Protocol 1

Preparation of culture media (1L).
1.- 5 g of yeast
2.- 10 g of sodium chloride.
3.- 10 g of triptone.
4.- Add water till 1 uL volume.

Protocol 2

Competent cell´s preparation

1.- Centrifug your media culture tube (25 mL) at 4,000 rpm during 15 min.
2.- Pour the supernatant and add 10 mL of cold MgCl2 solution. Centrifuge them at 4,000 rpm at 4 °C during 15 min.
3.- Pour the supernatant and resuspend the pellet in 40 mL of cold CaCl2 solution. Keep it in ice during 20 min and place them in eppendorf tubes. Centrifuge them at 4,000 rpm at 4°C during 15 min.
4.- Pour the supernatant and resuspend the pellet in 40 mL of cold 85mM CaCl2/15% glycerlol v/v solution. Centrifuge again at 2,100 rpm at 4°C during 15 min.
5.- Pour the supernatant and resuspend in 2 mL of cold 85bmM CaCl2/15% glycerol v/v solution.
6.- Aliquote in tubes of 100 uL previously frozen and store at -80°C.

Protocol 3

DNA extraction from Buffy coat:

1.- Centrifuge 5 mL of culture medium at 1,500 rpm for 10 min at 4°C.
2.- Carefully transfer 600 uL of buffy coat to a microtube.
3.- Add 1 mL of RBC buffer and mix throughly by vortex.
4.- Centrifuge at 4,500 rpm during 30 s and pour carefully so your pellet is not wasted.
5.- Repeat steps 2 and 3 adding just 500 uL from RBC buffer.
6.- Once separated, add 300 uL of lysis buffer to the pellet and mix by vortex.
7.- Add 150 uL of protein precipitation solution.
8.- Centrifuge at 11,000 rpm during 5 min; then, transfer the supernatant to a new microtube.
9.- Add 700 uL of cold isopropanol and mix by inverting until you see a white formation within the tube.
10.- Centrifuge at 11,000 rpm during 4 min and pour carefully taking care of the pellet at the bottom.
11.- Add 500 uL of cold ethanol 70% to wash and then centrifuge at 11,000 rpm during 4 min.
12.- Pour and let the ethanol evaporate at room temperature.
13.- Resuspend the DNA pellet with 12 uL of TE buffer; incubate all night at 4 °C.

Protocol 4

Electrophoresis:

1.- Prepare 1L of TAE 10X (48.4g of Tris base[ tris (hidroxymethyl) amminomethane], 11.4 mL of glacial acetic acid (17.4 M) and 3.7 g of EDTA then fill with deionized water to 1 L.
2.- Prepare a 50 mL solution of 3% agarose using TAE buffer (without using water).
3.- Heat the gel in the microwave in intervals of 30 s, 20 s and 10 s until the agarose is completely dissolved, taking care that it does not boil so much.
4.- Let cool down the gel (55-60°C) and pour in a base with the comb already on its place to form the charging wells.
5.- Wait until the gel solidifies.
6.- Remove the comb and place the base with the gel inside the electrophoretic chamber.
7.- Add TAE buffer until the gel is covered.
8.- Take 5 uL of each sample from the PCR and 1 uL of charging gel in a new PCR tube, mix well.
9.- Charge the samples inside the wells of the gel, adding a weight marcker.
10.- Close the electrophoretic chamber and run at 120 V during 30 min. Follow the displacement ofthe samples.
11.- Take out the gel from the chamber very carefully and place in the UV light to see the DNA strands.

Protocol 5

Competent cell transformation:

1.- Place 1 uL of the plasmid in a microtube, gently add 100 uL of your competent cells. Take another tube and place only 100 uL of competent cells, so you have a control group.
2.- Mix gently with a pipette and incubate during 30 min in ice.
3.- Ab ruptly place your cells in 42°C dry bath during 2 min and replace in ice whwn finished.
4.- Add 900 uL of LB medium and incubate at 37 °C for 30 min.
5.- Inoculate with 200 uL of your transformed cells in LB-Amp.
6.- Incubate overnight and store at 4 °C of freeze in 15-25% glycerol.

