Experiments

Introduction

The research we conducted involved assembling an adeno-associated virus phage (AAVP) plasmid that would generate phage particles. The following experiments were conducted in our efforts to construct our fdGPS2.1(RGD4C)-Amp plasmid, which include experiments that amplify DNA sequences, propagate viral progeny, and analyze our data.

Construction of the plasmid

PCR - To generate DNA sequence needed for our plasmid construction, we amplified two different segments within the f3-55nm plasmid. Taq DNA polymerase (link), nucleotide bases (link), and specially crafted primers generated at IDT were used to amplify our samples as they underwent several heat cycles. The heat cycles generated millions of copies of both fragments we targeted, allowing us to analyze and then isolate the copies.

Figure 1: Fact sheet about Polymerase Chain Reaction (PCR) (image acquired from National Human Genome Research Institute)

Gel Electrophoresis - After generating our PCR products, we used gel electrophoresis to separate the DNA fragments over the length of an agarose gel, allowing us to assess the efficiency of amplification within the DNA segments. The PCR samples were mixed with purple loading dye and then loaded into a solidified agarose gel before passing a current through it. Once completed, the gel is soaked in a solution of 1x SYBR green DNA staining solution (S7563), and then visualized using either a UV transilluminator or Chemidoc imaging tool. Given that both fragments were different sizes, we could easily visualize them with respect to each other and to the 1 kb DNA ladder (N3232S).

Golden Gate Assembly - Once we generated our PCR samples and ordered our specially crafted sequences from IDT, we performed a Golden Gate assembly to ligate all sequences into one plasmid. was used to ligate several DNA sequences together to generate a plasmid. By using the Type IIS restriction enzymes BbsI-HF (R3539S), we generated cuts in several DNA sequences at the same time within one reaction tube. After this occurs, T4 DNA ligase (Mo202L) re-combines these sequences together during an incubation step to assemble the plasmid product.

Bacterial Transformation - As an initial test of the plasmid’s function, we transformed it into NEB 10-Beta competent cells (C3019I). This involved adding the bacterial cells and the newly constructed plasmid to the same tube and then heat shocking them in a water bath. After this step, the bacteria were left to incubate overnight on LB agar plates containing ampicillin to allow only cells with a viable plasmid to grow. To test the efficiency of transformation and growth of cells, we used varying concentrations of ampicillin to determine the lowest level of antibiotic required to isolate cells with the plasmid.

Figure 2: Workflow of a Bacterial Transformation (image acquired from Khan Academy)

Miniprep - After propagating our plasmid, we planned to isolate it using a Monarch miniprep kit (T1010S). This would involve spinning down an inoculum containing bacterial cells with the plasmid, forming a pellet. Isolating the plasmid from this pellet would involve several wash steps, ultimately generating a solution containing the plasmid suspended in DNA elution buffer (T1016L). This solution would then undergo analysis using a nanoquant tool (plate reader) to determine its purity (260/280 ratio) and its concentration.

Restriction Digest - Following the miniprep, we would run a restriction digest using known restriction sites found in our plasmid. If our plasmid formed successfully, the digest would generate two fragments that could be visualized during gel electrophoresis. To do this, we would run the sample using SacII (R0157S) and KpnI (R3142S) restriction enzymes to generate fragments 2685 base pairs and 4387 base pairs long. This would be accomplished by incubating the samples in a water bath to activate the restriction enzymes, followed by incubation at a higher temperature to deactivate them to prevent further digestion of DNA sequences.

Ancillary Experiments

Inoculation - To ensure we isolated the plasmid of interest from our transformation experiment, we extracted a single colony from a petri dish containing the transformed bacteria and added it to an LB broth solution containing antibiotics. The sample was then incubated overnight in a shaking incubator to generate a higher density of bacteria containing the plasmid.

DNA/PCR Cleanup - After observing the presence of our PCR products on agarose gel, we purified the samples using a Monarch DNA/PCR Cleanup Kit (T1030S). This involved filtering the DNA samples using several wash steps, removing any impurities such as excess nucleotides, primers, and DNA polymerase. The sample is then analyzed using the nanoquant tool to determine its purity and concentration beforing using it in future experiments.

Gel Extraction - During the initial stages of our experiments used to isolate DNA fragments that were separated on agarose gel. Using either a razor blade or scalpel to cut the gel slice out, the sample was then dissolved using a Monarch Gel Extraction Kit (T1020S) purchased from NEB and then washed several times to produce a purified DNA sample.

Protocols used during Experimentation

PCR

Gel Electrophoresis

Golden Gate assembly

Bacterial Transformation

Miniprep

Restriction Digest