069-sra02-fp-domenech-sanchez-npse2013

Design of a Collection of Plasmids as a Tool for Genomic Teaching
Activities
1Antonio Doménech-Sánchez, 2Rafael Bosch
Microbiología (Departament de Biologia) and IUNICS, Universitat de les Illes Balears (Spain) [email protected]; [email protected] Abstract
Due to the improvement in sequencing techniques and the decrease of the associated costs in the last years, genomics is nowadays a leading technology for microbiologists and other live-science researchers. Thus, undergraduate students should start to be in contact with genomic sciences. There are many web facilities that allow students and researches to start the genomic game. Although this, we believe that automatic genomic annotation and analysis tools are good for researchers but they still remain confusing for students, because they are not prepared to understand the basement of genomic technology. One of the most common strategies in the field consists in teaching the genomic basic-analysis using well-characterized DNA fragments. However, frequently these well-characterized DNA fragments are easily found in databases and, therefore, “lazy-and-smart” students are able to finish the genomic analysis (i.e. gene annotation) of the problem DNA-molecule just by comparison with a single well-characterized DNA fragment. In these cases, the objective of the teaching activity is not fulfilled. To avoid this, we have in-silico constructed 12 different bacterial plasmids with sizes ranging from 40,220 to 48,304 bp in length. All of them harbor the replication machinery extracted from different well characterized plasmids. As for the physiological point of view, all plasmids harbor genetic determinants for the degradation of aromatic hydrocarbon. We have combined naphthalene, salicylate, and/or benzoate upper-pathways with catechol meta- and orto-degradation pathways. All degradation pathways’ genes are contained in mobile structures (composite transposons and integrons) that students will be able to identify. Additionally, plasmids contain antibiotic and/or heavy metal resistance genes, also harbored in mobile structures. Time-line acquisition of mobile elements should also be elucidated. The presented material constitutes a valuable tool for teaching activities related to genomics. 1. Background

Genomics is one of the most evolving technologies in the last decade. Initially, genome sequencing
was a tedious and very expensive technology, only affordable by a few groups or consortiums.
However, the scenario has dramatically changed in the last years: sequencing technology has
spectacularly improved, with the associated costs decreasing in a significant manner. Therefore, the
genomic approach is more accessible to institutions and scientists nowadays. As a consequence,
genomics is nowadays a leading technology for microbiologists and other live-science researchers. In
parallel, different applications and databases have been developed to store and manage this huge
amount of information. Altogether, the new scenario has modified the scientists’ approaches to
investigate their hypothesis. Thus, many microbiologists now formulate microbiological questions and
interpret research results in the context of genomic information.
This progress should not be limited only to basic sciences, but must be accordantly incorporated to
teaching activities. Educators need to become familiar to this new knowledge, adjust to the new
situation and develop innovative ways to assist students in the immersion to the novel genomics
sciences. Different teaching strategies have been used in the last years to attain this objective [1-3].
One of the most common strategies in the field consists in teaching the genomic basic-analysis using
well-characterized DNA fragments. However, frequently these well-characterized DNA fragments are
easily found in public databases and, therefore, “lazy-and-smart” students are able to finish the
genomic analysis (i.e. gene annotation) of the problem DNA-molecule just by comparison with a single
well-characterized DNA fragment. In these cases, the objective of the teaching activity is not fulfilled.
To avoid this, the material to be characterized should not be a fragment directly obtained from or
identical to one molecule present in public databases. Besides, DNA fragments specifically designed
for these activities are essential for students to gain the knowledge of genomics information
management. The aim of the present study was to design a collection of plasmids with information
specifically useful for genomic teaching.
2. Plasmid design

All the designed plasmids are constituted by one core and two related metabolic pathways (Figure 1).
In table 1 the origin of each genetic cluster used for the plasmid assembling process is described. In
silico sequence assembling was performed using the BioEdit v. 7.1.9 software [4]. Genetic information
was obtained directly from the GenBank database at the NCBI [5].