Lab archives// Mar 12, 2014

In order to grow our bacteria, we inoculated with 100 uL of E. Coli DH5-alpha in 50 mL of LB medium, and 100 uL of E. Coli TOP in 50 mL of LB medium. We left both samples incubating overnight

Lab archives// Mar 20, 2014

We inoculated E.Coli NEB10-beta in 50 ml of LB medium.

Lab archives// Mar 27, 2014

We prepared the medium in which our bacteria will grow, for that we dissolved solid LB with agarose, then we add it into 10 plates, we waited a few minutes for it to solidify and finally we added 500 uL of our antibiotic that is chloranphenicol to each of those plates.

Lab archives// Apr 01, 2014

we transformed luciferaze from DNA to cells by incerting them with change of temperature

Lab archives// Apr 02, 2014

We made a litter of agar and 500 L of growth medium, and we sterilized them along with crystal media disperssion balls

Lab archives// Apr 03, 2014

Today we prepared buffers for home made mini preps and did a cell re culture.

Lab archives// Apr 07, 2014

We added 700 mL of water in a beaker and then we prepared 23 grams of powdered agar that we added slowly while we moved the beaker. This in order to help the dissolve the powder. After that, we added other 300 mL to the solution and finally we passed it to an Erlenmeyer matrass. Also we prepared 500 mL of growth medium. First, we measured and added 5 grams of tryptone, 5 grams of yeast and 10 grams of sodium chloride. Those ingredients were added slowly to a graduated cylinder with 350 mL of wáter while the flask was moved. After finishing the addition we added other 150 mL of water. The solution was then transfered to another Erlenmeyer flask. Both Erlenmeyer flasks and a glass bottle with crystal media diperssion balls were sterilized with temperature and pressure.

Lab archives// Apr 02, 2014

300 ul of ampicillin at 100mg/ml chloramphenicol
3 Plates with agar
Promoters: BBa_K258005 (pO2), BBa_176500 (pFe) BBa_J52008 (luc-3)
The plates were impregnated with ampicillin promoting bacteria in the following manner
|pO2 | Plate | 150 ul Ampiciline | K258005 |
------------------------------------------------------
|pFe | Plate | 150 ul Ampiciline | 1765000 |
------------------------------------------------------
|Luc-3 | Plate | Chloranphenicol | J52008 |

Apr 08, 2014

**/Today we realized the mini prep for the oxygen promoter. /**
--500 ul of Ampiciline //this is for the oxygen promoter
--50 ul of oxygen promoter
--45 ml of LB medium
--at room temperature 15 minutes at 1500 rpm in the centrifuge.
--500 ul of chloramphenicol // this is for the red liciferase
--50 ul of red luciferase
--45 ml of LB medium
--The new LB medium was prepared with //LB medium
--10 g yeast
--5 g salt
--15 g Tryptone MINI PREP First 400 ul of Genomic Lysis Buffer to the 40 ml overnight [bacteriaand oxygen promoter] Then the mixture is transferred to a Zyne-Spine Collumn in a Collection Tube. Centrifuge 10,000 rpm /1 min Transfer the Zyno Spin Column to a new Collection Tube. Add 200 micro liters of Pre Wash Buffer. Centrifuge 10,000 rpm /1 min Add 500 uL of of g-DNA Wash buffer to the spin column. Centrifuge 10,000 rpm /1 min Transfer the spin column to a clear micro centrifuge tube. Add 50 uL of DNA Elution Buffer . Incubate 2-5 minutes to at room temperature. Centrifuge at top speed per 30 seconds. Then store at -80 °C

Apr 22, 2014

We prepared 2 overnight cultures (45ml of LB medium): GFP E0040 pSB1A2 with 100 uL of ampicillin. M-cherry (RFP) BBa_J04450 pSB1C3 with 500 uL of chloramphenicol.

