Team:Harvard/Project

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=Project Outline=
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__NOTOC__
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[[File:HARV Pipeline.PNG|frameless|915px|center]]
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=Massively Multiplexed Zinc Finger Protein Engineering=
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Zinc finger proteins are specialized proteins that bind to DNA. Due to their ability to target highly specific DNA sequences, zinc finger proteins offer great potential for gene therapy and personalized medicine: recently, they were shown to be effective in conferring HIV resistance and treating hemophilia in mice. In the past, however, designing new zinc finger  proteins - a necessity for individualized gene therapy - has been prohibitively expensive and time consuming. For more information about zinc fingers, see [https://2011.igem.org/Team:Harvard/Project#Zinc_Finger_Background here]. '''Our goal is to create zinc finger proteins that bind to DNA triplets that haven't been bound before: triplets for which no zinc finger protein currently exists. '''
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=Abstract=
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Gene therapy is a powerful approach for the treatment of disease; however, current therapies using viral vectors carry significant risk of tumorigenesis due to their use of non-specific gene insertion. To meet this challenge, we engineered zinc finger proteins (ZFPs), which are tailored to bind to DNA with high specificity, enabling precise genome editing. Our group has developed a foundational technology for synthesizing nearly 55,000 unique ZFPs using chip-based DNA synthesis based on bioinformatic analysis and for identifying the best binder using a novel genomically encoded 1-hybrid genetic selection scheme in a massively multiplexed fashion. Further, we employed multiplex automated genome engineering (MAGE) for facile editing of the E. coli genome enabling rapid modification of ZFP target sites, gene knockouts and silent codon substitutions. These tools allow for low-cost creation of ZFPs targeting any endogenous human gene, which will increase the accessibility of customized genome editing for gene therapy.
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__toc__
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=Zinc Finger Background=
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===What are Zinc Finger Proteins (ZFPs)?===
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====Function====
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ZFPs are found commonly in nature as a class of special transcription factors that bind to DNA, thus regulating gene expression. Zinc finger function was first studied using zinc finger protein Zif268.
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[[File:HARVZinc diagram.png|frameless|right|350px]]
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For our project, team members created and tested thousands of zinc fingers at a cost feasible for most labs. To do so, we harnessed three novel synthetic biology technologies: [https://2011.igem.org/Team:Harvard/Technology/Chip_Synthesis chip-based synthesis][[#References|[1]]], which allows for thousands (even millions) of DNA strands to be synthesized concurrently; multiplex automated genome engineering ([https://2011.igem.org/Team:Harvard/Technology/MAGE MAGE])[[#References|[2]]][[#References|[3]]]; and [https://2011.igem.org/Team:Harvard/Lambda_Red lambda red recombineering] [[#References|[4]]][[#References|[5]]], both of which make direct edits of the bacterial genome possible and can replace the used of small, cumbersome plasmids.
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====Structure====
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See our [https://2011.igem.org/Team:Harvard/Results results] page: using these methods, '''we found up to 15 novel zinc fingers''', which are still being characterized.
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ZFPs consist of smaller subunits called "fingers" which each contain a zinc finger binding helix that binds to unique DNA sequences. These fingers are linear and linked together by the "zinc finger backbone", a series of approximately 21 amino acids.
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*Cis2His2 ZFPs have three main structural components:
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** Zinc finger binding helix
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** Linker region
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** Zinc ion that is coordinated by two cysteine residues and two histidine residues.
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===Helpful Zinc Finger Links===
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See [https://2011.igem.org/Team:Harvard/Project/Details here] for our official abstract, and our [https://2011.igem.org/Team:Harvard/Results/Acknowledgements acknowledgments] page.  
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[http://en.wikipedia.org/wiki/Zinc_finger '''Zinc Fingers on Wikipedia''']
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*A more detailed introduction to zinc fingers.
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[http://compbio.cs.princeton.edu/zf/ '''Predicting DNA Recognition by C2H2 Zinc Finger Proteins''']
