Team:UANL Mty-Mexico/Safety/Organisms

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Factory Visit
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ORGANISMS
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<p align="justify"><b>BIOCIENCIA BIOSECURITY PROCESS</b><br><br>In order to learn about biosecurity processes in companies related with our project, we visited BioCiencia S.A de C.V, a private company where, molecular biologists and agronomists, parasite engineers work, they have extensive experience, theoretical and in practice with national agriculture problems. They have a traditional diagnostic laboratory and a molecular plant where they are able to detect pathogens (caused by fungi, bacteria, viruses, viroids, nematodes and Mycoplasmas), using the most advanced technologies at a national and international level such as ELISA, PCR (Polymerase Chain Reaction), RT-PCR (reverse transcription-PCR), Immuno-capture RT-PCR and hybridization of nucleic acids with no radioactive probes, among others. Their laboratory is approved by SAGARPA, (Secretaría de agricultura, ganadería, desarrollo rural, pesca y alimentación) (Key approval no. 98-719 -001-F).<br><br>It was a pleasant surprise to discover that, a company that uses molecular biology in the detection of plant diseases was in our community. Their function is closely related to our project because while we develop a bioinseticide to control the plague Premnotrypesvorax, that attacks potatoe corps; they are part of the 13 plant pathology and entomologyindustries in México that check and approve fruit quality, vegetables and legumes, that are put in supermarkets to be sold.<br><br>BioCiencia was very accessible company, when they let us visit their laboratories. They were very at the moment of showing their laboratories and processes; they also explained the security standards that their installations need to satisfy in order to be approved as an official SAGARPA laboratory. Talking about the diagnostic process, first they receive phytosanitary samples that previously fulfilled necessary requirements to enter the laboratory, without taking into account if they are sick or healthy. Then, the samples are passed to the third part of the laboratory, which is divided in three other parts, the sample stays there until they are processed, liquefied or macerated. The samples are passed to the ISO format to have a better organization. After that they make the diagnose to see what kind of plague, bacteria or virus they are dealing with, that is directly affecting the plants health, they do this by two methods, the most common, PCR, for bacteria and virus cultures, and ISA, for the detection of proteins. All the culture media is sterilized.<br><br>Biosecurity is defined as the set of preventive measures designed to maintain control of occupational risk factors from biological, physical or chemical issues, preventing harmful impacts. The importance of security in the laboratory goes farther, because a failure in the process can cause the loss of the material and the whole work. To preventing this and in case of the client wanting a review on the product, the laboratory keeps samples for 30 days.<br><br>this area of work has a great impact, because it reduces the risk of new plagues to enter national territory. In order to be certificated by the SAGARPA and EMA, Mexican Accreditation Entity, the installations are cheeked each year up to a 40% and each four years to a full 100%. Besides fulfilling the basic requirements of every laboratory, as the usage of appropriate safety equipment and following the registry on each instrument, it has to satisfy the requirements of the ISO/IEC 17025, the Mexican Institute of Standardization and Certification A.C., and the general requirements for the competence of testing and calibration laboratories, that is the main ISO/CASCO standard used by testing and calibration laboratories. The contents of ISO/IEC 17025 standard itself with five elements which are: Scope, Normative References, Terms and Definitions, Management Requirements and Technical Requirements. The two main sections in ISO/IEC 17025 are Management Requirements and Technical Requirements. Management requirements are primarily related to the operation and effectiveness of the quality management system within the laboratory. Technical requirements include factors which determine the correctness and reliability of the tests and calibrations performed in laboratory.<br><br>Laboratories use ISO/IEC 17025 to have the standardized quality system aimed at improving their ability to consistently produce valid results. In the case of BioCiencia, the material they use is obtained from AGDIA, the World Leader in Plant Pathogen Test Kits, and NEOGEN. And all the results obtained from the studies they make, are put monthly in a DVD; they also need to have a common server and a hard disk drive, which, depending on the importance of the document, is the amount of people that has access to it. Noteworthy that all the staff working in BioCiencia has at least a master’s degree.  Another important point of this company is, that the results from the plants diagnostic, are strictly confidential to the customer. <br><br>The visit to BioCiencia made us realize the importance of having an effective security process, as well as the fulfillment of its normativity, as an essential part of the general process. If both are not satisfied, neither will be completed acquired, thus we cannot consider any kind of process complete. Security optimizes the process, time, materials, human resources, and reduces the margin of error inside the laboratory. We realized that for our own project we will need to follow what we learned in this visit in order to continue improving our work.</p><p><b>BioCiencia website:</b> http://www.biociencia.com.mx/inicio.html<br><b>General Director:</b> Dr.Ramiro Gonzalez Garza</p>
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<p align="justify"><div class="Estilo8"><b>BACTERIOPHAGE P1</b></div><br><br>
 +
 
