KCNJ3_pGHE

Methods

Beginning this process, a plasmid map of our target plasmid, KCNJ3 in the pGHE backbone, was created using the TAAR1_pGHE plasmid (vector, ampicillin resistant) and the KCNJ3_pANT7 (insert), purchased from the Arizona State Plasmid Repository (DNASU). The desired action is for the coding sequence of KCNJ3 to replace the TAAR1 protein coding sequence in the vector plasmid using Polymerase Chain Reaction (PCR) and High Fidelity (HiFi) DNA assembly (Figure 1).

Figure 1:

(Top); TAAR1_pGHE, vector plasmid. This is the source of the pGHE vector. (Bottom); KCNJ3_pGHE, target plasmid. This plasmid is the target goal for this mutagenesis project. From this mRNA can be transcribed for expression in an oocyte system.

Subcloning primers for PCR were designed from this plasmid map using the New England Bio-labs (NEBuilder) primer tool, four total. A KCNJ3 forward HiFi primer (attccccgggATGTCTGCACTCCGAAGG), 28 nucleotides (nt) in length and a KCNJ3 reverse HiFi primer (gacccttctaTGTGAAGCGATCAGAGTTC) of 29nt in length, this will result in amplification of the KCNJ3 protein coding sequence for pGHE. For amplification of the pGHE backbone for KCNJ3, a KCNJ3_pGHE reverse primer (gtgcagacatCCCGGGGAATTGATCTGC) 28nt in length, and a KCNJ3_pGHE forward primer (tcgcttcacaTAGAAGGGTCAAGACAATTCTG) 32nt in length. These primers are arranged for a ~10-15bp overlap between the vector and insert sequences and will overlap in pairs like so: KCNJ3 forward/OPRM1_pGHE reverse, and KCNJ3 reverse/KCNJ3_pGHE forward (Figure 2). Protocol to help with this found here.

Figure 2:

(Top); The overlap between KCNJ3_FWD and pGHE_REV primers near the beginning (3') of the KCNJ3 protein coding sequence in pGHE (target plasmid). (Bottom); The overlap between pGHE_FWD and OPMR1_REV primers near the end (5') of the KCNJ3 protein coding sequence in pGHE (target plasmid).

Insert and vector sequences were amplified through overlapping (sticky ended) PCR using the Q5 mutagenesis kit from NEB.Two PCR reactions were carried out, each using one set of previously mentioned primers (KCNJ3 forward and KCNJ3_pGHE reverse/ KCNJ3 reverse and KCNJ3_pGHE forward). These reaction mixtures were mixed according to the Q5 kit (Figure 3), and run under the same cycling conditions in the same thermo-cycler (Figure 4). The first reaction mixture amplified the pGHE vector and will contained the source of pGHE vector (TAAR1_pGHE) and the primers of this vector that are specific for KCNJ3, designed as mentioned previously (KCNJ3_pGHE forward and reverse). The second reaction mixture will amplify the KCNJ3 coding sequence and will contain the KCNJ3 DNA template (purchased from DNASU, KCNJ3_pENTR), and the primers for this coding sequence in pGHE (KCNJ3 froward and reverse). PCR protocol found here.

Figure 3:

Specifications for the composition of PCR reaction mixtures using Q5.

Figure 4:

Cycling conditions for PCR Q5 mutagenesis.

Linear PCR products are then analyzed by gel electrophoresis. Agarose gels were made using ethidium bromide and successful linear PCR products visualized and confirmed by presence of single bands and by length after 45 minutes under 160V in a standard electrophoresis tray (Figure 5). The successful reaction mixtures were then allowed to digest with 1ul of Dpn1 restriction enzyme to remove unreacted parental plasmid DNA. Protocol for running and analyzing a gel can be found here. Successful linear PCR products were then ligated together in an overlapping style using the NEB HiFi DNA assembly master mix. This reaction was mixed according to the specifications of the HiFi kit for two fragment DNA assembly and incubated in a thermo-cycler for one hour at 50 degrees Celsius (Figure 6). This will produce our circular target plasmid.

Figure 6:

Recomendations for HiFi DNA assembly reaction mixtures using the HiFi DNA master mix from NEB.