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'''To achieve this result, our project had three main steps:'''
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*A program useful for predicting how well a given amino acid sequence will bind to a given DNA sequence
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[http://www.zincfingers.org/default2.htm '''The Zinc Finger Consortium''']
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==1. Design== 
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*Information & helpful resources for zinc fingers
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'''Use a bioinformatics approach to predict 55,000 zinc finger sequences.'''
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[http://www.jounglab.org/ '''Joung Lab''']
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As the structure and binding interactions of zinc fingers are not yet understood, our project utilized bioinformatics and computational analysis of the limited existing data to make “educated guesses” of what amino acid sequences will bind to our desired target sequence.
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*Information about Dr. Joung's extensive work with zinc fingers
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[[File:HARVTGG.png|thumb|left|Our generated sequences to bind the DNA triplet TGG.]]
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=Project Overview=
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Team members chose [https://2011.igem.org/Team:Harvard/Project/Design#Selection_of_Target_Sequences 6 target sequences] in the human genome (finding these sequences using a [https://2011.igem.org/Team:Harvard/ZF_Binding_Site_Finder binding site finder tool] written by a team member) that no currently-existing zinc finger is able to bind to. The sequences were selected from genes that cause [https://2011.igem.org/Team:Harvard/Project/Design#Target_Site_Background_Information colorblindness, some types of cancer, and high cholesterol]. These targets were chosen after conducting an extensive literature search, with the most useful information coming from a previous zinc finger study called CODA[[#References|[6]]].
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Zinc fingers are specialized proteins that bind to DNA. Due to their ability to target highly specific DNA sequences, zinc fingers offer great potential for gene therapy and personalized medicine: recently, they were shown to be effective in conferring HIV resistance and treating hemophilia in mice. In the past, however, designing new zinc fingers--a necessity for individualized gene therapy--has been prohibitively expensive and time consuming.
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Team members emailed the OPEN consortium[[#References|[7]]] and Dr. Anton Persikov[[#References|[8]]] to acquire their respective databases of zinc fingers: Persikov had compiled the results of the past 20 years of zinc finger research (everything before OPEN), and OPEN had found many more since then. In all, around 1,700 unique DNA triplet/zinc finger helix pairs were discovered.  Team members then decided how to analyze this data for the best chance of binding our target sequences, and programmed their ideas into a [http://sourceforge.net/projects/harvardigem/ Python program] to generate 55,000 potential zinc fingers (55,000 is the number of sequences that can be built using chip-based DNA synthesis).  
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For our iGEM project, we are creating and testing thousands of zinc fingers simultaneously at a cost feasible for most labs. To do so, we harnessed two novel synthetic biology technologies: chip-based synthesis, which allows up to millions of DNA strands to be synthesized concurrently, and MAGE (multiplex automated genome engineering), which makes possible direct edits of the genome of organisms, rather than using the small, cumbersome plasmids that are common in labs today.  
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For a complete overview of how our zinc fingers were designed, see [https://2011.igem.org/Team:Harvard/Project/Design Design].  
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As the structure and binding interactions of zinc fingers are not yet understood, our project also utilizes bioinformatics and computational analysis of the limited existing data to make “educated guesses” to generate zinc fingers that could bind to DNA for genes that cause colorblindness, some types of cancer, and high cholesterol.  
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==2. Synthesize==
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'''Use chip-based DNA synthesis to make these 55,000 sequences simultaneously, then insert the oligos into E.coli.'''
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Thus, our zinc fingers and their clinical applications are a new technology, and while we hope that our new zinc fingers work, the more important goal is maximizing efficiency and decreasing cost while utilizing new technology: we anticipate that future iGEM teams will find great use for chip-based synthesis and MAGE.