 +
<p align="justify"><b>Organism description</b><br>
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Bacteriophage P1 has been widely used to construct new bacterial strains and was used extensively to map the Escherichia coli chromosome. P1 has served as a model organism
 +
for different aspects of phage and biology such as DNA restriction modification, site-specific recombination and plasmid replication (Łobocka MB, et al 2004).<br>
 +
 
 +
Understanding phage genetics allowed the develop- ment of major basic tools still in use in recombinant DNA technologies. Phage P1 exhibits the classical bacteriophage morphology with an icosahedral head, 220nm long inflexible tail with a complete tube surrounded by a contractile sheath, baseplate and six kinked tail fibers. The icosahedral head contains the phage genome. A variable part(encoded by an invertible segment of P1 DNA) of the tail fibers(1 to 2nm thick) determines the specificity of P1 adsorption on different hosts.<br>
 +
 
 +
P1, like lambda, made its mark early in molecular biology. The significant capacity of P1 for mediating generalized transduction led promptly to P1 becoming a workhorse of genetic exchange among strains of E. coli, a role it is still playing today. Moreover, because P1 can package slightly more than twice as much DNA as can, and packaging can be efficiently carried out in vitro, P1-based vectors are now in common use for cloning and in vitro packaging of eukaryotic DNA. The recognition that P1 is maintained as a plasmid prophage led to the identification of its plasmid maintenance functions (Łobocka MB, et al 2004).<br>
 +
 
 +
<p align="justify"><b>Properties</b><br>
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 +
<b>Plasmid 40784: BBa_J72113-BBa_J72152</b><br>
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 +
<ul><li>Gene/insert name: Phagemid + GFP</li>
 +
<li>Vector backbone: p15a, CamR</li>
 +
<li>Vector type: Bacterial Expression, SynBio</li>
 +
<li>Bacterial resistance(s): Chloramphenicol</li>
 +
<li>Growth strain(s): DH10B</li>
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<li>Growth temperature (℃): 37℃</li>
 +
<li>High or low copy: High Copy</li>
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<li>Terms and Licenses: UBMTA Ancillary Agreement for Plasmids Containing FP Materials. </li>
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<li>Comments: For detailed composition information, look up the backbone (BBa_J72113) and insert (BBa_J72152) on the Registry of Standard Biological Parts. There is a G insert at 5860 of depositor's seq, in between the "phagemid" and GFP, T301C, T459C, these do not effect function. Addgene has sequenced a portion of this plasmid for verification. Full plasmid sequence is available only if provided by the depositing laboratory.</li>
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</ul>
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<p align="justify"><b>Plasmid 40780: BBa_J72110-BBa_J72103</b>
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 +
<ul><li>Gene/insert name: AraC-pBad + coi + cin + repL + pacA</li>
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<li>Insert size: 4722</li>
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<li>Vector backbone: p15a, AmpR, CamR</li>
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<li>Vector type: Bacterial Expression, SynBio; Phagemid</li>
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<li>Bacterial resistance(s): Ampicillin</li>
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<li>Growth strain(s): DH10B</li>
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<li>Growth temperature (℃): 37℃</li>
 +
<li>High or low copy: High Copy</li>
 +
<li>Terms and Licenses: UBMTA Ancillary Agreement for Plasmids Containing FP Materials.</li>
 +
<li>Comments: For detailed composition information, look up the backbone (BBa_J72110) and insert (BBa_J72103) on the Registry of Standard Biological Parts. T459C and T301C in AraC, C4096T, deletion T4313, C4425T, G insert at 4912, do not effect function. Addgene has sequenced a portion of this plasmid for verification. Full plasmid sequence is available only if provided by the depositing laboratory.</li>
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</ul><br>
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 +
<p align="justify"><b>Potential risks</b><br><br>
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 +
Although most bacteriophages do not represent a threat to human health (unless they are carrying virulence factors), the use of recombinant viral particles in some instances might raise some biosafety concerns by bringing and potentially disseminating new genetic traits among bacterial populations. Aspects that should be considered are the expected survival of the phage, its multiplication and dissemination in the identified ecosystems, and the anticipated interaction among the phage and the organisms likely to be present in the exposed ecosystems.<br>
 +
The genetic information encoded by the bacteriophage, its ability to transfer its genome among bacterial hosts, its host spectrum as well as its persistence in the environment are properties that should be assessed. From a biosafety point of view, the introduction of new genetic material into the bacterial gene pool may have positive, negative, or neutral outcomes depending on the genetic marker introduced; thus, the nature of this genetic marker is of great importance.<br>  
 +
The survival of a bacteriophage outside a host is extremely variable and depends on the nature of the phage itself. It is also highly influenced by surrounding environmental conditions, such as pH, ionic strength, temperature, soil structure, adsorption property, or sunlight.
 +
For the bacteriophage P1 to survive in the environment, they need to infect susceptible bacteria hosts to replicate and propagate themselves. The host range of a bacteriophage, defined by which bacterial strains can be infected, depends on the host cell surface receptor (proteins, lipopolysaccharide, or other cell surface components) recognized by this phage. Many phages are known to be highly specific for their receptors and are therefore characterized by a narrow host range, limiting their infectivity to a single species or to specific bacterial strains within a species.<br>
 +
Phages are obligate parasites, and their use will inevitably involve the manipulation of their bacterial hosts. Consequently, assessing the risk of the activity necessitates the evaluation of the susceptible bacteria involved. Risks associated with their manipulation depend mainly on the nature of the inserted genetic material and the bacterial hosts used to propagate these vectors and should be assessed on a case-by-case basis. In this laboratory where we use non-pathogenic E. coli laboratory strains, in this case, both phage P1 and the bacterial strains do not represent any risk to human health with the correct manipulation techniques, and therefore the risk of such an activity could be considered negligible, also because the genetic material used in this project is not pathogenic, thus is not considered hazardous.<br>
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<p align="justify"><div class="Estilo8"><b> <i>E. coli</i> - m1061 (DH10B derivative) </b></div><br><br>
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<p align="justify"><b>Organism description</b><br>
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Escherichia coli is a member of the family Enterobacteriaceae and is a Gram-negative rod which can be motile by peritrichous flagella or nonmotile. Escherichia is also a facultative anaerobe which has both a respiratory and a fermentative type of metabolism, and commonly occurs in the intestinal tract of humans and other animals.<br>
 +
 
