Team:Washington/Gram Positive/Build

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=Build (Gram Positive)=
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=Building Mutant CapD_CP=
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To build the mutant proteins, we follow the path of the central dogma. First, we created DNA that contains our mutations. Second, we induced our transformed cells containing the desired DNA to express the mutant proteins. Lastly, we harvested the proteins by lysing open the cells and filtering out non-desired cell components.
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==Mutate DNA==
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[[Image:Washington_Kunkel_summary.jpg|center|thumb|800px|A brief summary of the Kunkel mutagenesis protocol: Generate ssDNA, annealing and synthesizing the rest of the plasmid]]
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==='''Order Oligonucleotides'''===
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To mutate our wild-type gene, we used the [https://2010.igem.org/Team:Washington/Protocols/Kunkel Kunkel mutagenesis protocol]. Kunkel’s is a site-directed mutagenesis, requiring knowledge of wild-type sequences. After the desired mutation is modeled using [http://fold.it/ FoldIt], we order a mutation's oligonucleotides from [http://www.idtdna.com/ Integrated DNA Technologies]. Oligonucleotides are short segments of nucleotide primers which contain one or more mutations and will anneal to single-stranded DNA (ssDNA) of a plasmid containing the CapD expression gene. The result should be a double stranded plasmid which holds the [http://fold.it/ FoldIt]-designed mutation.
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==='''Generate ssDNA'''===
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In order to anneal oligonucleotides, CapD expression gene ssDNA must be obtained by transforming CJ236 cells with a plasmid containing the CapD gene. These cells use Uracil instead of Thymine, and are unable to destroy Uracil containing DNA. Colonies are then picked and M13K07 helper phage is introduced. The phage will use the cells to reproduce and copy the plasmid containing the CapD expression gene to daughter phages. The phage will produce one strand of DNA using reverse transcriptase, creating ssDNA. We use the Miniprep protocol to harvest the ssDNA from the phage.
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==='''Annealing to ssDNA'''===
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Received oligonucleotides are inactive, lacking phosphates which induce activity. Adding phosphates by kinasing readies them for annealing to ssDNA. The oligonucleotide binds to a specified location on the ssDNA except where the mutation is. This creates an area where the oligonucleotide will not bond to the template ssDNA.
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[[Image:Washington_Kunkel_pt_2.jpg|center|thumb|800px|After being transformed, the cell will replace the uracil with thymine. In the process, the complete mutant plasmid is generated.]]
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==='''Synthesize the Plasmid'''===
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Using DNA polymerase, the rest of the missing strand is synthesized. Finally the area in the plasmid where the mutation is fixed because the former ssDNA strand that contains uracil is destroyed by the cell we transform our mutant plasmid into. Since the cell uses the other strand (the strand with the Oligonucleotides) to fix the DNA, the result is a complete mutant plasmid. Plasmids are then sent for sequencing by [http://www.genewiz.com/ GENEWIZ] to confirm mutations.
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=='''Grow Protein'''==
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[[Image:Washington_Image_not_found.jpg|thumbnail|200px|Image of lac operon, lac inhibitor, capD gene]]
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==='''Transform E. coli with mutant plasmid'''===
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E. coli is transformed with our mutant plasmid. This completes mutant plasmid because these cells are able to remove uracil and replace it with thymine based on the complementary strand (which contains the mutation).
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==='''Grow cells'''===
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Inoculated E. coli is grown in terrific broth (TB) until 600nm optical density reaches desired range.
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==='''Protein Production'''===
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By introducing Isopropyl β-D-1-thiogalactopyranoside (IPTG), an allolactose mimic, we induce E. Coli to produce our protein. IPTG binds with the lac inhibitor protein and activates the lac operon, turning on the CapD gene and causing production of our mutant protein.
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==Harvest Protein==
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[[Image:Washington_lyse.png|thumbnail|500px|The lysis process]]
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==='''Spin down cells'''===
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Using a centrifuge, cells and media are spun to separate cells from media.
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==='''Lyse cells'''===
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The supernatant (media) is emptied and the cells at the bottom are lysed open. The result is a slurry containing all the cell’s proteins and DNA. Lysis is then spun down and the supernatant, containing all the proteins, is collected. Among the proteins is CapD.
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==='''Purify proteins'''===
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To purify the protein, we run the supernatant collected from lysis through a column containg TALON resin cobalt beads. CapD's designed histidine tags bind to the beads, whilst everything else flows through. Finally, the CapD is eluted with imidazole, a histidine without a backbone, which outcompetes the affinity to bind. The result is our purified mutant CapD.
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==Generating Mutant DNA==
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[[Image:Washington_Kunkel_summary_revised2.jpg|center|thumb| 760px| Kunkel Mutagenesis Protocol: Generate single stranded dU-DNA, Anneal primers, polymerization, Synthesize Mutant Plasmids and replace uracil with thymine to complete]]
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After we came up with the desired mutant protein designs, we employed the [[Team:Washington/Protocols/KunkelCapD|Kunkel Mutagenesis]] method to generate the desired mutant DNA. Kunkel mutagenesis is a three step process. First, we obtained ssDNA of wild-type CapD_CP gene from transformed cells that contain the CapD_CP gene and lack the enzyme to destroy uracil. The presence of uracil is used later to obtain the correct DNA strand. The second step involved annealing our mutation-containing primers to the ssDNA and polymerizing the strand that contains our desired mutations. The result is a double-stranded DNA, consisting of a wild-type CapD_CP strand and a mutations-containing (desired) strand. Lastly, to obtain a dsDNA that consists of only the desired strands, we transformed it into another type of cell that contains enzymes to destroy the uracil-containing strand. Once the uracil-containing wild-type strand is destroyed, the complementary strand is synthesized, resulting in a dsDNA that contains only our desired mutations.[[#References | [1]]]
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==Protein Expression and Purification==
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[[Image:Washington_lyse_revised4.png|thumbnail|700px|left|Protein Purification Process]]
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Once we obtained cells transformed with our desired mutant DNA, we induced the cells to express the mutant protein. By introducing Isopropyl β-D-1-thiogalactopyranoside (IPTG), an allolactose mimic, we induce E. Coli to produce our protein. IPTG binds with the lac inhibitor protein and activates the lac operon, turning on the CapD_CP gene and causing production of our mutant protein. For this step, we used two different protocols: [https://2010.igem.org/Team:Washington/Protocols/50mLPurificationCapD small scale] and [https://2010.igem.org/Team:Washington/Protocols/1LPurificationCapD large scale]. The concepts described below are the same for both protocols. To harvest our mutant proteins, we needed to first lyse open the induced cells and then purified out our proteins (refer to small/large scale protocol). For the purification procedure, we employed Talon beads. CapD_CP's designed histidine tags bind to the beads, whilst everything else flows through. The result of this process is our purified mutated CapD_CP proteins.
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==References==
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1. T.A. KUNKEL, P NATL ACAD SCI USA 82, 488 (JANUARY 1985, 1985)
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'''&larr; [[Team:Washington/Project/Baker/Design|Designing the Gram(+) Therapeutic]]'''
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'''&larr; [[Team:Washington/Gram Positive/Design|Designing the Gram(+) Therapeutic]]'''
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'''[[Team:Washington/Project/Baker/Test|Testing the Gram(+) Therapeutic]] &rarr;'''
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'''[[Team:Washington/Gram Positive/Test|Testing the Gram(+) Therapeutic]] &rarr;'''
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Latest revision as of 20:37, 27 October 2010