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Once our team generated these 55,000 sequences, we needed a way to bring them from a computer's memory into the real world. Using [https://2011.igem.org/Team:Harvard/Technology/Chip_Synthesis chip synthesis] of DNA (generously contributed by Agilent Technologies, a sponsor of iGEM, in partnership with our mentors in the Church Lab), all of these sequences were built simultaneously, then sent to us in a single 50µL tube.
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=Detailed Project Description=
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For more information about how the chip was created, see the original 2010 paper by Kosuri et al[[#References|[1]]]. Cost of chip synthesis is around $.00091 per DNA base, compared to ~$0.40–1.00 per base with current commercial pricing[[#References|[1]]]. Thus, chip synthesized DNA has the potential to be up to a factor of 1,000 times cheaper than current mainstream methods of DNA synthesis.
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Gene therapy is a powerful approach for the treatment of disease, and holds great potential for success.  While gene therapy has progressed significantly since its conception, much of its projected potential remains untapped, while existing therapeutic advancements are still fraught with serious problems. Many of these roadblocks to existing therapies are caused by non-specific gene insertion (as through viral vectors), which hinders therapeutic viability and can lead to unintended side effects including cancer.  In recent attempts to overcome this obstacle, however, one particular approach has shown much promise. This approach utilizes engineered zinc finger proteins, which have been shown to edit the genome with dramatically increased accuracy (Perez et al 2008, Li et al 2011). Zinc finger proteins are naturally-occurring protein domains composed of multiple individual zinc finger subdomains. Although the individual subdomains (“fingers”) occur in a variety of structural motifs, the classic Cys<sub>2</sub>His<sub>2</sub> (C<sub>2</sub>H<sub>2</sub>) motif is among the best characterized and can be observed in a variety of transcription factors. The C<sub>2</sub>H<sub>2</sub> motif (see Fig. 1) consists of a beta-sheet conjugated to an alpha helix, and it is structurally coordinated through interactions between two cysteine and two histidine residues with a single zinc ion. [[File:HARVZFmotif NoBG.png|thumb|right|'''Fig. 1''' C<sub>2</sub>H<sub>2</sub> zinc finger motif. Single finger shown with coordinating Zn<sup>2+</sup> ion.]] Many eukaryotic transcription factors utilize C<sub>2</sub>H<sub>2</sub> zinc finger domains, which possess a unique capacity to bind to specific DNA sequences. Zinc finger proteins have received increasing attention in recent research for their DNA-binding ability and specificity, which offers a solution to non-specificity in gene therapy and could thus bring the scientific community a step closer to realizing many novel therapeutic applications.  Recent studies, for instance have demonstrated that DNA-binding zinc fingers coupled with Type IIs restriction endonucleases such as Fok1 can be harnessed as effective tools to genetically treat hemophilia and confer HIV resistance in mouse models (Perez et al 2008, Li et al 2011).  
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Chip synthesis results in a pool of single-stranded oligos, designed with primer tags, which allow for the amplification of specific sub-pools: in our team's case, we used 6 sub-pools, one for each of our target sequences.  
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<br />
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For a complete overview of how our zinc finger DNA went from chip to being expressed in E.coli, see [https://2011.igem.org/Team:Harvard/Project/Synthesize Synthesize].
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While many different natural zinc finger proteins can be employed for synthetic biology-based tasks, engineered zinc finger proteins are most commonly composed of an array of three zinc fingers based on Zif268. Zif268 is a well-characterized mammalian early response transcription factor also known as EGR-1 that was discovered in mice. Zif268 has three C<sub>2</sub>H<sub>2</sub> finger subunits, and each finger binds a specific 3-nucleotide base pair (bp) triplet on a DNA strand. Composed of an array of three fingers, Zif268 binds to three consecutive DNA triplets that in total comprise a 9-bp binding site. This 3-finger domain, 9-bp specificity is a commonly occurring theme in zinc finger literature, although it is by no means a rule. Natural and engineered zinc finger arrays may contain fewer or more zinc fingers and bind DNA sequences of corresponding length and specificity. While any given zinc finger might bind a certain 3-bp DNA sequence with exquisite specificity, even the most specific 3-bp binding event is of little practical value for gene therapy, as a given 3-bp sequence may appear countless times within a given genome. Thus, for practical use in drug delivery or gene therapy, zinc fingers must be engineered to bind more specifically to DNA sequences.  Such increased specificity can be achieved through the creation of multi-finger arrays that bind to specific 9-bp sequences. With this in mind, research has focused on creating modular zinc finger subunits, which can be combined into multi-finger arrays. Three-finger arrays, for instance, are tailored to bind to a specific 9-bp sequence through the selection of appropriate zinc fingers to bind to each DNA triplet.  