Plasmid core included the plasmid mechanisms for maintenance and functionality: the mobilization
region (mob genes) encoding specific relaxosome components and the origin of transfer (oriT); genes
codifying for the different incompatibility groups; the transfer operon (tra genes), which encodes
proteins which are useful for the propagation of the plasmid from the host cell to a compatible donor
cell or maintenance of the plasmid; one integron as a mobile element, including the integrase gene
(intI) and the proximal recombination site (attI); several insertion sites (attC) as putative targets for
new insertion events; and a variety of resistance genes for antimicrobials or toxic compounds.
Figure 1. Illustration of the plasmids described in this study. A: the pE set of plasmids with core- components from Enterobacteria. B: the pP set of plasmids with core-components from Table 1. Gene clusters used for the plasmid construction. Lenght (bp)
Coded functions
Origin (GenBank accesion number, coordinates)
Acidithiobacillus caldus (DQ810790, 600-2910) Salmonella enterica (FJ980445, 858-918) Pseudomonas stutzeri (AY129392, 2205-2274) S. enterica pSPCV plasmid (NC_012124, 14600-15750) S. enterica pSPCV plasmid (NC_012124, 25400-27000) S. enterica pSPCV plasmid (NC_012124, 41700-44200) Plasmid segregation and replication proteins P. putida NAH7 plasmid (AB237655, 1-4500) P. aeruginosa (X14793, 2076-1636/1042-1) Burkholderia cepacia (M82979, 111-1439) B. cepacia (M82979, 1439-882/422-111) P. stutzeri (AF039534, 7100-14570) Trans .regulator & catechol orto-degradation pathway B. mallei (CP000525, 1210808-1211692) P. syringae (AE016853, 4857700-4860650) Transcriptional regulator and salicylate hydroxylase P. putida (AB237655, 43100-45800) Transcriptional regulator and salicylate hydroxylase B. xenovorans (CP000272, 233100-235300) mini-Tn4001PStetM vector (GQ420676, 300-2300) P. putida (AJ344068, 89000-92700) P. putida (AB434906, 92300-94500) toluene/xylene degradation upper-pathway P. putida (AB238971, 63498-68900) P. putida (AB238971, 57300-58400) P. putida (AB238971, 81642-85547) P. stutzeri (NC_015740, 1636694-1637647; NC_009434, 1784916-1785869) P. fluorescens (NC_007492, 2677058-2680948); P. putida (NC_002947, 3581838-3585746) P. stutzeri (NC_015740, 1636694-1637647; NC_009434, 1784916-1785869) P. fluorescens (NC_007492, 2677058-2680948); P. putida (NC_002947, 3581838-3585746) Pseudomonas sp. (AB257758, 1-9671; AF039533, 1-9669; AF491307, 14334-24206; AY208917, 54140-64012) Pseudomonas sp. (AB257758, 1-9671; AF039533, 1-9669; AF491307, 14334-24206; AY208917, 54140-64012) Pseudomonas sp. (AB257758, 1-9671; AF039533, 1-9669; AF491307, 14334-24206; AY208917, 54140-64012) Pseudomonas sp. (AB257758, 1-9671; AF039533, 1-9669; AF491307, 14334-24206; AY208917, 54140-64012) 50 5'-TGAATAGTTAAGCTGTCAGGAAGCCGTAATATCATCGTGTGAATAGTTAA-3' 50 5'-TGAATAGTTAAGACCATCATCATCTCATCATTAGAGCTTGAATAGTTAAG-3' 49 5'-CTGGGTGACGGAAATTTCTGGGATTCCGGCTTACAACCCCTTACGTTTC-3' Table 2. Phenotypes of the pE-series plasmids. Phenotype
Gene cluster
pE-series plasmids
pEMB41625 pEMN48304 pEMT47036 pEOB40779 pEON47087 pEOT46922 Catechol and metilcatecol degradation by extradiol cleavage to TCA Catechol and metilcatechol degradation by intradiol cleavage to TCA Table 3. Phenotypes of the pE-series plasmids. Phenotype
Gene cluster
pP-series plasmids
pPMB34809 pPMN41488 pPMT40220 pPOB34563 pPON41026 pPOT40706 Catechol and metilcatecol degradation by extradiol cleavage to TCA Catechol and metilcatechol degradation by intradiol cleavage to TCA Plasmid core included the plasmid mechanisms for maintenance and functionality: the mobilization
region (mob genes) encoding specific relaxosome components and the origin of transfer (oriT); genes
codifying for the different incompatibility groups; the transfer operon (tra genes), which encodes
proteins which are useful for the propagation of the plasmid from the host cell to a compatible donor
cell or maintenance of the plasmid; one integron as a mobile element, including the integrase gene
(intI) and the proximal recombination site (attI); several insertion sites (attC) as putative targets for
new insertion events; and a variety of resistance genes for antimicrobials or toxic compounds.