Building Mutant CapD_CP

To build the mutant proteins, we follow the path of the central dogma. First, we created DNA that contains our mutations. Second, we induced our transformed cells containing the desired DNA to express the mutant proteins. Lastly, we harvested the proteins by lysing open the cells and filtering out non-desired cell components.

Generating Mutant DNA

Kunkel Mutagenesis Protocol: Generate single stranded dU-DNA, Anneal primers, polymerization, Synthesize Mutant Plasmids and replace uracil with thymine to complete

After we came up with the desired mutant protein designs, we employed the Kunkel Mutagenesis method to generate the desired mutant DNA. Kunkel mutagenesis is a three step process. First, we obtained ssDNA of wild-type CapD_CP gene from transformed cells that contain the CapD_CP gene and lack the enzyme to destroy uracil. The presence of uracil is used later to obtain the correct DNA strand. The second step involved annealing our mutation-containing primers to the ssDNA and polymerizing the strand that contains our desired mutations. The result is a double-stranded DNA, consisting of a wild-type CapD_CP strand and a mutations-containing (desired) strand. Lastly, to obtain a dsDNA that consists of only the desired strands, we transformed it into another type of cell that contains enzymes to destroy the uracil-containing strand. Once the uracil-containing wild-type strand is destroyed, the complementary strand is synthesized, resulting in a dsDNA that contains only our desired mutations. [1]

Protein Expression and Purification

Protein Purification Process


















Once we obtained cells transformed with our desired mutant DNA, we induced the cells to express the mutant protein. By introducing Isopropyl β-D-1-thiogalactopyranoside (IPTG), an allolactose mimic, we induce E. Coli to produce our protein. IPTG binds with the lac inhibitor protein and activates the lac operon, turning on the CapD_CP gene and causing production of our mutant protein. For this step, we used two different protocols: small scale and large scale. The concepts described below are the same for both protocols. To harvest our mutant proteins, we needed to first lyse open the induced cells and then purified out our proteins (refer to small/large scale protocol). For the purification procedure, we employed Talon beads. CapD_CP's designed histidine tags bind to the beads, whilst everything else flows through. The result of this process is our purified mutated CapD_CP proteins.

References

1. T.A. KUNKEL, P NATL ACAD SCI USA 82, 488 (JANUARY 1985, 1985)

← Designing the Gram(+) Therapeutic       Testing the Gram(+) Therapeutic →

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