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<br />
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==3. Test==
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Different studies have suggested different models of binding and varying levels of success for zinc finger modularity. Thus, through an approach utilizing data-mining, binding models, and comprehensive bioinformatics, in conjunction with next-generation oligo synthesis technology, we hope to develop a method for engineering zinc finger proteins that can bind to any arbitrarily-selected DNA sequence. The success of such an engineering method would, in turn, facilitate clinical advancements by promoting highly-specific, targeted gene therapies, and would promote personalized medicine by allowing the production of zinc finger-based therapies tailored to unique genomic sequences.
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'''Use a genomic metabolic selection system to test which zinc finger sequences successfully bind DNA.'''
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[[File:HARVOne-hybrid_diagram.png|thumb|left|Our metabolic selection system. The cell’s ability to synthesize histidine is eliminated: however, homologous gene His3 has been placed on the genome, with transcription regulated by zinc finger arrays binding upstream. The cells undergo selection in minimal media. Thus, without a zinc finger binding event, the cell is unable to synthesize histidine and dies.]]
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==References==
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After creating and expressing 55,000 novel zinc finger sequences, our team needed to determine which ones effectively bind to their respective target sites. By tying zinc finger binding to cell survival (using an efficient selection system) all cells without successful binders would die, and thus living colonies indicate a viable zinc finger.  
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#Li H, Haurigot V, Doyon Y, Li T, Wong SY, Bhagwat AS, Malani N, Anguela XM, Sharma R, Ivanciu L, Murphy SL, Finn JD, Khazi FR, Zhou S, Paschon DE, Rebar EJ, Bushman FD, Gregory PD, Holmes MC, High KA (2011). In vivo genome editing restores haemostasis in a mouse model of haemophilia. ''Nature'', doi: 10.1038/nature10177 [Epub ahead of print].
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#Perez EE, Wang J, Miller JC, Jouvenot Y, Kim KA, Liu O, Wang N, Lee G, Bartsevich VV, Lee YL, Guschin DY, Rupniewski I, Waite AJ, Carpenito C, Carroll RG, Orange JS, Urnov FD, Rebar EJ, Ando D, Gregory PD, Riley JL, Holmes MC, June CH (2008). Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. ''Nature Biotechnology'', 26(7), 808-16.
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Team members constructed a one-hybrid selection system based off a metabolic system designed by Meng et al[[#References|[9]]] which tied zinc finger binding to histidine production. When grown in media without histidine, the cells can only survive if a zinc finger-omega subunit of RNA polymerase (also knocked out in the strain) fusion protein binds successfully and initiates creation of histidine.
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Where our team's selection departs from the one described by Meng and others is its use of a genome-based rather than plasmid-based system.  Not only did team members knock out HisB, PyrF, and rpoZ ourselves using the newly developed techniques of [https://2011.igem.org/Team:Harvard/Technology/MAGE MAGE] and [https://2011.igem.org/Team:Harvard/Lambda_Red lambda red], we also inserted the zinc finger binding site construct directly into the genome instead of expressing it in the cell on a vector.
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For a complete overview of how our selection system was designed, tested, and optimized, see [https://2011.igem.org/Team:Harvard/Project/Test Test].
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<br><br><br>
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=Results=
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See our [https://2011.igem.org/Team:Harvard/Results results] page for a summary of our most significant findings.
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'''By submitting the necessary [[Team:Harvard/Results/Biobricks|strains and parts to the registry]], and publishing these [[Team:Harvard/Protocols|easy-to-follow protocols]] along with our [[Team:Harvard/Results/Tools|source code]], it is our hope that future iGEM teams will also use these techniques in their own synthetic biology projects. '''
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=Technological Applications=
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Our zinc fingers and their clinical applications are a new technology that maximize efficiency and decrease cost. The novel methods we employed in our project have the potential to revolutionize synthetic biology practices, and the way that future iGEM competitions are conducted. To learn more about the technological applications of our project, please see our [https://2011.igem.org/Team:Harvard/Technology '''Technology'''] page.
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<!-- remove clinical applications since its already on bioinformatics page
 