Two different plasmid cores were constructed: pE and pP. The former was designed by sequences
from the Enterobacteriaceae family, whereas the latter was constructed from sequences from the
Pseudomonadaceae family (see Table 1). The pE plasmid core contained: i) the transfer operon from
plasmid pR100; ii) the IncFII incompatibility group determinant; iii) the mob genes from pCE10B
plasmid; iv) the integrase IntI1 from Salmonella enterica; v) a kanamycin-resistance cassette obtained
from p5E-acta1 vector; vi) a tetracycline-resistance cassette from mini-Tn4001PStetM vector; and vi)
and a ampicillin-resistant cassette from pTJ1 vector. Additionally, up to five attC insertion sites were
also distributed among the core. Finally, some non-coding sequences were included at the beginning
and the end of the core. For the pP plasmid core, no transfer genes were included. The initial non-
coding sequence was followed by the Inc-P9 incompatibility group genes from P. putida NAH7
plasmid, the PstIncIQ integrase from P. stutzeri and two toxic compounds resistance cassettes (one
for arsenic and one for potassium tellurite, from Acidithiobacillus caldus and the pBTBSh-6 vector,
respectively). These resistance determinants were flanked by attC insertion sites. The core was
completed with a sequence fragment without coding information.
The main metabolic pathway coded for catechol degradation activities. This route was inserted by a
transposon-like structure into one of the resistance genes present in the core and, therefore, the
resistance gene was inactivated. This structure consisted in a composite transposon derived from Tn3
minitransposon, where the operon genes for cathecol degradation were flanked by mini-Tn3
sequences. Non-coding regions were inserted between the operon and the left and right mini-Tn3
sequences for insertion purposes (see below).
Two different catechol degradation pathways have been used in the present study: the meta-pathway
and the orto-pathway. Nevertheless, only one of the two pathways was carried by each plasmid. In the
case of the plasmids from Enterobacteriaceae (pE), the meta-pathway (M) was inserted into the
kanamycin-resistance cassette of the core, thus originating the set of plasmids we have assigned as
pEM; alternatively, the orto-pathway (O) was instead introduced into the sequence of the tetracycline-
resistance determinant of the core, and then the pEO set of plasmids were obtained. Regarding to the
plasmids from Pseudomonadaceae (pP), the meta-pathway was included into the arsenic-resistance
cassette, giving the pPM set; and the orto-pathway was inserted into the sequence of the potassium
tellurite-resistance determinant to obtain the pPO set. Consequently, four different sets of plasmids
able to metabolize the cathecol molecule were obtained.
To enhance the metabolic diversity of our plasmids, a second metabolic pathway was included into the
above plasmids. In all cases, the second metabolic route was inserted into the non-coding regions of
the composite transposon containing the cathecol degradation pathway (Figure 1). An insertion event
was simulated by the presence of insertion sequences located each side of the metabolic route.
Finally, a collection of plasmids with different metabolic capacities were obtained. Their phenotypes
are shown in tables 2 and 3.
This collection represents a useful tool for genomic teaching activities, including annotation, detection
of replication/segregation and mobilization genes, insertion events and phenotypic characterization.
3. Acknowledgments
This study was supported by a grant (“Projectes d’innovació i millora de la qualitat docent 2012”) from the University of the Balearic Islands. References
[1] Campbell AM (2003) Public access for teaching genomics, proteomics, and bioinformatics. Cell [2] Ditty JL et al. (2010) Incorporating Genomics and Bioinformatics across the Life Sciences [3] Quevedo Garcia et al. (2011) Teaching Strategies to Incorporate Genomics Education into Academic Nursing Curricula. J Nurs Educ 50(11):612-619. [4] http:// http://www.mbio.ncsu.edu/bioedit/bioedit.html [5] http://www.ncbi.nlm.nih.gov

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