Bio-ARROW - SmartForm - Research Description
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This section is for describing your research from a biosafety perspective. It is not appropriate to copy and paste the research description from a grant proposal, animal protocol, or other document because we need different information.
Remember to enter information about the work that is done outside of your main laboratory (i.e. core laboratories). Describe what materials are used in the core facility and who performs the procedures (i.e. your staff, core staff, etc.).
For protocols with multiple projects, please number each project in the description. Add the biosafety level, location if applicable, relevant biosafety concerns including the risk to people, details on recombinant activities and factors important in risk assessment for each project.
If flow cytometry is used, please describe what kind of cells you plan to analyze or sort including viability (alive or fixed) and how the cells are manipulated (how they are generated, transfected, transduced, labeled, etc.).
Here are two examples of well written research descriptions:
Protocol A:
Coronavirus (CV) infections frequently cause disease. Our goal is to define aspects of virus-induced inflammatory responses that contribute to disease.
- We have created and continue to modify PCR-based methods for the identification various corona viruses from patients. To that end, we grow a variety of respiratory viruses to use as standards for developing diagnostic tests. We use commercial cloning vector pGEM-T Easy (Promega) for TA cloning of PCR products for molecular typing of clinical isolates. We will use the same methods for CV-BB strains.
- pGEX, pGEM and pET-based plasmids contain human coronavirus genes. Protein expression is under the control of the inducible tac promoter or T7lac and hybrid CMVie enhancer fused to the chicken β-actin promoter (pTriEx). These plasmids are used for expression of viral proteins in E. coli. They have the ampicillin-resistance gene for growing and selection in E.coli. Viral proteins are necessary for studying host-shutoff mechanisms of virus in eukaryotic cells and production of virus-specific antibodies.
- We are going to validate the CV receptor candidate gene(s) identified by overexpression in non-permissive cells by down-regulation of RNA expression (RNA interference). Since the differentiated human epithelial cells susceptible to viral infection cannot be transfected efficiently by lipofection or electroporation, we would like to transduce them with lentivirus vectors (HIV-1) encoding short-hairpin (sh) RNAs targeting candidate gene(s). We are planning to use commercially available third-generation Lentivirus vectors that are replication defective due to segregation of vector and packaging cassettes in several (at least 3-4) plasmids and deletion of viral accessory genes to minimize the risk of production of replication-competent lentivirus. The lentivirus will be produced in small quantities (laboratory scale) and applied only to cultured airway cells in vitro. (See attached appendix for figure) This will be done at BSL2 containment.
- Flow Cytometry—We are interested in the identification of coronavirus susceptible cell type in human cells. We will transfect cell cultures with an in vitro-transcribed RNA product that encodes for a full-length CV genome containing a fluorescent protein (e.g. Green or Enhanced Blue Fluorescent Protein (GFP, RFP-A respectively)) gene insert. Individual vectors are replication competent, but replicate at about one log lower efficiency than parent strains, and only infect about 1% of cells in a given Cell-1 culture. Fluorescence-activated cell sorting and return of cells will be used to sort GFP- or RFP-A -positive cells from the total cell population. Afterward, total RNA will be extracted from sorted cells for determination of cell type-specific transcripts via qRT-PCR. All work performed with cells before and after sorting will employ BSL2 safety precautions.
Protocol B:
Overview: We are currently synthesizing and evaluating new chemical ligands to modulate bacterial communication pathways (i.e., quorum sensing) in gram-negative and gram-positive bacteria. We are most interested in the modulation of communication pathways in pathogenic bacteria.
- Protocol for quorum sensing genotypic reporter strains:
BSL: 1-2
Risk: Slight risk to immunocompromised people if using BSL2 strains
Recombinant: Yes
Location: BSC in BSL2 lab
Special Details: N/A
These biosensor strains in our inventory are used to evaluate some form of quorum sensing-dependent gene expression. They do not produce activating ligands, but contain a plasmid with a functional LuxR-family protein cloned together with a cognate target promoter (usually the promoter of the cognate luxI synthase) or functional Gram-positive receptor protein, which positively regulates the transcription of a reporter gene (i.e., bioluminescence, beta-galactosidase, beta-lactamase, green-fluorescent protein, etc.). These strains are used to study the effects of designed synthetic ligands on Gram-positive and Gram-negative quorum sensing pathways, including S. aureus (AgrC), A. tumefaciens (TraR), P. aeruignosa (LasR, RhlR, QscR), R. palustris (RpaR), V. fischeri (LuxR) and A. baumannii (AbaR). The bacterial stocks are grown up overnight in a shaking incubator according to standard growth protocols with appropriate antibiotics. We perform assays in multititer plates or on agar plates by using overnight cultures, adding appropriate amounts of potential signal molecule analogs, and then measuring OD600 and beta-galactosidase, luminescence, or fluorescence activity.