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=References=
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'''1.''' Sriram Kosuri, Nikolai Eroshenko, Emily M LeProust, Michael Super, Jeffrey Way, Jin Billy Li, George M Church. (2010). Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips. ''Nature Biotechnology'', 28(12):1295-9. [http://www.nature.com/nbt/journal/v28/n12/full/nbt.1716.html]
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=Clinical Applications of Zinc Fingers=
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'''2.''' Harris H. Wang, Farren J. Isaacs, Peter A. Carr, Zachary Z. Sun, George Xu, Craig R. Forest, George M. Church. Programming cells by multiplex genome engineering and accelerated evolution. (2009). ''Nature'', 460(7257):894-8. [http://www.nature.com/nature/journal/v460/n7257/full/nature08187.html]
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==Colorblindness ''(Red Opsin)''==
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'''3.''' Isaacs FJ, Carr PA, Wang HH, Lajoie MJ, Sterling B, Kraal L, Tolonen AC, Gianoulis TA, Goodman DB, Reppas NB, Emig CJ, Bang D, Hwang SJ, Jewett MC, Jacobson JM, Church GM. (2011). Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. ''Science'', 333(6040):348-53. [http://www.sciencemag.org/content/333/6040/348.full]
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===<u>Goal</u>: Produce functional red opsin photoreceptor proteins in the eye===
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===<u>Method</u>: Insertion of functional red opsin gene (''OPN1LW'' [http://genome.ucsc.edu/cgi-bin/hgGene?hgg_gene=uc004fjz.3&hgg_prot=P04000&hgg_chrom=chrX&hgg_start=153409724&hgg_end=153424505&hgg_type=knownGene&db=hg19&hgsid=206379197 <sup>1</sup>]) upstream of normal locus in patient lacking the gene===
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====Journal Articles====
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*http://www.nature.com/nature/journal/v461/n7265/abs/nature08401.html
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*http://www.nejm.org/doi/full/10.1056/NEJMc0903652
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====In the News====
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*http://www.nature.com/news/2009/090916/full/news.2009.921.html
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*http://www.scientificamerican.com/podcast/episode.cfm?id=gene-therapy-cures-colorblind-monke-09-09-16
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*http://www.msnbc.msn.com/id/32879284/ns/health-health_care/t/gene-therapy-fixes-color-blindness-monkeys/
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*http://www.wired.com/wiredscience/2009/09/colortherapy/
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<br />
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'''4.''' Yu, D., H. M. Ellis, et al. (2000). An efficient recombination system for chromosome engineering in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 97(11): 5978-5983.[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC165854/]
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----
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==Inherited High Cholesterol ''(Familial Hypercholesterolemia)''==
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'''5.''' Mosberg JA, Lajoie MJ, Church GM. Lambda red recombineering in Escherichia coli occurs through a fully single-stranded intermediate. ''Genetics'' 2010;186:791-799.[http://www.genetics.org/content/186/3/791]
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===<u>Goal</u>: Produce functional LDLR protein to remove LDL cholesterol from the blood===
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===<u>Method</u>: Insertion of functional ''LDLR'' gene upstream of nonfunctional allele===
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*http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001429/
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*http://www.genome.gov/25520184
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*http://emedicine.medscape.com/article/121298-overview#a0199
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<br />
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'''6.''' Jeffry D Sander, Elizabeth J Dahlborg, Mathew J Goodwin, Lindsay Cade, Feng Zhang, Daniel Cifuentes, Shaun J Curtin, Jessica S Blackburn, Stacey Thibodeau-Beganny, Yiping Qi, Christopher J Pierick, Ellen Hoffman, Morgan L Maeder, Cyd Khayter, Deepak Reyon, Drena Dobbs, David M Langenau, Robert M Stupar, Antonio J Giraldez, Daniel F Voytas, Randall T Peterson,Jing-Ruey J Yeh, J Keith Joung. Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA)(2011). ''Nature Methods'' 8, 67–69. [http://www.nature.com/nmeth/journal/v8/n1/full/nmeth.1542.html]
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==Cancer ''(''Myc'' Oncogene)''==
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'''7.''' Morgan L. Maeder, Stacey Thibodeau-Beganny, Anna Osiak, David A. Wright, Reshma M. Anthony, Magdalena Eichtinger, Tao Jiang, Jonathan E. Foley, Ronnie J. Winfrey, Jeffrey A. Townsend, Erica Unger-Wallace, Jeffry D. Sander, Felix Müller-Lerch, Fengli Fu, Joseph Pearlberg, Carl Göbel, Justin P. Dassie, Shondra M. Pruett-Miller, Matthew H. Porteus, Dennis C. Sgroi, A. John Iafrate, Drena Dobbs, Paul B. McCray Jr., Toni Cathomen, Daniel F. Voytas, J. Keith Joung. Rapid “Open-Source” Engineering of Customized Zinc-Finger Nucleases for Highly Efficient Gene Modification (2008). ''Molecular Cell'' Volume 31, Issue 2, 25 July 2008, Pages 294-301.[http://www.sciencedirect.com/science/article/pii/S1097276508004619]
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===<u>Goal</u>: Knock out the oncogenic protein product and stop cancerous proliferation===
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===<u>Method</u>: Targeted disruption (deletion) in mutated oncogene===
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*http://www.ncbi.nlm.nih.gov/gene/4609
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'''8.''' Anton Persikov, PhD. [http://www.princeton.edu/~persikov/publications.html]
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*http://omim.org/entry/190080
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</div>-->
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'''9.''' 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. [http://www.nature.com/nbt/journal/v23/n8/pdf/nbt1120.pdf]
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=Technological Applications=
 
