Bio-ARROW - SmartForm - Research Description
Bio-ARROW help
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 as different information is needed.
Here
are some things to consider when writing your research description:
Describe what you are doing:
·
What microbes, cells,
animals, plants, genes, plasmids, toxins, and systems are you using?
·
In what combination
are the plasmids, genes, systems, cells manipulated?
·
What procedures and
equipment are used?
Identify special considerations for a project or activity:
·
Do you have a specific
space that you use for an experiment?
·
What containment equipment
used? (e.g. BSC, fume hood)
·
Will the activity
require different PPE?
·
Will the activity
require a specific piece of equipment?
·
Will activity require
specific training?
·
Change in risk to
person, environment, public, or animals?
·
Special hazard
communication?
Identify the recombinant materials:
·
What are the
constructs? How are your plasmids constructed? Are there multiple plasmids
used? How is it recombinant? (genes, system, microbes)
·
What modifications are
made and are there any expected changes in pathogenicity, virulence, host
range, or antibiotic resistance caused by the modifications?
State collaborations with another lab or a core facility:
·
On or off campus?
·
What activities will
your lab perform?
·
What activities will
the collaborator/core perform?
·
What materials will
you provide to the collaborator/core? (e.g., fixed, material type,
modifications)
·
What modifications
were performed on the materials prior to giving/sending them to the
collaborator/core?
·
What modifications
will the collaborator/core perform on the materials?
·
Will any materials be
returned to you from the collaborator/core?
·
Are there any special
hazard communication, disposal, spill clean-up, or other special considerations
for the materials shared with collaborator/core?
Below
are examples of well written projects within research descriptions:
Note:
The protocol example, all names, rooms, and activities
portrayed in this protocol are fictitious. No identification with actual
research protocols, places, buildings, and products is intended or should be
inferred.
Example Project 1: Bacterial cloning
Biosafety
Level: BSL1
Location:
Room 1140 (not core lab, BSL1 lab)
Recombinant:
Yes
Risk:
Low, E. coli strains not known to be pathogenic to immunocompetent people,
not known to be a risk to the environment or animals; no toxin or oncogenic
properties known or recombinantly
E. coli K12 and BL21 will be utilized to
propagate bacterial plasmids (pET-based). These plasmids will be used for
expression of various proteins in E. coli. Plasmids have the
ampicillin-resistance gene for growing and selection in E.coli. We
prepare these plasmids through standard cloning methods, including transforming
the bacteria, expanding the cultures and isolating plasmid DNA and/or proteins
from the bacterial cultures. No toxin genes or oncogenes will be
expressed.
Example Project 2: Natural antibiotic
resistance
Biosafety
Level: BSL2
Location:
Room 410 (BSL2 room, not core lab)
Recombinant:
yes, including antibiotic resistance
Risk:
A pathogen safety data sheet is available for S. aureus and attached under
Upload additional documents.
Risk
mitigation: use of BSC for all manipulations (except growth chamber/incubation
of cultures), no culture plates/broth cultures are handled on bench top
To understand the mechanism of
pathogenicity, Staphylococcus aureus USA 300 and clinical
isolates of S. aureus will be studied. DNA/RNA is extracted
and then used for PCR, sequencing, and metagenomics. Genes of interest will
also be identified using random mutant libraries consisting of transposons that
confer chloramphenicol resistance.
S. aureus is a human pathogen and will be handled at BSL2.
Containment will be used as described in the ‘Containment’ section. Since
strains of S. aureus can have natural occurring drug
resistance, clinical isolates will be tested for antibiotic resistance. The
susceptibility profiles of the isolates are attached. S. aureus USA
300 is known to be methicillin resistant.
Example Project 3: Yeast
Biosafety
Level: BSL1
Location:
Room 443 (cultures); Room 656 (shared equipment space for DNA screening, PCR,
genomic work)
Recombinant:
Yes
Risk:
Work involves stable introduction of antibiotic resistance genes. A pathogen safety
data sheet is available for Saccharomyces and attached under Upload additional
documents.
Risk
mitigation: work may be performed in a BSC or on the bench
The molecular function of genes will be tested
in Saccharomyces species. Several auxotrophic and drug-resistance
markers will be introduced on plasmids or through stable integration of DNA
into the genome. Conventional molecular biology approaches as well as
CRISPR/Cas9 are used. rDNA techniques include genome-wide screens,
gene overexpression, gene knock-down, as well as specific DNA deletions,
additions, and point mutations. Precautions used for experiments that use gene
drive are described in the ‘Recombinant Materials’ section.