Apr 24, 2014

2 Transformations: Bacteria NEB 10 Beta, DH5alpha 2 Mini Preps: GFP, RFP //Transformation and preps:-- Centrifugation at 2500 rpm for 15 min.--1ul DNA in 100 uL of bacterial culture (luciferase). --Filtering GFP, RFP bacteria. --Added 400 ml of Lysis Buffer Genomyc each crop by columns. --Centrifugation at 10,000 * g for 1 min. --Last column to another tube and DNA Prewash + 200 uL Buffer. --Centrifuged at 10,000 * g for 1 min. --Add 500ul of DNA Wash Buffer, centrifuged at 10,000 * g for 1 min. --Transfer to clean tube, add 50ul of DNA Elution Buffer. --Incubate for 2-5 min at room temperature, centrifuged at maximum speed for 30 sec.

Apr 28, 2014

Today was prepared LB media. It also was done the sterilization of 1 mL and 200 uL tips, water and ependorfs. After the sterilization of the LB media was done, we cultivated in it the bacteria carrying the oxygen promoter, RFP, and GFP. Then we left the bacteria growing in the incubator at 37°C overnight.

May 05, 2014

Plating bacteria with GFP, RFP, promoter Oxygen and iron promoter plasmids. Everyone in their respective environments and specific antibiotics (ampicillin and chloramphenicol). Tubes with bacteria was added and placed them in LB medium incubator to grow more product if necessary.

May 08, 2014

We did a centrifuge at 4800 rpm for 20 min for the plasmids had been in overnight which had oxygen promoter, GFP and RFP to rush after we separate the aqueous phase.

May 19, 2014

We did inoculation of GFP, RFP and Oxigen Prmoter

May 20, 2014

We started minipreps preparation of GFP, RFP and Oxigen Promoter. We added 1.5 ml of culture to each Eppendorff (4 for each promoter) and we centrifugated it at 13.2 rpm for 10 minutes, we did the same procedure 2 times. Also, we sterilized two Erlenmeyer flasks and 40 Eppendorffs.

May 20, 2014 //Mini prep preparation

1. We added 1.5 mL of the cultures to their corresponding Eppendorffs and centrifugated them at 3400 rpm during 10 minutes other three times.

2. Once we had a pellet we discarded the supernatant and we added 300 uL of TEG buffer (made of 100 mM of Tris-HCl pH8, 2 mM EDTA and 20% glucose)

3. We added 700 μL of NS solution (0.2 M NaOH, 1% w/v SDs) and we mixed by inversion.

4. We added 400 μL of 3 M sodium acetate pH 5.3, and we mixed by inversion.

5. We centrifugated the Eppendorffs at 3400 rpm during 15 minutes.

6. We transfered the supernatant to other Eppendorffs and we added 50 uL of cold isopropanol and mixed by inversion.

7. We centrifugated the Eppendorffs at 3400 rpm during 15 minutes.

8. We discarded the supernatant and washed each pellet with 70% ethanol and we let it dry for 15 minutes.

9. We resuspended the pellet in 500 uL of TE buffer and then we added 10 mg/mL RNAseA.

11. We precipitated the DNA with 100 uL of ethanol in each Eppendorff.

12. We centrifugated for 1 minute at 3400 rpm.

13. We got rid of the supernatant.

14. Finally, we resuspended the pellet in 50 uL of TE buffer

UNAM procedures// Digestion

|BSA | 1 uL |

|DNA | 2 uL |

|Buffer (2) | 1 uL |

|Buffer Xba I+Pstl | 3.3 uL |

|fe Xba I+Spef | 3.3 uL |

|H₂0 | 5.4 uL |

Gel agarose

1 kb NEB Ampyciline -> Pentneryl

| ---------------Bristol - Myers Squibb

| ---------------1 gram/ 3 mL

| ---------------inyectable

|_pO ₂ | 3.3 uL

|1 kb marker 5.4 uL

Homemade Mini prep Preparation

1.- Overnight bacterial culture of 5 mL in falkons of 50 mL

2.- Centrifugate in an eppendor of 1.5 mL and throw supernadant

3.- Add 152 uL of Buffer 1 + RNAse (11 micro-liters /1 buffer micro-liter ) **Buffer 20% Glucouse

