Team:Washington/Gram Positive/Build

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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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==Generating Mutant DNA==
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==GENERAL==
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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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Formatting
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==SECTION BASED==
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===Kunkels===
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===Grow===
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===Harvest===
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=Mutate DNA=
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[[Image:Washington_Image_not_found.jpg]]
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Image of kunkels overview, all steps in image format
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OUTLINE
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Order Olglionucleotides with the desired mutation
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To mutate our wild-type gene, we will be using the Kunkel’s mutagenesis protocol. Kunkel’s is a site directed mutagenesis, requiring the wild type sequence of the desired gene to be known. After the desired mutation is modeled using Foldit, we order oligonucleotides of that mutation from IDT. Oligonucleotides are short segments of nucleotide primers that contain our mutation and will anneal to ssDNA of a plasmid contiang the CapD gene. The result should be a double stranded plasmid that has the mutation we designed in Foldit.
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Create ssDNA of wild type gene
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In order to anneal the oligonucleotides containing the desired mutations, we must have ssDNA of the CapD gene. To do this, we transform CJ236 cells with a plasmid containing the CapD gene. The colonies are then picked and M13K07 helper phage is introduced. The phage will use the cells to reproduce and in the process, copy the plasmid containing the CapD gene into daughter phages. This is allowed to happen overnight. The phage will reproduce one strand of DNA using reverse transcriptase, creating the desired ssDNA. We use the miniprep protocol to harvest the ssDNA from the phage.
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Allow Olglios to annele to the ssDNA
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The oligonucleotides we receive are inactive, lacking phosphates that allow [something to happen]. We add phosphates by kinaseing the oligonucleotides. The oligonucleotide is ready to anneal to the ssDNA. The oligonucleotide will bind to the specified location on the ssDNA with a nick where the mutation is.
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Use polymerase and DNTPs to synthesize the rest of the gene
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Using DNA polymerase, the rest of the missing strand is synthesized. Finally the nick in the plasmid where the mutation is fixed by replacing the nucleotides on the ssDNA strand of the plasmid, completing the mutant plasmid. We send in the plasmid for sequencing to verify that it the mutagenesis worked.
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=Grow Protein=
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[[Image:Washington_Image_not_found.jpg]]
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Overview
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Transform E. coli with the mutant plasmid
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E. coli is transformed with our mutant plasmid.
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Grow and OD until desired OD is reached
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We grow our inoculated E. coli in terrific broth and measure optical density until we reach the desired optical density.
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Induce bacteria to produce our protein
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To induce the E. coli to produce our protein, we introduce Isopropyl β-D-1-thiogalactopyranoside (IPTG) which mimics allolactose. IPTG binds with the lac inhibitor protein and activates the lac operon on the plasmid. This turns on the CapD gene and the E. coli starts producing our mutant protein.
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Image of lac operon, lac inhibitor, capD gene
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[[Image:Washington_Image_not_found.jpg]]
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Grow for 24 hours
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We allow the cells 24 hours to grow our protein.
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=Harvest Protein=
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[[Image:Washington_Image_not_found.jpg]]
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Overview
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Spin down cells
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Using a centrifuge, the cells and the growth media are spun to separate the cells from the growth media.
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Lyse cells
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The supernatant (media) is emptied out and the cells at the bottom are lysed open. The result is a liquid containing all of the cell’s proteins and its DNA. This lysis is then spun down and the supernatant is collect, this contains all of the cell’s protein. Among the collection of proteins is CapD.
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Purify cells
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To purify the protein, we run the liquid collected from lysis through a column containg TALON resin Nickel beads. The proteins are able to bind to the beads, while everything else drips through. CapD is marked with a His tag so it is able to specifically bond to the nickel beads. Since the other proteins are unable to do this, they are washed out. Finally, the CapD is eluted out by imidazole, a histadine without the backbone, which outcompetes the CapD. The result is our mutant CapD ready to be tested.
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[[Image:Washington_Image_not_found.jpg]]
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Image of how it bonds to the beads
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concentrate as needed
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<!---------------------------------------PAGE CONTENT GOES ABOVE THIS---------------------------------------->
<!---------------------------------------PAGE CONTENT GOES ABOVE THIS---------------------------------------->
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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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{{Template:Team:Washington/Templates/Footer}}
{{Template:Team:Washington/Templates/Footer}}

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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