- Protocol for Arabidopsis studies:
BSL: 1
Risk: None
Recombinant: No
Location: BSL1 lab space at minimum - plant growth shelves are in BSL2 space
Special Details: N/A
In order to evaluate the effects of small molecule modulators of quorum sensing on eukaryotes we surface sterilize seeds of wild type Arabidopsis thaliana Col-0 (2% bleach, 70% EtOH) or Medicago truncatula A17 (2% bleach, 70% ethanol, sulfuric acid wash) and germinate them on solid media supplemented with gibberelic acid (a germination stimulant). Post germination these seeds are transferred to plates containing the compound of interest at a predetermined concentration. Seedlings are then grown for up to 21 days then evaluated for growth effects in response to AHL exposures.
- Protocol for genetic manipulations:
BSL: 1-2
Risk: Slight risk to immunocompromised people if using BSL2 strains
Recombinant: Yes
Location: BSC in BSL2 lab
Special Details: N/A
Most strains, with the exception of some P. aeruginosa and E. coli strains harboring plasmids, are acquired from other research labs. To transform E. coli, we use electroporation and chemical competent cells. For the P. aeruginosa strains, plasmids are transferred from E. coli via conjugation or transformed using chemical competent cells into a strain of interest. In addition to using plasmids from other research groups, new plasmids are created using standard molecular cloning techniques (PCR, restriction digestion, ligation, etc.).
- General protocol for reporter assays in Staphylococcus aureus and Staphylococcus epidermidis
BSL: 2
Risk: Slight risk to immunocompromised people from BSL2 strains
Recombinant: Yes
Location: BSC in BSL2 lab
Special Details: N/A
Peptide stock solutions were diluted with DMSO in serial dilutions (either 1:3, 1:5, or 1:10), and 2 uL of the diluted solution are added to each of the wells in a black 96-well microtiter plate. An overnight culture of S. aureus gfp strain iss diluted 1:50 with fresh TSB (pH 7.35). A 198-uL portion of diluted culture is added to each well of the microtiter plate containing peptide. Plates are incubated at 37 degC for 24 h. Fluorescence (EX 500 nm / EM 540 nm) and OD600 of each well are then recorded using a plate reader and IC50 values are calculated.
- Procedure for growing C. elegans
BSL: 1-2
Risk: Slight risk to immunocompromised people if using BSL2 strains
Recombinant: Yes and no
Location: BSC in BSL2 lab
Special Details: N/A
C. elegans is grown on NGM plates with E. coli as the food source. For each experiment, one worm is transferred to a new plate and grown for 4 days. The entire population is washed from the plate to a falcon tube and bleached for 30 seconds. The falcon tube is centrifuged to pellet the unhatched eggs. The supernatant is removed and the eggs are washed with water and pelleted a couple of times. The eggs are left to hatch in M9 for 6 h, then poured to a new plate. The worms are left to grow for 48 h, then washed from the plate using M9 and 20-30 worms are placed on new plates containing P. aeruginosa strains. The worms are grown for 9 days in the plates and the live/dead ratio as measured every 24 h.
- Toxic Shock Syndrome Toxin-1 ELISA Assays
BSL: 2
Risk: Risk from TSST-1, slight risk to immunocompromised people if using BSL2 strains
Recombinant: Yes and no
Location: BSC in BSL2 lab
Special Details: N/A
TSST-1 toxin was used in ELISA assays as a standard (similar to BSA in Bradford assay) for quantifying TSST-1 production by S. aureus. Dilute overnight culture of S. aureus in fresh media containing peptide and incubated for 24 hours. Rabbit anti-TSST-1 IgG was added to a 96-well ELISA (Enzyme Linked Immunosorbent Assay) plate and incubated with shaking for 18 hours at 37deg. The ELISA plate was then washed, blocked with BSA, and washed again. Bacterial cultures were centrifuged and supernatants diluted in rabbit serum, followed by further dilution in PBS-Tween. TSST-1 purchased from Toxin Technologies, Inc. was used as a standard. The dilutions and the TSST-1 was added to the ELISA plate and incubated for 2 hours. Following incubation, plates were washed again and anti-TSST-1 IgG/HRP conjugate was added and, following another incubation, substrate was added. Absorbance was measured at 405nm. Toxins are not purified from any strain.