Example Project 4:
Viral Vectors
Biosafety Level: BSL2
Location: room 1109, Room 1109a
Recombinant: yes
Risk
mitigation: use of BSC for all manipulations, no cell cultures or virus are
handled on bench top
Research includes use
of RG2 agents. Inadvertent exposure to virus has the potential to cause
infections in laboratory workers. We are aware that with the use of
retroviral vectors poses a residual risk of viral genome integration in the
host chromosomal DNA and emphasize this when instructing new lab members. To
minimize exposure and the potential for transmission, these agents are handled
in a BSC and personnel follow the laboratory exposure control and disinfection
protocols.
To
understand the function of the oncogene PI3K, we will use CRISPR to modify the
point mutation within PI3K to remove the mutation (thus restoring gene
function). It is not anticipated that by removing the point mutation in
PI3K that this would cause an increase in risk to people working with the cell
line.
To
do this we will use one of several viral vector systems (see construct section
for details). The viral vectors we use are rendered defective by using
multi-plasmid systems. The vector-systems used contain deletions in essential
replication and structural genes, so that the resulting vectors support only
one round of integration and are unable to further replicate.
We
use the second generation HIV based viral vector (as listed in construct
section) which is a three plasmid system. The tat and rev genes are on a
separate plasmid from the gag and pol genes. Accessory genes vif, vpu, vpr and
nef have been removed. 3’ LTR is present and replication competent virus
generation would require several recombination events.
We
also use a third generation HIV based lentiviral vector system as a four
plasmid system. The rev gene is on a separate plasmid from gag and pol.
Accessory genes vif, vpu, vpr and nef have been removed. The 3’ LTR-SIN has
been deleted and replication competent virus generation would require four
recombination events.
For
both the second and third generation, the envelope gene is VSV-G as it enters a
variety of cells. HEK293 is used as the packaging cell line. The second and
third generation systems will not be mixed together to prevent the formation of
replication competent viruses. Both systems will be used with cell lines listed
in the ‘Cell Culture’ section with genes listed in the ‘Genes and DNA/RNA
fragments’ section.
The
moloney murine leukemia virus (MMuLV)-derived retroviral vector is also
utilized and may be modified so that it is able to enter human cells through
the VSV-G envelope gene. It is a two plasmid system and PT67 and HEK293 are
packaging cell lines (providing gag and pol). This vector system will only be
used with HEK293 cells with gfp and Akt or mTOR.
We
express CRISPR guide RNAs and CAS from a single construct, and homology
dependent repair cassettes from two separate plasmids. The sequences of
guide RNAs and homology repair cassettes are specific for the
species-origin of suitable host cells for any given virus under
study. These include human-derived cells. The CRISPR system is a
single vector system and does not contain homologous DNA surrounding either the
Cas9 or the guide RNAs. No gene drive issues with this system as this is for
cell culture only.
Since
the PI3K is a known oncogene, persons working with the HIV based viral vector
systems and CRISPR will be apprised of the possibility that the vector has the
capability to enter a human cell. They will be provided information on this
research in case they would like to follow up with a medical professional for
questions or in the event of an exposure. (Specified in the ‘Emergency
Response’ sections and ‘Occupational Health Considerations’ sections of this
protocol)
Example Project 5: Cell Culture and
Animals
Example Project 5A:
Biosafety
Level: BSL2
Location:
room 1533 (BSL2 lab, not shared space);
Recombinant:
yes
Risk:
human cell, BBP; siRNA
Risk mitigation: use
of BSC for all human material manipulations (except cell incubation), no cell
cultures are handled on bench top.
In order to study genes involved with
tumor progression in squamous cell carcinoma (SCC) in vitro we will utilize
immortal mouse and human tumor tissue culture lines that we have generated
(mSCC and hSCC respectively). To assess the relevance of genes of
interest (GOI) as possible tumor promotor genes in these cells we will add
siRNA to knock-down the GOI. Scrambled siRNA will be used as a control.
Knocking down a tumor promotor gene should allow for decreased growth of
the cells. Once we have genes that we feel are relevant we will confirm
them in primary cells from fresh tumors. We will also study the mechanism in
more detail by upregulate the selected tumor promotor as further described in
5B. This work will all be done at BSL2 in a BSC. Fixed samples will
be transported to UWCCC Experimental Pathology Laboratory for histology.
Once cells are lysed protein work can be performed on the bench.
Example Project 5B:
Biosafety
Level: BSL2
Location:
room 1533 (BSL2 lab, not shared space
Recombinant:
yes
Risk:
human cell, BBP; siRNA
Risk mitigation: use
of BSC for all human material manipulations (except cell cultures), no cell
cultures are handled on bench top.
