Number of Pages: 10
Palacky University Regulations for working with genetically modified organisms
a) Name (or Names), Surname, Business Firm, Residency and Identification Number (if
assigned), Entitled Persons (natural person entitled for business).
Not referred.
b) Name or Business Firm, Residency and Identification Number (if assigned), Entitled
Persons, If Legal Person then Name, Surname and Residency of Corporate Authority.
Name of the Organization: Palacky University in Olomouc, public university.
Residency and Identification Number: Olomouc, Křížkovského 8, PSČ 771 47
IČO: 61989592
Legal Position: public university
Rector: prof. RNDr. Lubomír Dvořák, CSc.,
tel.: 585631001 E-mail: [email protected]
Permanent Residency: Olomouc, Thomayerova 28, PSČ 772 00
c) Name (or Names), Surname and Residency Address of Buildings or Properties
Owner, if the Owner is not Identical with Entitled Person then Name, Surname or
Business Firm Name, Residency and Identification Number (if assigned) or Name,
Surname and Residency of Corporate Authority if natural person.
Not referred.
d) Name, Surname, Permanent Residency, Phone Number or Fax Number and e-mail
Address of Expert Advisor.
RNDr. Oldřich Navrátil,CSc Institute of Experimental Botany AV ČR researcher,
tel. 225 106 433, fax 225 106 456, e-mail [email protected]
Trvalý pobyt : U Havlíčkových sadů 3, 120 00 Praha 2, tel. 222 511 028
e) Hazard category for dealing with genetically modified organisms designated for
a workplace if that dealing is enclosed.
Hazard category I and II:
f) Person Responsible for Workplace Operation:
Head of the Cell Biology and Genetics Department
doc. RNDr. Milan Navrátil, CSc.
Tel: 585 634 900
E-mail:
[email protected]
Zikova 28, Olomouc 779 00, Tel.: 585 434 463
Head of the Department of Biochemistry:
Prof. Mgr. Marek Šebela, PhD.
Tel: 585 634 920
E-mail:
[email protected]
Řepčínská 69, Olomouc 779 00, Tel.: 585 415 608
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g) Pattern, purpose and a description of technical items securing enclosure of the area (if
enclosed dealing prescripted)
Particular laboratory complexes are enclosed by doors without a handle (with a round handle).
Cultivation rooms and boxes are under permanent lockout. The keys have those workers of
PřF UP in Olomouc-Holice who use that precincts and become acquainted with GMO
regulations. Keys records are in competence of head of an appropriate division.
h) List of obligatory procedures used at workplaces and their description
Workplaces are determined for research work. Basic approaches are plasmid vectors
construction for bacterial cells transformation, transformation into bacteria Escherichia coli,
Agrobacterium tumefaciens and yeast Saccharomyces cerevisiae and Pichia pastoris, plant
cells transformation by Agrobacterium tumefaciens, transgenic plants regeneration, transgene
detection in transgenic plants, effects of transgenes in the transgenic plants detection and
other actions related to transgenic plants utilization for research problems solving. New DNA
constructs with different transgenes for plants transformation are developing (listed below).
Cloning into cloning and expression vectors with aim to sequence and study functional
expression of the genes of interest in bacterial or yeast expression systems is one of the main
processes.
Main experimental processes used at workplaces are methods of molecular genetics, genetic
engineering, tissue cultures cultivation, virus biotechnology and classic genetics. As the
methods are progressing continuously it is impossible to list all the methods used at
workplaces. Main processes are based on basic bioengineering methods as DNA, RNA
isolation, PCR, RT-PCR, digestion by restriction endonucleases, electrophoresis, cloning,
library construction, plasmid clones selection, Southern blotting, PCR sequencing, methods
for reporter genes determination as GUS, ipt etc. Some of the tissue culture methods are used,
for example plants cultivation in vivo, callus forming, plants regeneration from callus,
transgenosis via Agrobacterium.
Methodical processes are modified depending on initial material and aims of the experiments
with eventual renovation based on new knowledge described in literature or according to
researchers’ inventions.
i) List of Workers that become acquainted with GMO regulations on their workplaces
Cell Biology and Genetics Department:
Doc. RNDr. Milan Navrátil, CSc.
RNDr. Martin Fellner, Ph.D.
RNDr. Renata Fialová, Ph.D.
RNDr. Petr Nádvorník, PhD.
Mgr. Dana Šafářová, Ph.D.
Mgr. Sebastien Gadiou
Mgr. Renata Hloušková
Mgr. Kateřina Čermáková
Mgr. Jana Petrusová
RNDr. Lenka Uvírová
RNDr. Pavla Válová
Department of Biochemistry:
Prof. RNDr. Ivo Frébort, CSc.
Doc. Mgr. Petr Galuszka, Ph.D.
Mgr. Hana Pospíšilová
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Mgr. Iva Sedlářová
Mgr. Šárka Vyroubalová
Mgr. Mária Šmehilová
j) Specification of species and amount of a genetic modified organism used on the
workplace
Accepters:
Bacteria:
Escherichia coli
JM109: genotypee: F’ traD36 proA+B+ lacIq ∆(lacZ)M15 / e14- (McrA-) ∆(lac-proAB)
endA1, source: New England Biolabs
TOP10: genotypee: F- mcrA ∆(mrr-hsdRMS-mcrBC) Φ80lacZ∆M15 ∆lacΧ74 recA1
araD139 ∆(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG. Source: Invitrogen
XL1-Blue: genotypee: supE44 hsd R17 recA1 endA1 gyrA46 thi relA1 lac- F´[proAB+
lacIq lacZ∆Tn10(tetr)], source: Stratagene
XL-1MRF’Blue: genotypee: (mcrA)183 ∆ (mcrCB-hsdSMR-mrr)173 endA1 supE44
thi-1 recA1 gyrA96 relA1 lac [FproAB lacIqZDM15 Tn10 (Tetr)], source: Stratagene
XL-10 Gold: genotype: TetR∆ (mcrA)183 ∆ (mcrCB-hsdSMR-mrr)173 endA1 supE44
thi-1 recA1 gyrA96 relA1 lac Hte [F‘ proAB lacIqZ∆DM15 Tn10 (TetR) Amy CamR]
source: Stratagene (approved by resolution 841/OER/05, date 4.5.2005)
BL21 StarTM (DE3) genotype: F- ompT hsdS(rB-mB-) gal dcm rne131 (DE3), source:
Invitrogen
BL21(DE3) genotype: F– dcm ompT hsdS(rB– mB–) gal λ(DE3), source: Stratagene
BL21(DE3)pLysS genotype: F– dcm ompT hsdS(rB– mB–) gal λ(DE3) [pLysS Cam‘],
resistance :chloramfenikol, source: Stratagene
AD494(DE3)pLysS genotype: Dara-leu7967 DlacX74 DphoAPvuII phoR DmalF3
F'[lac+(lacIq)pro] trxB::kan, resistance: chloramfenikol, source: Novagen
SG13009 [pREP4] genotype: derived from a strain K12, NalS strS rifS thi- lac- ara- gal+
mtl- F- recA+ uvr+ lon+ [pREP4 KanR], resistance: kanamycin, source: Qiagen
M15 [pREP4] genotype: derived from a strain K12, NalS strS rifS thi- lac- ara- gal+ mtlF- recA+ uvr+ lon+ [pREP4 KanR], resistance: kanamycin, source: Qiagen
DB3.1 genotype: F- gyrA462 endA1 glnV44 ∆(sr1-recA) mcrB mrr hsdS20(rB-, mB-)
ara14 galK2 lacY1 proA2 rpsL20(Smr) xyl5 ∆leu mtl1, source: Invitrogen
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TG1 genotype: derived from a strain K12 (lac-pro), supE, thi, hsd∆5/F' traD36, proAB,
lacIq, lacZ∆M15, Pharmacia
Agrobacterium tumefaciens
EHA105
LBA 4404
AGL1, genotype: derived from EHA101, recA::bla, T-region removed, Mop+ CbR,
source: John Innes Center, UK, resistance rifampicin, ampicilin
GV 3101, genotype: derived from EHA101, source: Frei University Berlin, resistance
rifampicin
Agrobacterium rhizogenes
15834
Yeast:
Saccharomyces cerevisiae
23344c genotype: MATa/MATα ura3-52
INVSc genotype: MATa/MATα his3∆1, leu2 trp1-289 ura3-52, source: Invitrogen
Pichia pastoris
X-33 source: Invitrogen
The most frequently used plasmid vectors in bacteria Escherichia coli, Agrobacterium
tumefaciens and in yeast Saccharomyces and Pichia:
(bacterial gene for ampicilin and kanamycin resistance)
pGEM®-T
®
pBSII SK+
(bacterial gene for ampicilin resistance )
pCR® 2.1-TOPO
(bacterial gene for ampicilin resistance )
pBR322
(bacterial genes for ampicilin and tetracyclin)
pUC119
(bacterial gene for ampicilin resistance)
pDrive
(bacterial gene for ampicilin and kanamycin resistance)
pCR
(bacterial gene for ampicilin resistance)
pCR Blunt II TOPO (bacterial gene for zeocin and kanamycin resistance)
pCR T7NT TOPO (bacterial gene for ampicilin and chloramphenicol resistance)
pTYB
(bacterial gene for ampicilin resistance)
pET101/D-TOPO (bacterial gene for ampicilin resistance)
pYES2
(shuttle vector for transfer between bacteria and yeast, ampicilin and
ura+ resistance)
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pDR197, 195
pGAPZ
pPICZ
pET15b,16b,32b
pQE30
(shuttle vector for transfer between bacteria and yeast, ampicilin and
ura+ resistance)
(shuttle vector for transfer between bacteria and yeast, zeocin
resistance)
(shuttle vector for transfer between bacteria and yeast, zeocin
resistance)
bacterial expression plasmid (ampicilin resistance)
bacterial expression plasmid (ampicilin resistance)
pBin-Hyg-TX
binary vector for plant transformation, kanamycin and hygromycin
resistance
pGREEN
binary vector for plant transformation, gene for kanamycin and
hygromycin resistance, gene for glucuronidase
pCAMBIA
binary vector for plant transformation, kanamycin and hygromycin
resistance, gene for glucuronidase
pH-TOP, pVKH18, pBIN-LhGR, pV-TOP, pBIN-LR-LhGR
binary vector for plant transformation, kanamycin and hygromycin
resistance
pBRACT214, pAHC25
• binary vector for plant transformation (kanamycin,
chloramphenicol, phosphinothricin resistance)
Plants:
Arabidopsis thaliana (Arabidopsis thaliana) ecotype: Columbia
Tobacco (Nicotiana sp.) cv. Samsun NN and a cell line induced on tobacco callus Nicotiana
tabacum cv. BY-2 (cultivar Bright Yellow – 2)
Barley (Hordeum vulgare) cultivar: Golden Promise, Scarlet, Viktor
Wheat (Tritium aestivum), cultivar: Kontesa
Donor Organisms:
Phytoplasms:
Aster yellows phytoplasma
Stolbur phytoplasma
Apple proliferation phytoplasma
Pear decline phytoplasma
European stone fruit yellows phytoplasma
Elm yellows phytoplasma
DNA inserts of these phytoplasms are kept frozen in plasmid vectors collection in Escherichia
coli bacteria.
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Plant Viruses:
Plum pox virus (PPV), Leek yellow stripe virus (LYSV), Onion yellow dwarf virus (OYDV),
Pea seed borne mosaic virus (PSBMV), Pea enation mosaic virus (PeMV)
cDNA inserts of these viruses are kept frozen plasmid vectors collection in Escherichia coli
bacteria.
Bacterial Viruses:
Bacteriophage lambda FIX2 (commercial genome library of barley, source: Invitrogen)
Yeast and Fungi:
Aspergillus niger, Saccharomyces cerevisiae, Schizosaccharomyces pombe
Plants:
Hordeum vulgare, Arabidopsis thaliana, Triticum aestivum, Zea mays, Lycopersicon
esculentum
In regards to diversification and continual modification among kept organisms it is impossible
to specify the exact number of particular GMO derived form accepters.
k) Organizing and technical provision of the workplace
Laboratories of the Department of Biochemistry and The Cell Biology and Genetics
Department of PřF UP in Olomouc are parts of PřF Olomouc-Holice precinct. These
laboratories are intended for research activities of the departments’ workers as well as for
students teaching. The Students work on their bachelor, diploma or doctoral thesis at the
departments. They also take a part in grant proposals solving.
Organizing and technical provision of the workplace is given by PřF UP structure and
their fragmentation for appropriate departments.
Laboratories Organization:
Dealing with GMO is allowed in following areas:
1. Cell Biology and Genetics Department (supervisor doc. RNDr. Milan Navrátil, CSc.)
(Within the meaning of the code 153/2000 about microbiology laboratories)
Laboratories No.:
Building D: 2nd floor: 23, 26, 34, 35;
Building D: 1st floor: cultivation room 1; cultivation boxes 1 a 2
2. Department of Biochemistry (supervisor prof. Mgr. Marek Šebela, PhD.)
(Within the meaning of the code 153/2000 relating to microbiology laboratories)
Laboratories No.:
Building D: 1st floor: cultivation room 6 and greenhouse box 1
Building E: 1st floor: SE VL1, SE VL2, SE SL, SE TL
One of the requirements for working in laboratories is limited access and lockout. Because of
this requirement the complexes of laboratories with the only single access were composed.
The access to the complexe will be secured by one door locked by round handle. Free access
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will have only those people that work in these laboratories and are allowed to have the keys.
These doors will be secured by electrical ring.
l) Accident or fire danger precautions including the Emergency Plan according to § 20
In case of fire the objects that were affected by fire are referred to be voided of genetically
modified organisms. After keep down the fire the persons responsible for accident effects
liquidation search the not completely damaged cultures and these sanitize according to
instructions described in the Emergency Plan. If it is necessary to carry out some of the
objects then the head of the department will decide which objects (if they could be
contaminated by genetically modified organisms) will not be carried out. In other cases the
liquidation will be processed according to the fire and emergency plans.
m) Workers responsibilities (working regulations following, areas sanitizing protocols,
equipment, safety protection and protective clothes decontamination).
Procedure for areas sanitizing and instruments decontamination are designed by head of the
department depending on activity character.
Liquidation is provided by a trained technical assistant. The technical assistant process the
liquidation following standard operating protocol taking records about amount of sanitized
waste.
Dealing with GMO hazard category II (bacterial expression strains) is maintained in
laboratories intended for this dealing only (SE SL, SE TL a SE VL). Access to these
laboratories is prohibited for basic laboratory techniques course students. The laboratories are
equipped by sink with incorporated antibacterial filter.
Protective clothes used in the hazard category II laboratories must be disinfected before
washing, rest of the protective clothes contaminated by genetically modified organisms will
be washed as infectious material. The same applies for protective coats of students working in
the laboratories of hazard category I. All the wasted clothes are collected and transported
separately to laundry MORAVA Prádelna a čistírna (Roman Melka), address: Tererovo nám.
897/5, 779 00 Olomouc. Used protective gloves will be liquidated according to protocol for
GMO liquidation.
n) System and frequency of controlling the areas, equipment and protective precautions
maintenance
Controlling will be kept concurrently with fire protection control provided by trained workers
four times per year. Control records will be maintained by division director.
o) Maintenance workers regulations
Workers of maintenance must follow instructions of the head of the department while
working in areas intended for dealing with GMO.
Workers of maintenance must use protective clothes labeled with the mark Biohazard. Use of
these protective clothes outside the building is forbidden.
p) Work safety and hygiene requirements given by legal enactments
Workers are due to use protective clothes in areas specified for recombinant DNA working as
well as students.
q) Waste, contaminated materials and objects regulations, protocol for genetically
modified organisms liquidation and the effectivity of the processes determination
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All GMO are inactivated by autoclaving and sequentially liquidated by Van Gansewinkles a.s.
company long-term contracted by Palacky University in Olomouc, contract no. 320814.
Dealing with these wastes is regulated by Palacky University in Olomouc according to
Environment Bureau of Olomouc Municipality Statement no. ŽP/8742/03/No. These wastes
are classified as waste hazard category 180103.Genetically modified plants are liquidated by
autoclaving in special polypropylene boxes. Leftovers of genetically modified bacteria are
liquidated by autoclaving, containers used for GMO cultivation are sanitized by disinfection
agent (bleach powder, chloramine, Ajatin in appropriate concentrations, 2% Incidur).
r) List of personal protective items and another tools determined by employer according
to legal enactment (Government edict no. 495/2001 Sb.). Protection is specified for each
particular process.
Use of a protective coat out of the area intended for dealing with GMO is strictly prohibited.
Latex gloves must be used for working with microorganisms and DNA, RNA electrophoresis.
s) Activities prohibited at the workplaces
Eating, drinking or smoking while working with genetically modified organisms is strictly
prohibited. Dealing with GMO is allowed according to specified regulations only, other
dealing is prohibited.
t) Regulations of making records about equipment operating, sanitizing and security
devices controlling
Every action with GMO must be recorded daily into a special protocol describing type of
action, date and date of the record. All workers must keep records of their work in a special
notebook.
u) Precautions to avoid the access of trespassers
Laboratory areas are divided into laboratory complexes and functional units working with
GMO. These areas are labeled. Part of the laboratory complexes are also cultivation rooms.
