site were two yeast genes: His3, a positive metabolic selector, and URA3, a negative selector to eliminate leaky promoters.  To create a selection strain for this system, we accordingly knocked out the endogenous  E. coli homologs (HisB and PyrF, respectively) using multiplex automated genome engineering (MAGE).  The PyrF MAGE oligo inserted two early in-frame stop codons by substituting 8T-->A and 16C-->G, while the HisB oligo deleted 6 in-frame nucleotides (694-699) coding for VE.  5 rounds of MAGE were performed in an EcNR2 E. coli line using both oligos simultaneously (see Protocols for exact procedure) and the resulting colonies underwent allele-specific PCR (Figure 1) and were sequenced at the HisB and PyrF loci (Figure 2).  We chose a colony that contained both the HisB deletion and the PyrF substitutions as the basis of our selection strain.   
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'''Fine-tuning the one-hybrid selection system'''
 
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After the one-hybrid His3-URA3 strain was constructed using lambda red and MAGE, we characterized its growth phenotype in preparation for the chip-synthesized zinc fingers.  Cultures with the Zif268 binding site either with and without the Zif268 protein were grown overnight in a plate reader to chart their growth under various conditions by measuring the absorbance levels at 600nm.  As expected, the strain without Zif268 grew normally in complete media but failed to grow in NM media without histidine (Figure 1A).  Zif268 successfully resuced the growth phenotype in NM (Figure 1B)  and reached similar saturation levels to cultures grown in NM+histidine.  Addition of 5-FOA killed the Zif268 cultures because of their expression of URA3 but did not have a great effect on the selection strain alone, showing that the zinc finger binding site promoter is not inherently leaky.  3-AT, the competitive inhibitor of His3, also fine-tuned selection: Zif268 cultures grew less as 3-AT concentration increased.  Overall, the strain showed the proper phenotypes and successfully illustrated selection for zinc finger binding.
 
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For the zinc fingers synthesized from the chip, the selection strain would need to be able to recognize low numbers of hits among a high level of background. Approximately 9000 different zinc fingers were designed for each binding site, and all 9000 are unlikely to successfully bind to the target, so we tested the sensitivity of the selection strain by diluting Zif268  into zinc finger plasmids that would not bind to the Zif268 site (Figure 2).  Overnight growth in a plate reader showed that our selection strain was sensitive enough to detect Zif268 when diluted  as low as one to one million in as high a concentration of  3-AT as 10mM.  This is more than sufficient to pick out one hit among the 9000 chip-synthesized zinc fingers.
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[[File: HARVMAGEresults1.png|none|500px|Figure 1: Allele-specific PCR]]
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We designed an additional one-hybrid selection system utilizing TolC, an SDS pump, under the control of a zinc finger binding site.  The strain was successfully made and showed the proper phenotypic rescue when Zif268 was present, but SDS concentrations were not as titratable as 3-AT, and it was not able to recognize hits among high background levels as well as the His3-URA3 system. Due to TolC’s inferior sensitivity, we decided to transform the chip-synthesized zinc fingers only into the His3-URA3 selection strain.
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References:
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[http://www.nature.com/nbt/journal/v23/n8/full/nbt1120.html Xiangdong Meng, Michael H Brodsky, Scot A Wolfe.  A bacterial one-hybrid system for determining the DNA-binding specificity of transcription factors. (2005) Nature Biotechnology, 23(8): 988-994.]
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Latest revision as of 14:48, 26 September 2011

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Overview | Biobricks | Source Code | Acknowledgments | Accomplishments

For a detailed explanation of how MAGE works, check out our MAGE technology page.

For a step-by-step procedure, see Protocols.

Building the selection strain: MAGE

Figure 2: Sequence Alignment of Selection Strain Clone with HisB and PyrF Reference Sequences

To test the zinc fingers constructed by chip synthesis, we used a one-hybrid system tying zinc finger binding to cell survival (Meng et al, 2005). Under the control of a zinc finger binding site were two yeast genes: His3, a positive metabolic selector, and URA3, a negative selector to eliminate leaky promoters. To create a selection strain for this system, we accordingly knocked out the endogenous E. coli homologs (HisB and PyrF, respectively) using multiplex automated genome engineering (MAGE). The PyrF MAGE oligo inserted two early in-frame stop codons by substituting 8T-->A and 16C-->G, while the HisB oligo deleted 6 in-frame nucleotides (694-699) coding for VE. 5 rounds of MAGE were performed in an EcNR2 E. coli line using both oligos simultaneously (see Protocols for exact procedure) and the resulting colonies underwent allele-specific PCR (Figure 1) and were sequenced at the HisB and PyrF loci (Figure 2). We chose a colony that contained both the HisB deletion and the PyrF substitutions as the basis of our selection strain.


Figure 1: Allele-specific PCR

References: [http://www.nature.com/nbt/journal/v23/n8/full/nbt1120.html Xiangdong Meng, Michael H Brodsky, Scot A Wolfe. A bacterial one-hybrid system for determining the DNA-binding specificity of transcription factors. (2005) Nature Biotechnology, 23(8): 988-994.]

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