Once we have selected genes that act as tumor
promotors in our cell lines we will screen multiple established tumor cell
lines from ATCC by using the siRNA as described in Project 5A. This
project will have all the same safety precautions as Project 5A.
Example Project 5C:
Biosafety
Level: ABSL-1
Location:
room 1703 (ABSL1 vivarium); Core facility rooms 344, 354
Recombinant:
yes
Risk:
human cell, BBP, siRNA
Risk mitigation: PPE
(gloves, eye protection, lab coat), use of BSC for all human material
manipulations (except cell cultures), no cell cultures are handled on bench
top.
The in vitro studies will be followed by
experimentation in vivo. We have established transgenic mouse lines that
produces SCC tumors. We will initiate tumor growth and then at
predetermined tumor size will give the siRNA or control scrambled siRNA via
tail vein injection. Tumor harvesting will be performed according to
predetermined criteria. This work will be done at ABSL1. Analysis
will include imaging of animals, tumor histology, and Western blot. The UW-Madison
Small Animal Imaging Facility (SAIF) core will be informed of the ABSL1
status. Animals will be transported according to our Transport protocol
and will be housed in quarantine after the imaging sessions. Histology
samples are fixed and therefore can be handled at BSL1 by UWCCC Experimental
Pathology Laboratory personnel.
Example Project 5D:
Biosafety
Level: BSL2, ABSL-2
Location:
room 1533 (BSL2 lab, not shared space); Core facility rooms (imaging)344, 354;
Sorting activities in room 7116, 7118
Recombinant:
yes
Risk:
human cell, BBP, animals administered human material; siRNA
Risk mitigation: PPE
(gloves, eye protection, lab coat), use of BSC for all human material
manipulations (except cell cultures), no cell cultures are handled on bench
top. Animal work (cage changes, surgery, administration of human materials)
performed in BSC. Needles used for animals at ABSL2: needle locking systems,
decontaminated prior to discard (see NIH Guideline Appendix M-II-B-g-(2).
We will assess relevance of the selected gene
for clinical situations using a Patient Derived Xenograph (PDX) model.
Human tumors will be received from an IRB approved protocol. These will
be implanted into the flanks of NSG mice to be propagated. According to
pre-set criteria, the tumors will be harvested in a BSC. A small portion
will be fixed and sent for histology, a small portion will be froze back, and
the remainder will be passaged into the flanks of Nude mice. When
passaged tumors reach experimental criteria the siRNA or scrambled siRNA will be
given to the Nude mice by tail vein injection. Scheduled imaging will be
performed on live mice at the UW-Madison Small Animal Imaging Facility (SAIF)
at ABSL2. Animals will be transported according to our Transport protocol
and housed in quarantine after imaging sessions. Tumors will be harvest
in a BSC according to pre-set criteria and samples sent for fixed histology and
used for protein analysis as described above. Sorting of live cells will be
performed at the UWCCC Flow Cytometry core by core staff.
When experiments require the use of core facilities, all
preparative steps in the procedures are as much as reasonably possible
performed in our own lab rather than at these core facilities. We provide
hazard communication to personnel at the core facilities on the experimental
set up to ensure they are informed about all risks, containment practices, and
inactivation procedures.
Example Project 6: Plants
Biosafety
Level: BSL2, BSL-2P
Location:
room 234 (BSL2 lab); room 234A (growth chamber room) BSL-2P
Recombinant:
Yes
Risk: Salmonella is a
pathogen to humans, a pathogen safety data sheet is available and attached
under Upload additional documents.
Risk
mitigation: use of BSC for Salmonella manipulations and plant manipulations
post inoculation (except for culture incubator and post plant inoculation in
growth chambers.
Our research goal is to determine the genes and mechanisms responsible for the colonization of plants by Salmonella. Various Salmonella mutants will be generated using standard genetic techniques (chemical mutagens and transposable elements), including point mutations and gene deletions in putative colonization genes. Complementation studies may be performed using the plasmids listed in the ‘Constructs’ section. Salmonella mutants may be tetracycline and/or kanamycin resistant. No increase in virulence is anticipated. Plants will be inoculated in a BSC with 106 CFUs Salmonella (wild type or mutant) per plant at different stages of growth. Plants will be grown in plant growth chambers located in a BSL-2P laboratory, see attached picture, under various light cycles and temperatures. Samples of plant tissues will be handled in the BSC for harvesting, homogenization, and analysis by plating and bacterial enumeration at various stages post inoculation. All transgenic plants are destroyed by autoclaving prior to flowering.
Still have questions about this section of the Biosafety Protocol? We are happy to help! (608)263-2037 biosafety@fpm.wisc.edu