This product was analyzed through gel electrophoresis and bacterial transformation. Gels were made using agarose with ethidium bromide and products were visualized and confirmed by the presence of multiple bands in a single lane and by length (Figure 7). These products were also confirmed by transformation in JM109 E. Coli cells on plates containing ampicillin by running the HiFi product transformation against a transformation of their linear PCR products only in a negative control. This method of analysis is advantageous as transformation is also required to increase the available volume of target plasmid DNA. Cells were purchased from ThermoFisher and inoculated in SOC liquid media for one hour with HiFi DNA products. Once plated onto ampicillin agar plates, colonies were given overnight (~14 hours) to grow. Due to the nature of bacterial cells, negative control bacteria did not grow because of the inability of the bacteria to uptake the linear PCR product DNA. Experimental control bacteria, containing circular HiFi DNA products, did grow as the bacteria uptake the circular product containing ampicillin resistance. This DNA was replicated by the cells and colonies grew (Figure 8). If any growth occurred in negative control transformation experiments, it was likely due to undigested, unreacted, parental DNA, PCR and HiFi reactions were repeated. Protocol for transformation found here.

Figure 5:

Successful products of PCR reactions are linear strands of DNA. This can be visualized on agarose gels by the presence of a single band in the lane of a gel. Here, circular samples are ran as well, against the product samples, as negative controls. The circular DNA strands will produce two bands in a single lane while the desired linear products of the PCR reaction produce only one band. This can be seen in lanes 5 and 6 for KCNJ3. Concentration of a DNA product can also be determined along with length.

Figure 7:

Successful products of HiFi DNA assembly reactions are circular strands of double stranded DNA. This can be visualized on an agarose gel by the presence of two bands in a single lane on the gel. Here in lane 2, two bands can be seen, indicating a successful reaction. However, it is common that the amount of circular DNA that is produced during the HiFi reaction is so small (due to the small amount of linear PCR product left after digestion by Dpn1), it will be unable to be seen using the standard volumes of DNA that are added to a well for electrophoresis. To correct for this, we found a larger volume of HiFi product can be added to the gel reaction mixture to produce visible bands and extra volume of HiFi product DNA subtracted from the volume of water normally added. The best verification for the success of a HiFi reaction is bacterial transformation of its products alongside a negative control transformation of its precursor, the respective linear PRC products. (See Figure 5 as well)

Figure 8:

Due to the nature of bacterial cells, negative control bacteria did not grow because of the inability of the bacteria to uptake the linear PCR product DNA. Experimental control bacteria, containing circular HiFi DNA products, did grow as the bacteria uptake the circular product containing ampicillin resistance. This DNA was replicated by the cells and colonies grew. When the DNA is replicated by the cells, it is done for the cells survival, to gain the resistance to the antibiotic, in this case ampicillin. In doing so, the entire DNA plasmid is replicated, and thus, our protein coding sequence for KCNJ3, resulting in a larger available volume of target plasmid DNA. This process can be visualized by the large difference in number of colonies on the agar plates. The plate on the left was the experimental plate, containing the HiFi DNA product and grew many colonies while the negative control plate on the right, containing the PCR products only (HiFi precursors) grew ~2 colonies.

After successful transformation 3 colonies were inoculated individually in 75ml of nutrient broth media containing ampicillin. The inoculation flasks were shaken at 170 rpm at 37 degrees Celsius for 16 hours. Once removed from the shaker, a maxi-prep and ethanol precipitation of the DNA was performed. Tubes containing media were spun in an ultra-centrifuge at 5,000 rpm for three minutes to form a pellet. Pellets were then digested with a tens buffer and washed with sodium acetate before being spun again to separate the aqueous layer, once removed RNase was added to digest unwanted RNA. This layer was washed with Phenol Chloroform (PCI) and chloroform, individually, and spun between washes to allow for the removal of the aqueous layer before the next wash. To the final aqueous layer, 100% ethanol (etOH) was added to precipitate a DNA pellet. A final wash was performed with 70% etOH, the surfactant drained, and tubes allowed to sit open, covered by a kim-wipe to dry. Pellets were re-suspended in an appropriate amount of Molecular Biology Grade (MBG) water and ran on an agarose gel to determine the concentration. Agarose gels were made with ethidium bromide for this analysis (Figure 5). Once a concentration was determined samples were prepared for analysis by sanger sequencing. Our primers used for sequencing were designed using the OPRM1 DNA coding sequence and genscript, an online tool. These primers were designed to bind approximately 120nt from the end (5') and beginning (3') of the KCNJ3 coding sequence and read forward and reverse respectively, into the vector region to confirm the presence of KCNJ3prt ;,,,,,,,,,,90[, in pGHE (Figure 9).