 +
E. coli belongs to the taxonomic family known as Enterobacteriaceae, which is one of the best-defined groups of bacteria. The strain E. coli K-12 is a debilitated strain which does not normally colonize the human intestine. It has also been shown to survive poorly in the environment, has a history of safe commercial use, and is not known to have adverse effects on microorganisms or plants. Because of its wide use as a model organism in research in microbial genetics and physiology, and its use in industrial applications, E. coli K-12 is one of the most extensively studied microorganisms.<br>
 +
 
 +
<p align="justify"><b>Properties</b><br>
 +
<ul><li>Blue/white selection</li>
 +
<li>While DH10B has been classically reported to be galU galK, the preliminary genome sequence for DH10B indicates that DH10B (and by their lineage also TOP10 and any other MC1061 derivatives)</li>
 +
<li>Genome sequence indicates that DH10B is actually deoR+</li>
 +
<li>Streptomycin resistant</li>
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<li>Leucine auxotroph</li></ul>
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 +
<p align="justify"><b>Potential risks</b><br>
 +
 
 +
 
 +
</p><p><b>BioCiencia website:</b> http://www.biociencia.com.mx/inicio.html<br><b>General Director:</b> Dr.Ramiro Gonzalez Garza</p>
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Revision as of 00:11, 18 October 2014

Safety
ORGANISMS

BACTERIOPHAGE P1


Organism description
Bacteriophage P1 has been widely used to construct new bacterial strains and was used extensively to map the Escherichia coli chromosome. P1 has served as a model organism for different aspects of phage and biology such as DNA restriction modification, site-specific recombination and plasmid replication (Łobocka MB, et al 2004).
Understanding phage genetics allowed the develop- ment of major basic tools still in use in recombinant DNA technologies. Phage P1 exhibits the classical bacteriophage morphology with an icosahedral head, 220nm long inflexible tail with a complete tube surrounded by a contractile sheath, baseplate and six kinked tail fibers. The icosahedral head contains the phage genome. A variable part(encoded by an invertible segment of P1 DNA) of the tail fibers(1 to 2nm thick) determines the specificity of P1 adsorption on different hosts.
P1, like lambda, made its mark early in molecular biology. The significant capacity of P1 for mediating generalized transduction led promptly to P1 becoming a workhorse of genetic exchange among strains of E. coli, a role it is still playing today. Moreover, because P1 can package slightly more than twice as much DNA as can, and packaging can be efficiently carried out in vitro, P1-based vectors are now in common use for cloning and in vitro packaging of eukaryotic DNA. The recognition that P1 is maintained as a plasmid prophage led to the identification of its plasmid maintenance functions (Łobocka MB, et al 2004).