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*The novel methods we employed in our project have the potential to '''revolutionize synthetic biology''' practices, and the way that future iGEM competitions are conducted. To learn more about the technological applications of our project, please see our [https://2011.igem.org/Team:Harvard/Technology '''Technology'''] page.
 
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Latest revision as of 02:53, 29 October 2011

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Overview | Design | Synthesize | Test | Zinc Finger Background | Protocols

Massively Multiplexed Zinc Finger Protein Engineering

Zinc finger proteins are specialized proteins that bind to DNA. Due to their ability to target highly specific DNA sequences, zinc finger proteins offer great potential for gene therapy and personalized medicine: recently, they were shown to be effective in conferring HIV resistance and treating hemophilia in mice. In the past, however, designing new zinc finger proteins - a necessity for individualized gene therapy - has been prohibitively expensive and time consuming. For more information about zinc fingers, see here. Our goal is to create zinc finger proteins that bind to DNA triplets that haven't been bound before: triplets for which no zinc finger protein currently exists.

For our project, team members created and tested thousands of zinc fingers at a cost feasible for most labs. To do so, we harnessed three novel synthetic biology technologies: chip-based synthesis[1], which allows for thousands (even millions) of DNA strands to be synthesized concurrently; multiplex automated genome engineering (MAGE)[2][3]; and lambda red recombineering [4][5], both of which make direct edits of the bacterial genome possible and can replace the used of small, cumbersome plasmids.

