Isolation, Purification and Characterization of Gêmeos and

Isinaayusferti, bacteriophages Isolated from Tropical Soils

Amanda M. Icazatti Burtell, Fernando Pachecho Ocasio, John Rivera Meléndez & Leyda Villagrasa Méndez

Dr. Michael RubinRISE Program

University of Puerto Rico at Cayey

Introduction

• What is a bacteriophage?• What host bacteria are being used?– Mycobacterium smegmatis– Bacillus cereus

• Where can bacteriophages be found?• What do bacteriophages need for living?• Types of life cycles: Lytic vs. Lysogenic cycle

• Why are bacteriophages important?Phages can be used in various fields of science such

as: - Genetics - Cloning and mutation of DNA and

studies in genome - Therapeutic uses for the creation of antibiotics

resistant to bacteria

• What is the purpose of this experiment?

Problem and Hypothesis

• Problem: Can novel and interesting bacteriophages be isolated from the tropical soils of Puerto Rico?

• Hypothesis: Both Mycobacteriphages and Bacilophages will be found on the tropical soils of Puerto Rico.

Procedure

Collection of SoilEnrichment Centrifuge

FiltrationStreakin Process: Purification X3

DilutionsSpot test

Empirical testWeb pattern

Preliminary Results

Sample Coordinates Details Depth Temperature Date Place

Sample #1AMIB

Lat: 18.21561L:on: -66.044790

Humid and Dry soil

3’’ 25˚C 02/02/14 Caguas, PR

Sample # 2AMIB

Lat: 18.234378Lon:-66.061491

Dry soil 2’’ 25˚C 02/17/14 Caguas, PR

Sample #3AMIB

Lat: 18.234604Lon:-66.06138

Humid and Dry soil

1’’ 27˚C 02/24/14 Caguas, PR

Sample #4AMIB

Lat: 18.213103Lat: -66.041709

Humid and Damp soil ’

3’’ 26˚C 03/04/14 Caguas, PR

Sample #5AMIB

Lat: 18.215715Lon: -66.044879

Dry soil 1’’ 25˚C 03/11/14 Caguas, PR

Sample #1FPO

CoordinatesLat: 18.11’15”NL:on: 65.57’54”

DetailsDry soil

Depth3”

Temperature24c

Date04/02/14

Place San Lorenzo, Pr

Sample #2FPO

Lat: 18.11’15”NLon: 65.57’53”W

Very dry soil.

4” 26˚C 18/02/14 San Lorenzo, Pr

Sample #3FPO

Lat: 18.7’6”NLon: 66.9’42”W

Humind Soil

3” 28˚C 18/02/14 Cayey, Pr

Sample #4FPO

Lat: 18.11’15”NLon: 65.57’53”W

Humid Soil

4” 22˚C 26/02/14 San Lorenzo, Pr

Sample #5FPO

Lat: 18.2’26”NLon: 66.35’35”

Humid Soil

6” 30˚C 02/03/14 Ponce, Pr

Gêmeos (bacilophage)• Coordenates: Lat: 23' 36" 23 S Long: 46' 41" 51 W• Temperature: 19˚C• Details: Dark and damp soil • Found in a populated area near a river• Sao Paolo. Brazil

Gêmeos: Preliminary Results

Sample Coordenates Details Depth Temperature Date Place

Sample #1LVM

Lat: 18.2333419Long:-66.0478104

Humid and plain soil

2” 26˚C Feb/3/14 Caguas, PR

Sample # 2LVM

LAT: 18.1192448LONG: -66.1667764

Dry and lumpy soil

1” 25˚C Feb/16/14

Cayey, PR

Sample #3LVM

Lat: 18.2333419Long:-66.0478104

Humid and plain soil

3” 27˚C Feb/23/14

Caguas, PR

Sample Coordenates Details Depth Temperature

Date Place

Sample #1JLR

Lat: 18ᵒN Long: 66ᵒW

Humid and saturated soil

1‘’ 31˚C Feb/3/14 Cidra, PR

Sample # 2JLR

Lat: 18ᵒNLong: 66ᵒW

Dry soil under trash disposal

Surface sample

32˚C Feb/21/14 Cidra, PR

Isinaayusferti (mycobacteriophage)Coordenates: Lat: 18˚ 14’ .0312 " N Long: 66˚ 2' 52.116" WTemperature: 27 ˚ C Place: Caguas, Puerto RicoDetails: Humid and plain soil. Found near the garbage in front of a house.

Isinaayusferti (mycobacteriophage)

Isinaayusferti: Preliminary Results

High Titer Phage Lisate

Conclusion

• The soil samples were collected and isolated using the enrichment method.

• A bacillophage was found in Sao Paolo, Brazil and was named Gêmeos.

• Third Purification Stock Filtrate (Gêmeos)• A mycobacteriophage found in Caguas, Puerto

Rico was named Isinaayusferti.• Concentrated Stock Phage (Isinaayusferti)

Acknoledgements

• RISE Program• Dr. Michael Rubin• Dr. Eneida Díaz• Gustavo Martínez• Giovanni Cruz• Christopher Quintanal