Mycobacteriophages:The Journey

Juan E. Maldonado WengAlejandra González TorresGustavo A. Martínez MuñizAngelín M. Figueroa

The Beginning:Mycobacteriaphages

Viruses that infect bacteriaSimilar to fingerprints, no identical phages have been identifiedGreat example of evolution and diversity“Natural predators” of bacteriaSo many, we want to analyze them all

The Beginning:The Reproduction

Types of ReproductionLyticLysogenic

Lifestyles:LyticTemperate

The Beginning:The Diagrams

http://diverge.hunter.cuny.edu/~weigang/Images/13-12_lysogeniccycle_1.jpg

The Beginning:The Diagrams

http://faculty.ccbcmd.edu/courses/bio141/lecguide/unit3/viruses/pen_lyt.html

http://faculty.ccbcmd.edu/courses/bio141/lecguide/unit3/viruses/lysosum.html

The Plan:The Collection

Sterilize conical tubeSoil collection from different citiesSpoon to transfer the soil sample in the conical tubeFill the sterilized tube Data Recorded

Date and time Air temperatureLocationDepthMoisture contentFeatures of the site

The Plan:Materials and Methods

EnrichmentFiltration

Sterilize filterTuberculin syringesPipettes

Plaque screeningMycobacterium smegmatis culture tubesPositive controlPhage bufferTop agar

The Plan:Materials and Methods

Purification (looking for phages)Three purificationMycobacterium smegmatis cultureAgar plate

Plaque streakTiter assay

To determine plaque-forming units concentration

The Plan:The Strategy

The Data

Gustavo's Collections

Collection # Date Time Air Temp. (Celsius) Depth Moisture Features GPS Latitude GPS Longitude

1 2/18/2013 5:44 P.M. 26.7 2-2.5 cm Dry/Moist Rural 18.21398 -65.995476

2 2/28/2013 4:13P.M. 30 3cm Saturated Rural 18.22168 -65.996278

3 3/10/2013 6:25 P.M. 22.2 10 cm Damp Rural 18.215628 -65.995694

4 3/17/2013 7:16 P.M. 26.1 1 cm Moist Rural 18.218559 -65.998019

The Data

Gustavo Collection #1Date: 02/18/2013Time 17:44/5:44 P.M.GPS Latitude: 18.21398GPS Longitude: -65.995476Air temp (approx.): 26.7 degrees CelsiusDepth: 2-2.5 cmMoisture content (approx.): dry/moistFeatures

RuralNext to water tank cement box, and ficus rootsNot sloped.

The Data

Gustavo Collection #2Date: 02/28/2013Time: 16:13/4:13 P.M.GPS Latitude: 18.221680 GPS Longitude: -65.996278Air temperature (approx.): 30 degrees CelsiusDepth: 3 cmMoisture content: saturatedFeatures

RuralIn a small field close to a riverFlat soil No constructions nearby

The Data

Gustavo Collection #3Date: 03/10/2013Time: 18:25/6:25 P.M.GPS Latitude: 18.215628GPS Longitude: -65.995694Air temperature (approx.):/ 22.2 degrees CelsiusDepth: 10 cmMoisture content: damp and coolFeatures

Rural areaIn front of a house

The Data

Gustavo Collection #4Date: 03/17/2013Time: 19:16/7:16 P.M.GPS Latitude: 18.218559GPS Longitude: -65.998019Air temperature (approx.): 26.1 degrees CelsiusDepth: 1 cmMoisture content: moist and coolFeatures

Rural areaIn a canalNext to a street

Juan's Collections

Collection # Date Time Air Temp.

(Celsius) Depth Moisture Features GPS Latitude GPS Longitude

1 2/9/2013 6:06 P.M. 28 Superficial Dry/Moist Urban 18.221986 -65. 432580

2 2/18/2013 12:03 P.M. 27 Superficial Dry-moist Urban 18.221952 - 65.432384

3 3/3/2013 3:29 P.M. 29 Superficial Dry Urban 18.221934 - 65.43166

4 3/11/2013 6:36 P.M. 27 Superficial Dry Urban 18.7575 - 66.84186

503/17/201

3 1:59 P.M. 27 Superficial Moist Urban 18.221876 - 65.432118

The Data

The Data

Juan Collection #1Date: 02/09/2013Time 6:06 P.M.GPS Latitude: 18.221986GPS Longitude:-65. 432580Air temp (approx.): 28 degrees CelsiusDepth: Superficial levelMoisture content (approx.): dry-moistFeatures

A park in urban areaClose to several treesLuquillo, Puerto Rico

The Data

Juan Collection #2Date: 02/18/2013Time 12:03 P.M.GPS Latitude: 18.221952GPS Longitude:- 65.432384Air temp (approx.): 27 degrees CelsiusDepth: Superficial levelMoisture content (approx.): dry-moistFeatures

A park in urban areaClose to several treesLuquillo, Puerto Rico

The Data

Juan Collection #3Date: 03/03/2013Time 3:29 P.M.GPS Latitude: 18.221934GPS Longitude: -65.43166Air temp (approx.): 29 degrees CelsiusDepth: Superficial levelMoisture content (approx.): dryFeatures

Urban area near a cable companyUnder a treeLuquillo, Puerto Rico

The Data

Juan Collection #4Date: 03/11/2013Time 6:36 P.M.GPS Latitude: 18.7575GPS Longitude: -66.84186Air temp (approx.): 27 degrees CelsiusDepth: Superficial levelMoisture content (approx.): dryFeatures

Country sideElevated areaCayey, Puerto Rico

The Data

Juan Collection #5Date: 03/17/2013Time 1:59 P.M.GPS Latitude: 18.221876GPS Longitude: - 65.432118Air temp (approx.): 27 degrees CelsiusDepth: Superficial levelMoisture content (approx.): moistFeatures

Urban AreaBackyardLuquillo, Puerto Rico

Mycobacteriaphage:The Find

Trial Name: Mirificus tempusCollected at San Juan, Puerto Rico Time 12:45 P.M.GPS Latitude: 18 28'2.96” NGPS Longitude: 66 6'23.92” WAir temp (approx.): 31.1 degrees CelsiusDepth: Superficial levelMoisture content (approx.): dry

The Second part of the Plan:Methodology

Enrichment:Stimulates the growth of phages with bacteria

Plaque Purification: We need our phages as clean as possible to be able to study them.

The ResultsGustavo - Samples were gathered:

Dark brown or near black soil; damp to moist; around or under vegetation and near bodies of water. In a mountainous, rural area. From a closed-access neighborhood which had been constructed 10-15 years earlier.Near houses or man-made constructions.At temperatures equal to or below 30 degrees Celsius.

Juan - Samples were gathered:Dry to moist soil; near or under trees; one sample was from rocky soil. In an urban area, then rural area. In parks, near houses or cable company.At temperatures equal to or below 30 degrees Celsius.

The Results

Alejandra - Samples were gathered:Loose, dry soil; in grass vegetation; In an urban area, in front of the Capitol of P.R. Close to the coastAt an approximate temperature of 31

The Results

Angelin - Samples were gathered:At the university’s athlete housing at CayeyLate at nightDamp moisture content; 2 cm underneath the grassAt temperature approximately 22 degrees Celsius.

The Results

The End of the Journey:The conclusions

MycobacteriophagesTo best find one:

Near the coastNear a busy cityMany more people

Finding more phages will lead to more concrete answers

The End

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