Experiments | Stony-Brook

Cloning

(1). Primers were designed using SnapGene's Gibson assembly feature to contain appropriate 5' overhangs for assembly, and trimmed following Xia et al paper for the correct overhang length and annealing temperature. Annealing Temperature of the primer was calculated using the NEB Tm Calculator. (use buttons below to see full protocols)

(2). Gene Fragments amplification and plasmid linearization were achieved by PCR with NEB Q5 2x master mix following manufacturer's protocol. The PCR reaction mixture is then analyzed on a 1% agarose gel to check for the correct length prior to plasmid assembly. Once we confirmed that the PCR product is the right length, PCR purification was carried out using Thermo Fisher GeneJET PCR purification kit following manufacturer’s protocol.

(3). Gel Extraction: Due to high amounts of nonspecific bands for our Ago2 PCR, we performed a gel extraction using a 1% agarose gel and the GeneJet Gel extraction kit from Thermo Fisher. PCR reaction was scaled up to 100uL, and 25 uL of the reaction mixture was loaded into each lane of the gel. DNA from Exercised gel containing our band of interest was extracted following manufacturer’s protocol, but eluted with 30uL of TE as opposed to 50 uL.

(4). Preparation of Competent Cells:

(i). Expression Strain MRE600 or BL21*: A 2 mL starter culture of MRE600 or BL21* is grown overnight at 37 C at 250 rpm with LB. Note that for expression of Ago2, we grew cells at 25 C. The culture is then inoculated the following day in 200mL of fresh LB and grown to an OD600 of 0.35-0.4. The cells are then harvested, centrifuged, and treated with a solution containing HEPES and CaCl2. After that, it is centrifuged and resuspended in a solution containing HEPES, CaCl2, and DMSO. Cells are stored at -80 until use. (see full protocol using the button below)

(ii). Cloning: For our initial assemblies (Promoter+RBS+GFP+Terminator in PACYC and Promoter+Ago2+Terminator in PJL1) involving products less than 5000 base pairs, we used competent DH5 alpha cells prepared the same way as MRE600 or BL21* described above. Future assemblies involving larger products failed with our in-house competent DH5 alpha, and we used the DH10B cells provided by NEB.

(5). T5 exonuclease-dependent assembly:
To assemble all our gene fragments into our plasmid backbone, we used the T5 exonuclease-dependent assembly method from Xia et. al paper because 1) the 2023 SBU iGEM team has had success and 2) for its low cost. This is a modified version of the Gibson assembly where gene fragments and backbones with appropriate overhangs are incubated with just a T5-exonuclease for 30 minutes. The resulting product is then transformed into the bacterium for the rest of the repair (see full protocol using the button below) This can be referred to as TEDA cloning.

Assays

(1). miRNA Heat shock Assay:
A major question we hoped to answer was whether or not a bacteria-based microRNA detector could be possible, feasible, and efficient. Through our countless literature reviews, we did not find any sources documenting whether it is possible for bacteria to take up miRNA, which is instead endogenous to eukaryotes. Therefore, we set out to design an experiment to qualitatively assess whether bacteria can uptake miRNA.
Briefly, competent E. coli cells prepared chemically with protocol mentioned above are heat-shocked with fluorophore labeled mi-RNA mimic ordered from IDT. These cells are then washed twice with SOC media and then three times with PBS. Samples are then analyzed with confocal. (See full protocol using button below)

(2). Fluorescence Plate Reader Readings:

(i). GFP Calibration Curve
A GFP Calibration curve was generated following iGEM's Plate Reader Fluorescence Calibration protocol (but with GFP protein).We obtained wildtype green fluorescent protein from SBU Karzai lab, and checked for its concentration using the nanodrop.

(ii). Fluorescent Reading of Bacterial Sample
5mL of bacterial samples were inoculated overnight at 37C and 250 rpm in LB supplemented with the appropriate antibiotics. The next day, cells are harvested and diluted to an OD below 1 for OD600 reading, washed three times in PBS, and resuspended in the appropriate amount of PBS to ensure the same cell density among all experimental and control samples. (See our full protocol using buttons below)

(iii). Fluorescent Reading of Cell-Free System
Cell-Free systems were set up using NEBExpress® Cell-free E. coli Protein Synthesis System with the T7 polymerase provided with the kit substituted with endogenous e.coli polymerase saturated with S70 also purchased from NEB. The final reaction mixtures were incubated for four hours at 37 C at 250rpm. For reactions assessing functionality of our full circuit and target miRNA (miR-326), 1 nmole ofmiR-326 mimics ordered from IDT were added before incubation along with 1uL of 2uM MgCl2 solution.

(3). Western:
The N-terminal of our Human Argonaute2 protein (Ago2) was fused to glutathione S-transferase. Because our protein is under an endogenous promoter, we opted for a western blot as the expression level of our protein cannot be identified with staining methods such as a coomassie staining.

Due to the limit of time, we were only able to test expression of our argonaute protein in MRE600. Briefly, a 2mL starter culture was inoculated overnight at 37 C and 250 rpm. The following morning, this starter culture is transferred to 200 mL of fresh LB, and grown at 25 C for 4hr, 8hr, and overnight.

Extracted protein samples are separated by SDS-PAGE and transferred to a nitrocellulose membrane by semi-dry electroblotting. Primary anti-GST Tag antibodies and a secondary peroxidase-conjugated anti-goat antibody were both kindly given by SBU Glynn Lab. Bound antibodies were visualized by an enhanced chemiluminescence system (ECL) according to the manufacturer's protocol (Thermofisher).