Seminario biologia molecular nuevo

Presentado por:

María Alejandra Nicholls Molina

Daniela Rios Carmona

Introduction• AMS emphasis on learning activities that promote scientific thinking

and critical reasoning.• Train students in all invaluable skills within the diagnostic laboratory.• Many programs have done research projects to generate novel

experimental data in the form of ALUREs.

• Done because an introductory microbiology course for second year students within a four year undergraduate science program.

• Prerequisite student knowledge.

Terminology• Difference between community and hospital acquired

infections

ALURE

• Authentic Large-scale Undergraduate Research Experience is a method in which the student himself drives a research project designed to generate novel experimental data.

• ALURE was done for: experience in technical laboratory skills, critical reasoning and problem solving skills within diagnostic microbiology

PCR

• Polymerase chain reaction is a tool used to focus in segments of the DNA and copy it billions times over; is used mostly to diagnose diseases, identify bacteria and viruses.

• In this study was used because they wanted to amplificate mouth swab DNA by conducting mouth swabs for bacterial DNA extraction

Relation

• Learning techniques (ALURE)• Culture• Extract genomic DNA• 16S rRNA (sequencing)• Identify Micro organisms on mouth swabs

General objective

• Develop confidence in undergraduate students in technical skills for laboratory, scientific reasoning, and skills in problem solving within diagnostic microbiology, by an ALURE in mapping the human oral microbiome to learn about microbial identification and the relative merits of culture dependent vs culture independent methods, distinguishing the bacterial composition of healthy and diseased oral cavities.

Materiales y Métodos

Sesión I: Creando habilidad• Microscopía• Cultivo• Gram• Platos de TSA• Kit de tinción de gram

incubación de muestras inoculadas a 37ºC durante 24

horas y almacenamiento a 4ºC hasta la sesión 2

Materiales y Métodos

Sesión II:muestreo de la microbioma oral humana• Extracción de DNAg de los hisopos

bucales• PCR (amplificación de genes 16S

RNAr usando los primers: 803F; 803Fd; 1392wR) electroforesis

Realizar hisopos bucales para la extracción de ADN bacteriano (por PCR y secuenciación de 16S RNAr) y la inoculación de medios selectivos y diferenciales de cultivo (agar sangre, agar sal manitol y placas de agar)

Materiales y Métodos• 2 semanas requeridas para optimizar

la amplificación por PCR de ADN de la muestra bucal, la secuenciación de ADN, y la agrupación en unidades taxonómicas operacionales (OTU). Imágenes electroforesis en gel y tablas OTU deben estar preparados para los estudiantes de la sesión 4.

• incubación de muestras inoculadas a 37ºC durante 24 horas y almacenamiento a 4ºC hasta la sesión 3

Materiales y Métodos

Sesión III: identificación de microbios orales (cultivo dependientes)• Identificación, que incluye kit

de coloración de gram, microscopía, mechero, platos de agar inoculados

Identificación de la microbiota bucal de los

cultivos basada en tinción de Gram, el crecimiento de colonias en medios de agar

selectivo y diferencial, pruebas bioquímicas y pruebas inmunológicas

Materiales y Métodos

• 1-2 días (dependiendo del tamaño de clase) para preparar la demostración de microorganismos, kits de pruebas bioquímicas e inmunológicas.

Materiales y Métodos

Sesión IV: Identificación de microbios orales (cultivo independientes)• Secuenciar• Roche 454 GS-FLX Titanium

platform• Acacia software• OTU (97% de la información

agrupada)• Base de datos de referencia

Greengenes

análisis e interpretación de los datos recogidos a través de identificación de cultivo dependiente e independiente de la microbioma oral, a través de cohorte de estudiantes

Materiales y Métodos

• 1-2 días para recopilar imágenes de electroforesis en gel y tablas OTU para todos los estudiantes dentro de cohorte.

Resultados

Explicación e interpretación de los datos

Calificación Justificación

Reprueba No muestra una correcta comprensión de la diferencia entre los métodos de identificación

Pasa Falta profundidad y detalles al describir los datos obtenidos

Pasa con alto nivel Además de la correcta descripción utiliza citas de otras bibliografías

Tabla 5. Criterios de evaluación

Uso de fuentes bibliográficas para evaluar críticamente los resultados

Tabla 6. Criterios de evaluación

Calificación Justificación

Reprueba Incorrecta comparación entre el estudio citado y el propio experimento

Pasa Comparación válida, omite el tamaño de la muestra y los datos demográficos de los voluntarios

Pasa con alto nivel Comparación válida, profunda discusión de los datos demográficos de la población

Conclusions

Author Citation Agreed or disagreed

Merkel, S. “Furthermore, a majority of students received a High Pass forthe criteria relating to explanation and interpretation of resultsand using sources for critical evaluation in both 2012 and 2013,which represent core competencies that emphasize scientificthinking and deeper conceptual understanding and align withlearning objectives 4 and 5”

Author Citation Agreed or disagreed

Hanauer, D.I, D.Jacob-Sera, M. L.Pedulla, S. G.Cresawn, R.W. Hendrix, and G.F.Hatfull.Lord, T., and T. Orkwiszewski.Wang, J. T. H., M. A. Schembri, M. Ramakrishna,E. Sagulenko, and J. A. Fuerst.Weaver, G. C., C. B. Russell, and D. J. Wink.

“The positive impactof this ALURE on student development of research skills isconsistent with previous reports in chemistry, biochemistry, andmicrobiology (12, 19, 26, 27), and this project has been able toprovide another documented case of integrative research experiencesthat are adaptable for large undergraduate classes”

Author Citation Agreed or disagreed

Petrosino, J. F., S. Highlander, R. A. Luna, R. A. Gibbs,and J. Versalovic.

“A similar experimentalapproach could be adopted in mapping the microbiomeacross different parts of the human body, in line with theholistic approach adopted by the Human MicrobiomeProject the culture-dependent tests and diagnosticstandard operating procedures would need to be adjustedaccordingly depending on common resident microbiota atthe respective body sites”

Author Citation Agreed or disagreed

Kroes, I., P. W. Lepp, and D. A. Relman.

“If next-generation sequencing technology is not availablefor potential adopters, 16S rRNA amplicons can be ligated intoplasmids before direct plasmid sequencing of the PCR fragments, which is feasible for smaller class sizes”

Conclusion #1

This type of study will help the future of science itself because it will provide to our society more prepared professionals, capable of critical reasoning, and more experience in the laboratory field, and so that more and more undergraduate students will make a difference in science research since such a young age.

Conclusion #2

The results that are shown prove that the majority of the undergraduate students have learned and achieved the main objectives of the study, and they didn’t do it poorly as it is perfectly clear that most of the student population has obtained a rating that exceeds excellence.

Conclusion #3

Also this type of studies that helps students improve in their laboratory techniques will help us to have every time less mistakes and have the right results so we can give more accurate diagnosis and treatments for community and hospital-acquired infections.

Conclusion #4

Most of us are not conscious of the amount of bacteria and microorganisms that naturally live in our body, with this study we were able to identify the different types of microbiome with a large rate of techniques.

Alejandra Nicholls Molina

Daniela Rios Carmona