See our results page: using these methods, we found up to 15 novel zinc fingers, which are still being characterized.

See here for our official abstract, and our acknowledgments page.

To achieve this result, our project had three main steps:

1. Design

Use a bioinformatics approach to predict 55,000 zinc finger sequences.

As the structure and binding interactions of zinc fingers are not yet understood, our project utilized bioinformatics and computational analysis of the limited existing data to make “educated guesses” of what amino acid sequences will bind to our desired target sequence.

Our generated sequences to bind the DNA triplet TGG.

Team members chose 6 target sequences in the human genome (finding these sequences using a binding site finder tool written by a team member) that no currently-existing zinc finger is able to bind to. The sequences were selected from genes that cause colorblindness, some types of cancer, and high cholesterol. These targets were chosen after conducting an extensive literature search, with the most useful information coming from a previous zinc finger study called CODA[6].

Team members emailed the OPEN consortium[7] and Dr. Anton Persikov[8] to acquire their respective databases of zinc fingers: Persikov had compiled the results of the past 20 years of zinc finger research (everything before OPEN), and OPEN had found many more since then. In all, around 1,700 unique DNA triplet/zinc finger helix pairs were discovered. Team members then decided how to analyze this data for the best chance of binding our target sequences, and programmed their ideas into a [http://sourceforge.net/projects/harvardigem/ Python program] to generate 55,000 potential zinc fingers (55,000 is the number of sequences that can be built using chip-based DNA synthesis).

For a complete overview of how our zinc fingers were designed, see Design.

2. Synthesize

Use chip-based DNA synthesis to make these 55,000 sequences simultaneously, then insert the oligos into E.coli.

Once our team generated these 55,000 sequences, we needed a way to bring them from a computer's memory into the real world. Using chip synthesis of DNA (generously contributed by Agilent Technologies, a sponsor of iGEM, in partnership with our mentors in the Church Lab), all of these sequences were built simultaneously, then sent to us in a single 50µL tube.

For more information about how the chip was created, see the original 2010 paper by Kosuri et al[1]. Cost of chip synthesis is around $.00091 per DNA base, compared to ~$0.40–1.00 per base with current commercial pricing[1]. Thus, chip synthesized DNA has the potential to be up to a factor of 1,000 times cheaper than current mainstream methods of DNA synthesis.

Chip synthesis results in a pool of single-stranded oligos, designed with primer tags, which allow for the amplification of specific sub-pools: in our team's case, we used 6 sub-pools, one for each of our target sequences.

For a complete overview of how our zinc finger DNA went from chip to being expressed in E.coli, see Synthesize.

3. Test

Use a genomic metabolic selection system to test which zinc finger sequences successfully bind DNA.

Our metabolic selection system. The cell’s ability to synthesize histidine is eliminated: however, homologous gene His3 has been placed on the genome, with transcription regulated by zinc finger arrays binding upstream. The cells undergo selection in minimal media. Thus, without a zinc finger binding event, the cell is unable to synthesize histidine and dies.

After creating and expressing 55,000 novel zinc finger sequences, our team needed to determine which ones effectively bind to their respective target sites. By tying zinc finger binding to cell survival (using an efficient selection system) all cells without successful binders would die, and thus living colonies indicate a viable zinc finger.

Team members constructed a one-hybrid selection system based off a metabolic system designed by Meng et al[9] which tied zinc finger binding to histidine production. When grown in media without histidine, the cells can only survive if a zinc finger-omega subunit of RNA polymerase (also knocked out in the strain) fusion protein binds successfully and initiates creation of histidine.

Where our team's selection departs from the one described by Meng and others is its use of a genome-based rather than plasmid-based system. Not only did team members knock out HisB, PyrF, and rpoZ ourselves using the newly developed techniques of MAGE and lambda red, we also inserted the zinc finger binding site construct directly into the genome instead of expressing it in the cell on a vector.

For a complete overview of how our selection system was designed, tested, and optimized, see Test.




