Experimental Methods

Protocol for Experiments

Summary of the Experiments

Synthesizing the Polymerases

Polymerase genes were synthesized and cloned into pET28a(+) by Twist Biosciences, with a His-Tag added to the C terminus. The proteins were synthesized using BL21(DE3) bacteria, with induction using IPTG. Proteins were purified using Ni-NTA spin columns. Polymerases were tested for activity in PCR.

Testing Polymerase Accuracy

A variant of the pUC19 plasmid is designed to have a gap in the coding or non-coding lacZα gene. Each polymerase is used to fill the gap, and the plasmid is transformed into NEB10 E. coli. Blue colonies represent plasmids that have been filled without errors, while white colonies represent plasmids that have been filled with errors.

Getting the Plasmid for Accuracy Testing

To get the plasmid for testing polymerase accuracy, several steps were done. The following assay is based on the paper “Plasmid-based lacZα assay for DNA polymerase fidelity: application to archaeal family-B DNA polymerase” (Jozwiakowski SK, Connolly BA):

Step 1: Site Directed Mutagenesis of pUC19
- To create the gap required for DNA polymerase to fill, pUC19 underwent PCR-based site directed mutagenesis to create nicking sites at the start and end of the gene
- After the first PCR, plasmid was transformed into NEB10 bacteria and mini-prepped to obtain enough sample for the second round of PCR
- The new plasmid is now called PSJ1
Step 2: Nicking PSJ1
- To create the gap, the enzymes NtBpu10I and NbBpu10I were used
- NtBpu10I creates nicks in the coding strand and NbBpu10I creates gaps in the non-coding strand
- However, the nicks don’t separate the nicked segment from the backbone, as the bases remain hydrogen bonded
Step 3: Creating and Using the Competitor DNA
- PCR is used to create copies of the lacZα gene, with one set introducing a terminal phosphate on the coding strand and another set introducing it on the non-coding strand
- Lambda exonuclease is used to degrade the phosphorylated strand, which leaves a single stranded strand of the lacZα gene
- For the PSJ1 nicked by NtBpu10I, the non-coding strand is added during thermocycling so that when the DNA strands separate due to heat, the non-coding strand binds to the nicked coding strand. This leads to their separation from the main backbone, leaving a gapped plasmid
- The opposite is done for the PSJ1 nicked by NbBpu10I
Step 4: Isolating the gapped plasmid
- To get rid of any plasmid which may not have a gap, the enzyme PstI is added to the samples. This enzyme selectively linearizes plasmids which don’t have a gap
- The enzyme Exonuclease V is added to degrade any DNA which isn’t in plasmid form, so all linearized DNA is degraded along with any other forms of linear DNA
- Gapped plasmid is purified using a PCR purification kit

DNA Polymerase Project Presentation.png

Expressing and Purifying Polymerases

To actually get the proteins in the lab, I had to express and purify them. Here was the process behind doing that:

Step	Explanation
Order sequences from Twist	Once I had the five polymerases I wanted to test, I ordered clonal genes for them through Twist Biosciences. The polymerase genes were cloned into the MCS of pET28(+), producing a protein with a 6x His-tag at the C terminus as the product
Transform plasmid into bacteria	Transform plasmid containing polymerase gene into BL21(DE3) E. coli and plate on selection plate containing kanamycin
Create a 3mL culture	Pick a colony and inoculate into 3mL LB media with 50µg/µl kanamycin
Create a 200mL culture	Add 2mL of overnight culture and add to 200mL of LB media with 50µg/µl kanamycin
Induce protein expression	Add IPTG to 0.1mM final conc. to the culture after 2.5 hours of incubation at 37°C
Collect and Lyse Cells	Centrifuge cells to harvest pellets and add 4mL of B-PER per gram of cells
Collect Supernatant	Centrifuge samples and collect soluble protein in supernatant while leaving cell debris
Purify Polymerases	Use His-Pur Spin Columns to purify proteins, with first elution fraction being used for further analysis

Testing for Activity

To test if the polymerases (if they expressed properly) have activity, throw them into a PCR reaction to see the results. The reactions were 50µl and contained: