Biobrick Safety
The team project will be using some non-virulent parts from a non-virulent strain of Streptococcus pneumoniae.
To avoid any possible infection, we will not work on any form of live S. pneumoniae, but we will only work on
its genomic DNA where we requested from another laboratory.We are also using lab strains E. coli routinely used
in cloning like DH10B and DH5alpha which are safe to operate.
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Species name (including strain)
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Risk Group
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Risk Group Source
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Disease risk to humans?
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Part number/name
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Natural function of part
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How did you acquire it?
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How will you use it?
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Notes
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E.coli (DH10B)
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Group 1
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http://tools.
invitrogen.co
m/content/sfs
/msds/2013/50
0015_MTR-NALT
_EN.pdf
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It may be harmful to people who inhale or swallow. In rare case, such as with susceptible person, it can cause eye and skin irritaion.
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We acquired from Professor King Lau Chow's lab.
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This organism is our chassis.
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E.coli (DH5alpha)
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Group 1
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https://micro
bewiki.kenyon
.edu/index.ph
p/DH5-Alpha_E
.coli
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It may be harmful to people who inhale or swallow. In rare case, such as with susceptible person, it can cause eye and skin irritaion.
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We acquired from Professor King Lau Chow's lab.
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This organism is our chassis.
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comD from Streptococcus pneumoniae
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part DNA that encodes a membrane-bound histidine-kinase receptor, ComD
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"We plan to use PCR to isolate the part from genomic DNA of its parent organism. Also, we have ordered part DNA from Universite de Toulouse, France but have not obtained yet."
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This part produces a membrane-bound histidine-kinase receptor, ComD which enables competence-stimulating peptide (CSP) to bind. This serves as a detector of Streptococcus pneumonia.
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There are no known risk of diseases associated with this gene.
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comE from Streptococcus pneumoniae
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part DNA that encodes a ComE which gets phosphorylated once competence-stimulating peptide (CSP) binds to the receptor, ComD
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We plan to use PCR to isolate the part from genomic DNA of its parent organism. In addition, We ordered the part DNA from the lab of Jean-Pierre Claverys and Patrice Poland in Toulouse. And we obtained three different types of comE; pKHS-comE WT, pKHS-comE (D58E) and pKHS-comE (R120S)
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This part produces ComE which gets phosphorylated and this phosphorylated ComE activates the promoter for transcription of next gene.
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There are no known risk of diseases associated with this gene.
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comX from Streptococcus pneumoniae
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part DNA that encodes an alternative sigma factor that directs the transcription of late competence (com) genes.
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"We plan to use PCR to isolate the part from genomic DNA of its parent organism. Also, we have ordered part DNA from Universite de Toulouse, France but have not obtained yet."
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This part produces a sigma factor that activates the promoter for transcription of late competence gene.
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There are no known risk of diseases associated with this gene.
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comW from Streptococcus pneumoniae
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part DNA that encodes ComW which is involved in activation and stabilization of ComX.
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We plan to use PCR to isolate the part from genomic DNA of its parent organism. Also, We have ordered part DNA from University of Illinois at Chicago and we are waiting for stock to come soon.
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This part produces ComW which enhances activation and stabilization level of ComX.
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There are no known risk of diseases associated with this gene.
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BBa_J01008
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Artificial trans-activating RNA that can unlock cis-repressing RNA to activate translation process by exposing RBS to the ribosomes.
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Oligo annealing
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We will use it to characterize various lock and keys
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Designed by Isaacs et al.
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BBa_J01010
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Artificial cis-repressing RNA that represses translation by forming a loop structure to disperse the RBS.
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Oligo annealing
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We will use it to characterize various lock and keys
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Designed by Isaacs et al.
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BBa_J01086
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Different version trans-activating RNA
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Oligo annealing
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We will use it to characterize various lock and keys
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Designed by Isaacs et al.
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BBa_J01080
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Different version of cis-repressing RNA
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Oligo annealing
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We will use it to characterize various lock and keys
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Designed by Isaacs et al.
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BBa_J23008
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Modified version trans-activating RNA BBa_J01010 that unlocks BBa_J23031
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Oligo annealing
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We will use it to characterize various lock and keys
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Designed by Berkeley 2006
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BBa_J23031
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Modified version of cis-repressing RNA BBa_J01080 the represses translation
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Oligo annealing
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We will use it to characterize various lock and keys
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Designed by Berkeley 2006
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BBa_K175032
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Trans-activating RNA that unlocks BBa_K175031
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Oligo annealing
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We will use it to characterize various lock and keys
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Designed by Delft 2009
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BBa_K175031
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Cis-repressing RNA that disperse B0032 to repress translation
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Oligo annealing
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We will use it to characterize various lock and keys
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Designed by Delft 2009
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Environmental and General Public Safety
Improper disposal of biological waste might result in leak of biological materials to the environment,
which might cause contamination, and improper disposal might also cause infection to those who process the wastes.
However, there is little biological risk associated with our project in the first place, as described above because
we are using innocuous bacteria strains that are unlikely to propagate in the environment. Despite being so, we
autoclave all solid biological wastes before they are discarded as landfill garbage.
We also treat liquid waste with 10% bleach to ensure that no biological waste could escape to the environment.
Commonly used chemicals are mostly buffers and therefore pose little threat and risk to the environment.
For chemicals that are hazardous, they could cause damage to other people if leaked out. We avoid these risks by
collecting these chemical wastes and sending them to a centralized chemical waste disposal
center within our university, where they will be processed by professionals in chemical waste treatment.
The only living biological material that could escaped from the lab would be our E. coli host for DNA cloning and
parts characterization, and there is little risk associated with their leakage because we are using DH10B as the
major cloning host in which the strain is auxotrophic for leucine, and therefore rendering it unlikely to propagation
outside laboratory environment. The strains of E. coli we use in lab, DH10B and DH5alpha, are also routinely used lab
strains that are innocuous and non-virulent.
Researcher Safety
Tacking risk of organism, only non-virulent part was extracted from genomic DNA of non-virulent S. pneumoniae strain
obtained from Professor Patrick Chiu Yat Woo at Hong Kong University.
For chassis, only Biosafety Level 1 organisms (DH10B and DH5alpha) are dealt in the lab.
All members are trained to deliver safe lab practices. In dealing with toxic chemicals such as DMSO, we wear lab coats
and gloves and execute under fume hood.
All experiments are conducted in a lab premise under authorized supervision.
Risks with handling toxic chemicals, including DMSO, phenol and chloroform,
which include chemical burns and damage to the health of team members in the long run, have been addressed.
We wear lab coats and gloves whenever we handle these chemicals to minimize exposure to these chemicals,
and we carry out bulk management of these chemicals inside a well ventilated fume hood.
Risks associated with UV exposure during gel documentation and excision are also minimized by wearing face shields
and gloves until the UV light is off.
University Safety
Health, Safety & Environment Office (HSEO) is responsible for safety regulations at our institution,
The Hong Kong University of Science and Technology, HKUST.
We, team members, have not directly discussed our project with the director of HSEO but, our supervisors have.
After discussing the risks, we decided to operate only on the genomic DNA of a non-virulent strain of Streptococcus pneumoniae and
we are not handling the live bacteria itself.
The biosafety guidline of our institution can be directed to:
http://www.ab.ust.hk/hseo/sm06/ch9.htm
The regulations that govern biosafety in research laboratories in Hong Kong can be directed to:
http://www.legislation.gov.hk/blis_pdf.nsf/6799165D2FEE3FA94825755E0033
E532/CD005BD8FCD84653482576EA0053077B/$FILE/CAP_607_e_b5.pdf
Safety Training Courses
This year's HKUST team members have received safety training over the summer. Topics that we have learned about
in safety training are as following:
Biological Safety Chemical Safety I/ Chemical Safety for Laboratory Users Chemical Safety II/ Hazardous Waste Management
The link to the laboratory safety training requirements of our institution (The Hong Kong University of Science and Technology, HKUST)
can be directed to:
http://www.ab.ust.hk/hseo/training.htm
Reference
Chapter 9 Biological Safety. (2006, November 1). Retrieved October 3, 2014, from
http://www.ab.ust.hk/hseo/sm06/ch9.htm
Health, Safety and Environmental Office. (2014, August 20). Retrieved October 3, 2014, from
http://www.ab.ust.hk/hseo/training.htm
Cap 607 Genetically modified organisms. Retrieved October 3, 2014, from
http://www.legislation.gov.hk/blis_pdf.nsf/6799165D2FEE3FA94825755E0033E532/CD005BD8FCD84653482576EA0053077B/$
FILE/CAP_607_e_b5.pdf
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