Results

See our results page for a summary of our most significant findings.

By submitting the necessary strains and parts to the registry, and publishing these easy-to-follow protocols along with our source code, it is our hope that future iGEM teams will also use these techniques in their own synthetic biology projects.

Technological Applications

Our zinc fingers and their clinical applications are a new technology that maximize efficiency and decrease cost. The novel methods we employed in our project have the potential to revolutionize synthetic biology practices, and the way that future iGEM competitions are conducted. To learn more about the technological applications of our project, please see our Technology page.

References

1. Sriram Kosuri, Nikolai Eroshenko, Emily M LeProust, Michael Super, Jeffrey Way, Jin Billy Li, George M Church. (2010). Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips. Nature Biotechnology, 28(12):1295-9. [http://www.nature.com/nbt/journal/v28/n12/full/nbt.1716.html]

2. Harris H. Wang, Farren J. Isaacs, Peter A. Carr, Zachary Z. Sun, George Xu, Craig R. Forest, George M. Church. Programming cells by multiplex genome engineering and accelerated evolution. (2009). Nature, 460(7257):894-8. [http://www.nature.com/nature/journal/v460/n7257/full/nature08187.html]

3. Isaacs FJ, Carr PA, Wang HH, Lajoie MJ, Sterling B, Kraal L, Tolonen AC, Gianoulis TA, Goodman DB, Reppas NB, Emig CJ, Bang D, Hwang SJ, Jewett MC, Jacobson JM, Church GM. (2011). Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science, 333(6040):348-53. [http://www.sciencemag.org/content/333/6040/348.full]

4. Yu, D., H. M. Ellis, et al. (2000). An efficient recombination system for chromosome engineering in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 97(11): 5978-5983.[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC165854/]

5. Mosberg JA, Lajoie MJ, Church GM. Lambda red recombineering in Escherichia coli occurs through a fully single-stranded intermediate. Genetics 2010;186:791-799.[http://www.genetics.org/content/186/3/791]

6. Jeffry D Sander, Elizabeth J Dahlborg, Mathew J Goodwin, Lindsay Cade, Feng Zhang, Daniel Cifuentes, Shaun J Curtin, Jessica S Blackburn, Stacey Thibodeau-Beganny, Yiping Qi, Christopher J Pierick, Ellen Hoffman, Morgan L Maeder, Cyd Khayter, Deepak Reyon, Drena Dobbs, David M Langenau, Robert M Stupar, Antonio J Giraldez, Daniel F Voytas, Randall T Peterson,Jing-Ruey J Yeh, J Keith Joung. Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA)(2011). Nature Methods 8, 67–69. [http://www.nature.com/nmeth/journal/v8/n1/full/nmeth.1542.html]

7. Morgan L. Maeder, Stacey Thibodeau-Beganny, Anna Osiak, David A. Wright, Reshma M. Anthony, Magdalena Eichtinger, Tao Jiang, Jonathan E. Foley, Ronnie J. Winfrey, Jeffrey A. Townsend, Erica Unger-Wallace, Jeffry D. Sander, Felix Müller-Lerch, Fengli Fu, Joseph Pearlberg, Carl Göbel, Justin P. Dassie, Shondra M. Pruett-Miller, Matthew H. Porteus, Dennis C. Sgroi, A. John Iafrate, Drena Dobbs, Paul B. McCray Jr., Toni Cathomen, Daniel F. Voytas, J. Keith Joung. Rapid “Open-Source” Engineering of Customized Zinc-Finger Nucleases for Highly Efficient Gene Modification (2008). Molecular Cell Volume 31, Issue 2, 25 July 2008, Pages 294-301.[http://www.sciencedirect.com/science/article/pii/S1097276508004619]

8. Anton Persikov, PhD. [http://www.princeton.edu/~persikov/publications.html]

9. 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. [http://www.nature.com/nbt/journal/v23/n8/pdf/nbt1120.pdf]

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