4.- Mix via Buffer and add 150 uL of Buffer 2 *Wait 4-5 minutes*

(1.5mL)Buffer 2 -> 880 uL H₂O,20uL NaOH 10 N

---------------------------------------100 uL SDS 10%

5.- Mix via inversion and add150 uL Buffer 3

------------------------------Sodium Acetate 3M pH 5

6.-Centrifuge 5 minutes.

7.-Take the supernadant to antoher tube. Add 1 mL ETOH Absolute

8.-Centrifuge by 5 minutes and decant

9.-Wash with 500uL ETOH 70 %

10.-Centrifugate 1 minute

11.-Decant and dry at 37 celsius centigrades (30 minutes)

12.-Resuspend in 15 uL of H₂O

Ligation

Inserto 7mL

Plasmid 3mL

Buffer 10x 1.5mL

Ligase 0.5 mL

H₂O 3 mL

Total = 15 ml

E.Coli cells transformation.

To transform E.Coli cells with an exogen DNA, 200 uL of competent cells were taken they were mixed with 1-20 ug(Micrograms) of DNA that was incubated on ice for 30 minutes. This makes posible the absorbtion of the exogen DNA to the cells surface, after that, the cells are incubated at 42Celsius Degrees during 90 seconds, for then incubate them during 1 minute again in ice. THis termal shock makes posible the entrance of the plasmid to the inside of the cell. 600 uL of LB medium were added and then incubated during 60-90 minutes at 37 Celisus Degres, to do posible the segregation of the exogenic plasmid during the celular division (3 to 5 generations). Finally, Alicuote of 100-200 uL were cultivated by dimention in the LB agar medium, which ere incubated at 37 Celsius Degrees. After 15-20 hours of incuvation colonies could be observed

Ligations

1# Grow h mL in overnight

2# Do minipreps

3# EcoRI and PstI restrictions.

--for Fe EcoRIand SpeI

O₂+ GFP ---1,2,3 - 931

Fe+GTP ---4,5,6 -1,828

O₂+ RFP ---7,8,9,10,11,12,13 -890

Fe+RFP ---14,15,16,17,18,19 -1,787

GFP -> 761 bp EcoRI Po+I // 728 bp XbaI SpeI

RFP -> 714 bp Xba - SpeI // 747 bp EcoR - Ps+I

pFe -> 1,067 bp EcoRI - SpeI

_pO₂ -> 178 bp EcoRi SpeI // 145 bp Xba - Ps+I

O₂ + GFP 1/1 - 1/2 - 3/3

FO ₂ + GFP 4/1 - 5/2 - 6/3

O₂ + RFP 7/1 - 8/2 - 9/3 - 10/4 - 11/5 - 12/6 - 12/13

Fe + RFP 13/1 - 14/2 - 15*16/3 - 17/4 - 18/5 -19/6

Digestion

2 --- BSA --- 1uL

4 --- DNA --- 2uL

3 --- (M) Buffer // Enz (0.3 uL each one)--- 1uL

5 --- EcoRI --- 0.3 uL

--- PstI --- 0.3 uL

1 --- H₂O --- 5.4 uL

Total == 10 uL rxn

GFP 761 bp

RFP 742 bp

pFe 1,067 bp

pO₂ -15.5 bp

pO₂ + GFP 931 bp

pFe + GFP 1,828 bp

pO₂+ RFP 890 bp

pFe + RFP 1,787 bp

Gel 1 = 8/9/10/Lodder 1kb/11/12/13

Gel 2 = 15/16/17/Lodder 116/18/19

Gel 3 = 1/2/3/4/Lodder 1kb/5/6/7