Gram Positive Build

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Kunkels

Grow

Harvest

Mutate DNA

OUTLINE

Order Olglionucleotides with the desired mutation

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.

Create ssDNA of wild type gene

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.

Allow Olglios to annele to the ssDNA

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.

Use polymerase and DNTPs to synthesize the rest of the plasmid

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.

Grow Protein

Overview

Transform E. coli with the mutant plasmid

E. coli is transformed with our mutant plasmid.

Grow and OD until desired OD is reached

We grow our inoculated E. coli in terrific broth and measure optical density until we reach the desired optical density.

Induce bacteria to produce our protein

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.

Image of lac operon, lac inhibitor, capD gene

Grow for 24 hours

We allow the cells 24 hours to grow our protein.

Harvest Protein

Overview

Spin down cells

Using a centrifuge, the cells and the growth media are spun to separate the cells from the growth media.

Lyse cells

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.

Purify cells

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.

Image of how it bonds to the beads