Figure 9:

Sequencing primers designed for OPRM1. R = Reverse, F = Forward.

The GeneWiz company was utilized for the purchasing of sequencing primers and sequencing. Sequencing primers and re-suspended DNA were prepared in a pre-mix sample for the beginning (3') and end (5') of the OPRM1 coding sequence to confirm the presence of OPRM1 in pGHE, resulting in two sequencing samples for each resuspended pellet of DNA, specifications are provided by Genewiz (Figure 10). Results were analyzed with Snapgene to visualize the overlap in the sequenced region (Figure 11).

Figure 10:

Genewiz pre-mixed sample specifications. Template DNA is the re-suspended pellet DNA being sequenced.

Figure 11:

Highlighted is the sequenced area near the beginning (3') of the OPRM1 coding sequence. The highlighted portion covers both the vector of pGHE and in insert OPRM1, confirming its presence in pGHE.

Linearization of the sequenced DNA is required for subsequent in vitro transcription into mRNA. For OPRM1_pGHE, this was completed using the Nhe1 restriction enzyme, specific for a target sequence in pGHE. The reaction mixture was incubated at 37 degrees Celsius, overnight to allow for complete digestion (Figure 12). This reaction was confirmed using an agarose gel with ethidium bromide to visualize the single band against its circular, maxi-prep product. DNA in vitro transcription was carried out using the mMESSAGE mMACHINE T7 Ultra kit purchased from ThermoFisher. The initial reaction solution was mixed in accordance with the kit and incubated for two hours at 37 degrees Celsius (Figure 13). 1ul of Turbo DNase restriction enzyme was added following the incubation period, digesting any unreacted DNA. Poly(A) tailing was achieved using reagents from the same kit under an additional one hour incubation period at 37 degrees Celsius (Figure 14).

Figure 12:

Volumes for DNA linearization reaction and restriction enzyme specifics.

Figure 13:

Specification of required reagent volumes for in vitro DNA transcription using the mMESSAGE mMACHINE T7 Ultra kit.

Figure 14:

Specification of required reagent volumes for Poly(A) Tailing of the RNA product using reagents from the mMESSAGE mMACHINE T7 Ultra kit.

This reaction was stopped using 10ul of a sodium acetate solution. A phenol : chloroform clean-up was preformed to purify the transcription product. This was done with an initial wash using acidic PCI, spun at 13,000 rpm to separate the aqueous layer which was then removed and washed with chloroform, spinning repeated. The subsequent aqueous layer was again removed and isopropanol added, this mixture was set in -20 degrees Celsius for 15 minutes. The mixture was then spun at 4 degrees Celsius and 13,000 rpm for an additional 15 minutes, forming an RNA pellet. Supernatant was drained and 70% etOH added for a final wash and spin cycle. The tube was drained and allowed to dry. The dried pellet was resuspended and stored in a -80 degree Celsius freezer, ready for injection into an oocyte for electrophysiology experimentation (Figure 15).

Figure: 15

Results of an electrophysiology experiment using an oocyte injected with a solution composed of 1% OPMR1 and .009%CFTR(R104C) RNA in Frog Ringers (FR) solution. Dimerization occurs between opioid and beta adrenergic receptors when the opioid receptor is bound by and agonistic ligand. This causes activation of Cystic Fibrosis Transmembrane Regulatory (CFTR) channel. Here, [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO), a known Mu-opioid receptor agonist, causes increase in transmembrane conductance when applied. Conductance was brought back to baseline after FR was applied to the system.

pGHE+hOPMR1

RNA Gel

The bands are roughly in the correct position. Since KCNJ3 has 501 amino acids in its structure, there would be 1506 base pairs including the stop codon. By looking at the gel the fourth band on the ladder is the 1500 base pair marker, and the KCNJ3 band is around that band. It is a little higher since there is the polyA tailing and the 5’ and 3’ untranslated regions.

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