Properties
Plasmid 40784: BBa_J72113-BBa_J72152

  • Gene/insert name: Phagemid + GFP
  • Vector backbone: p15a, CamR
  • Vector type: Bacterial Expression, SynBio
  • Bacterial resistance(s): Chloramphenicol
  • Growth strain(s): DH10B
  • Growth temperature (℃): 37℃
  • High or low copy: High Copy
  • Terms and Licenses: UBMTA Ancillary Agreement for Plasmids Containing FP Materials. 
  • Comments: For detailed composition information, look up the backbone (BBa_J72113) and insert (BBa_J72152) on the Registry of Standard Biological Parts. There is a G insert at 5860 of depositor's seq, in between the "phagemid" and GFP, T301C, T459C, these do not effect function. Addgene has sequenced a portion of this plasmid for verification. Full plasmid sequence is available only if provided by the depositing laboratory.

Plasmid 40780: BBa_J72110-BBa_J72103

  • Gene/insert name: AraC-pBad + coi + cin + repL + pacA
  • Insert size: 4722
  • Vector backbone: p15a, AmpR, CamR
  • Vector type: Bacterial Expression, SynBio; Phagemid
  • Bacterial resistance(s): Ampicillin
  • Growth strain(s): DH10B
  • Growth temperature (℃): 37℃
  • High or low copy: High Copy
  • Terms and Licenses: UBMTA Ancillary Agreement for Plasmids Containing FP Materials.
  • Comments: For detailed composition information, look up the backbone (BBa_J72110) and insert (BBa_J72103) on the Registry of Standard Biological Parts. T459C and T301C in AraC, C4096T, deletion T4313, C4425T, G insert at 4912, do not effect function. Addgene has sequenced a portion of this plasmid for verification. Full plasmid sequence is available only if provided by the depositing laboratory.

Potential risks

Although most bacteriophages do not represent a threat to human health (unless they are carrying virulence factors), the use of recombinant viral particles in some instances might raise some biosafety concerns by bringing and potentially disseminating new genetic traits among bacterial populations. Aspects that should be considered are the expected survival of the phage, its multiplication and dissemination in the identified ecosystems, and the anticipated interaction among the phage and the organisms likely to be present in the exposed ecosystems.
The genetic information encoded by the bacteriophage, its ability to transfer its genome among bacterial hosts, its host spectrum as well as its persistence in the environment are properties that should be assessed. From a biosafety point of view, the introduction of new genetic material into the bacterial gene pool may have positive, negative, or neutral outcomes depending on the genetic marker introduced; thus, the nature of this genetic marker is of great importance.
The survival of a bacteriophage outside a host is extremely variable and depends on the nature of the phage itself. It is also highly influenced by surrounding environmental conditions, such as pH, ionic strength, temperature, soil structure, adsorption property, or sunlight. For the bacteriophage P1 to survive in the environment, they need to infect susceptible bacteria hosts to replicate and propagate themselves. The host range of a bacteriophage, defined by which bacterial strains can be infected, depends on the host cell surface receptor (proteins, lipopolysaccharide, or other cell surface components) recognized by this phage. Many phages are known to be highly specific for their receptors and are therefore characterized by a narrow host range, limiting their infectivity to a single species or to specific bacterial strains within a species.
Phages are obligate parasites, and their use will inevitably involve the manipulation of their bacterial hosts. Consequently, assessing the risk of the activity necessitates the evaluation of the susceptible bacteria involved. Risks associated with their manipulation depend mainly on the nature of the inserted genetic material and the bacterial hosts used to propagate these vectors and should be assessed on a case-by-case basis. In this laboratory where we use non-pathogenic E. coli laboratory strains, in this case, both phage P1 and the bacterial strains do not represent any risk to human health with the correct manipulation techniques, and therefore the risk of such an activity could be considered negligible, also because the genetic material used in this project is not pathogenic, thus is not considered hazardous.

E. coli - m1061 (DH10B derivative)


Organism description
Escherichia coli is a member of the family Enterobacteriaceae and is a Gram-negative rod which can be motile by peritrichous flagella or nonmotile. Escherichia is also a facultative anaerobe which has both a respiratory and a fermentative type of metabolism, and commonly occurs in the intestinal tract of humans and other animals.
E. coli belongs to the taxonomic family known as Enterobacteriaceae, which is one of the best-defined groups of bacteria. The strain E. coli K-12 is a debilitated strain which does not normally colonize the human intestine. It has also been shown to survive poorly in the environment, has a history of safe commercial use, and is not known to have adverse effects on microorganisms or plants. Because of its wide use as a model organism in research in microbial genetics and physiology, and its use in industrial applications, E. coli K-12 is one of the most extensively studied microorganisms.

Properties

  • Blue/white selection
  • While DH10B has been classically reported to be galU galK, the preliminary genome sequence for DH10B indicates that DH10B (and by their lineage also TOP10 and any other MC1061 derivatives)
  • Genome sequence indicates that DH10B is actually deoR+
  • Streptomycin resistant
  • Leucine auxotroph

Potential risks

BioCiencia website: http://www.biociencia.com.mx/inicio.html
General Director: Dr.Ramiro Gonzalez Garza