Experiments
Project 
EXPERIMENTS
DNA EXTRACTION AND POLYMERASE CHAIN REACTION (PCR)
We obtained early stage OPC DNA by first freezing the samples in liquid nitrogen and grinding them followed by genomic DNA extraction using the HotSHOT protocol which leaves the samples ready to be added to the PCR-reaction. The concentration of our extracts was determined using a Nanodrop Spectrophotometer.
Primers were designed for the genes of interest in a way that the resulting amplicons contain the regions of the genes we are trying to target with our siRNAs.
This enables us identify if the target gene regions are indeed present by sequencing the amplicons.
PCR was performed using Invitrogen™ Platinum™ SuperFi™ PCR Master Mix or Phusion® High-Fidelity PCR Master Mix.
GEL ELECTROPHORESIS
Gel electrophoresis was used to separate and visualize our DNA fragments (obtained from the PCR) based on their size to see the success of amplification, as a first proof of the presence of the target fragments. In general, gel electrophoresis is conducted by injecting DNA sample of interest into wells and then using an electric current to pull the DNA through the gel. DNA fragments have a negative charge hence they will always be pulled towards the positive end of the electric current. The fragments are separated on the basis of their length and are compared to a reference (ladder).
The gel electrophoresis protocols used were taken from the creators of the product; Invitrogen™ TrackIt™ 1 Kb Plus Ladder (Pub. No. MAN0000843).
SEQUENCING
The OPC target genes amplified by PCR, were isolated and further amplified in a second PCR round in order to obtain a sufficient fragment concentration to be sequenced by Macrogen. This is necessary to ensure that the target sequences are exactly the same as the ones that were found on databases. Only this way, we can ensure that the siRNAs can specifically and efficiently bind to their selected target.
PLASMID ASSEMBLY
GIBSON ASSEMBLY
L4440 plasmids were isolated with the QIAprep® Spin Miniprep Kit from Qiagen. Afterwards, L4440 was linearized with PciI and NgoMIV in CutSmart® Buffer or NEBuffer™. The linearized plasmid was isolated from an agarose gel using QIAprep® Spin Miniprep Kit. Following this step, the expression cassette gBlock was added to the linearized plasmid in a 2:1 ratio and assembled using NEBuilder HiFi DNA Assembly Reaction Protocol. The resulting vector was transfected into high copy number DH5α cells. pUC19 plasmid was transfected to the cells as a positive control. Cells were grown to an exponential phase and plated onto ampicillin containing selection plates and incubated overnight. Finally, single colonies were picked and grown in overnight cultures.
PLACING THE INSERTS
The shRNA coding DNA-sequences were ordered as ssDNA oligonucleotides from IDT. The single strands were designed to form EcoRI and BamHI overhangs complementary to the ones created in L4440 by digest. The oligos were annealed in IDT oligonucleotide annealing buffer at 94°C for 3 min and then slowly cooled down to room temperature in order to ensure annealing despite potential secondary structures formed by the hairpin coding DNA.
CREATING THE FINAL STRAIN
The L4440 plasmids containing the expression cassette were isolated with the QIAprep® Spin Miniprep Kit from Qiagen. The plasmids were digested with the restriction enzymes EcoRI and BamHI in CutSmart® Buffer. Ligation protocol was used to insert the shRNA coding DNA-sequences into the plasmid. First, the plasmids were transfected into high copy number DH5α cells and spread on ampicillin containing selection plates. Afterwards, from successfully grown colonies, liquid cultures were made and the plasmids with the final construct were isolated. These plasmids were transfected into the final HT115(DE3) strain and spread on ampicillin+tetracycline containing selection plates.
INDUCTION AND DETECTION OF shRNAs
shRNA production was induced by addition of IPTG (0.4mM) to induce the expression of our shRNAs.
This procedure was followed by a total RNA extraction of the cells.
HATCHING CATERPILLARS
To properly test the function of the shRNAs they must be tested on the target. In this case that would require OPCs that are in their earliest stages, which is when the pesticides would be sprayed on the leaves and subsequently kill them. For this we required live, young caterpillars that we could observe consume the shRNAs in our laboratory.
The hatching protocol consisted of very few steps to properly maintain the climate conditions required for natural hatching of the eggs. The main device used was a climate chamber; in this machine we were able to mimic the conditions required for natural hatching of the eggds, such as temperature, humidity, and hours of light/dark exposure. We found that the optimal temperature was 18°C with approximately 12 hours of light in a day. The humidity was not a factor in the OPC egg hatching hence it was kept at room humidity.
In further testing, we are planning to hatch additional caterpillars and equip the climate control chamber with oak branches sprayed with OakShield to test the influence of our pesticide on the caterpillars when ingested.
DESIGN
ENGINEERING
LAB BOOK