Access into each of the units is possible only through doors that can be unlocked by keys. The
keys are in competence of trained employees. They can allow students working at PřF UP to
access the unit. Cultivation rooms and boxes are locked and marked with the label Biohazard
on their door. Only certified workers wearing protective clothes with the label Biohazard have
access into these areas. These protective clothes can be weared only in these areas.
v) In case of GMO introduction into the environment.
Not referred.
w) Time limitation of these regulations.
The regulations for working with genetically modified organisms are valid until cancellation
of Palacky University rector.
Guidance:
The regulations for working with genetically modified organisms must have the appropriate
certified number of pages. Removal or damage of the pages is forbidden. The regulations for
working with genetically modified organisms must be maintained according to § 3 sec. 7 b)
code 78/2004 Sb.
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Expert advisor pronouncement:
The workers have long-lasting experience with dealing with GMO. The regulations for
working with genetically modified organisms contains all necessary appurtenances needed for
dealing with all species of genetically modified organisms.
Prague, November 11, 2007
RNDr. Oldřich Navrátil, CSc.
Laboratory regime:
Selected laboratories are specified for working with microbial cultures, plant cultures treated
with genetically modified (GM) bacteria Agrobacterium. Plasmids are kept in bacterial strain
Escherichia coli or in yeast Saccharomyces cerevicae, Pichia pastoris. Experiments with
transgenic bacteria, plant tissues and sterile or non-sterile cultivated transgenic plants are
investigated for scientific purposes. That is the reason why all regulations for working in
microbiological laboratories with transgenic plant material must be adhered.
Liquid waste and GMO liquidation: Pouring out any kind of liquid waste and GMO without
previous sanitizing is strictly prohibited. Agars and liquid cultures can be liquidated after
autoclaving or other feasible treatment only (liquid cultures can be sanitized by bleach powder
treatment). Solid waste containing GMO as well as other equipment that was contaminated by
GMO must be inactivated in sterilizing boxes by steam sterilization. Waste water containing
GMO must be inactivated by bleach powder or chloramine. Eating, drinking or smoking in
laboratories is strictly prohibited. In laboratories where dealing with GMO is allowed is
necessary to wear protective coat. It is necessary to use protective gloves while working with
GMO.
Cultivation rooms regime:
Selected cultivation rooms are specified for working with microbial cultures, plant tissues and
plant cultures containing inserted DNA. These plants are cultivated under sterile and nonsterile conditions. That is the reason why all regulations for working in microbiology
laboratories with transgenic plant materials must be adhered.
The cultivation rooms are marked with a sign “Zákaz vstupu nepovolaným osobám“, which
means that nobody can access the area but trained people working in that area. Access to these
areas is allowed with a protective coat only. The same regulations as in article Laboratory
Regime apply for liquid or solid waste and waste water. Transport of genetically modified
plants between laboratories and cultivation rooms is allowed only in enclosed containers.
GMO material transportation among the areas and workplaces of PřF UP in Olomouc
intended for dealing with GMO
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Bacterial cultures transportation: bacterial cultures in glass blasks can be transported in
enclosed containers only.
Transportation of Flowering plants, plants with seeds or with vegetative parts growing in soil:
Transportation allowed in enclosed containers only.
GMO transportation out of the workplace PřF UP Olomouc - Holice
Transportation allowed in enclosed containers only.
Regulations:
• All transgenic plants must be regarded as potentionally dangerous transgenic material
until confirming they are not.
• It is necessary to deal with all plant tissue cultures grown in vitro as suspensions or agar
cultures co-cultivated with bacteria A. tumefaciens (and similar strains) as with bacterial
cultures. All instruments and equipment used for dealing with these cultures must be
liquidated by autoclaving.
• All workers must change their clothes, keep records of their work for 10 yrs, certify the
experiments and know the Emergency Plan.
• All experiments must be labeled and marked appropriately and kept from trespassers and
uninformed persons.
Elaborated by Doc. RNDr. Milan Navrátil, CSc.
Olomouc, November 11, 2007
prof. RNDr. Lubomír Dvořák, CSc.
rektor UP
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