Team:UTP-Panama/Week 10

Week 10: August 8 to 13

August 8

WET LAB

Place: INDICASAT
Work Session #36 (morning)
Transformation of Competents Cells with pSB1A3, for ensure that our creation of competents cells work.

OBJECTIVE: Preparation of culture medium. 1. JM 109 strain used for cloning plasmids and growth. This is a commercial strain that comes in a kit, were planted in a solid LB medium and then treated to make them competent.

OBJECTIVE: Transforming Competent Cells. 1. Proper markings were made. 2. One of the tubes were scored as negative control. 3. Place 2 of plasmids and resuspended in 50. 4. The nomenclature was as follows: p-1  plate 1 1G orientation on the plate 5. The positive control was diluted 1 / 10 before taking him to the plate. 6. Was allowed to stand for 30 minutes on ice. 7. Thermal shock applied to facilitate the entry of the plasmid to the bacteria, it to 2.5 º C for 60 seconds. 8. Incubated for 5 min on ice and for the bacteria to recover from heat shock SOC 200 added to each tube including the negative control, were left incubating at 37 ° C with vigorous stirring (1.5) for 2 hours.

August 9

WET LAB

Place: INDICASAT
Work Session #37 (morning)
The Transformation works!!! Preparation of 4 falcons tube with trasnformed cells (for growth). (Objetives or Title):

OBJECTIVE: Creating Competent Cells. 1. Prepare the three buffers to be used. B1 100 mL 100 mM MgCl2 B2 100 mL 100 mM CaCl2 100mL () () () = 11.098g (100x10-3) = 0.1006 g 100mL () () () = 9.5211g (100x10-3) = 0.1047 g 2. Prepare a stock solution for the buffer (100mL of 1M solution) C1V1 = c2v2

V1 = 10mL 100mL to accommodate up 3. This solution was filtered using a syringe and special filter, everything worked within the extraction chamber. 4. From the stock solution of B1 were prepared 50 mL of 1M solution. C1V1 = c2v2

V1 = 5mL to 50mL with water accommodate up sterilazed 5. To this solution were used falcon tubes and sterile water. 6. All solutions were kept on ice. 7. Centrifugation of the culture. 4000 rpm for 16 min. 8. The supernatant was removed and discarded in chlorine. 9. Add 1 mL of the pellet and resuspended MgCl2, expect 20 minutes and centrifuged again this time for 10 min. 10. For the B2 solution was prepared 50mL 85mm from its stock solution.

V1 = 4.25 mL to 50 mL volumetric flask with sterile water until

11. Add 20 ml of CaCl 2 and left incubating for 20 min. 12. Centrifugation was carried 13. We added in a 1000 ml eppendorf tube Buffer 3 which have: • 850 L of Buffer???? • 150 uL glycerol molecular • be kept on ice

August 10

Work Session #38 (morning)
Place: INDICASAT Project Design Group: session of selection & analisys of the Gatech and Bristol Biobricks models and funtioning. We started to study the possibility of use the CspA promoter to improve gene expression under cold shock stress.

WET LAB

Work Session #39 (morning)
Minipred of our Transformed Cells.
(Objetives or Title):

OBJECTIVE: Dirty Mini Prep 1. We 50mL Buffer P1 50mL (100μg/mL) (1mg/1000 g) = 5mg 5mg (ul / 5 g) (1000 ug / mg) = 1000 uL = 1mL 2. We falcom centrifuge tubes which contained approximately 500 L of our growing bacteria and Lb. Centrifugation at 4000 rpm for 5 minutes. The nomenclature for these tubes was RFP (Red Fluorescent Protein). 3. While centrifuged prepare 50mL of P1 and RNAse (5μg/μL) (1mL) both were held in cold, then mix that were labeled and kept in cold.

Note1: there was not enough RNAse (RNAse needed to 1mL of 5μg/μL) 100l use only which means that our P1 will 10μg/mL RNase concentration.

Note2: Falcom tubes containing bacteria that were not centrifuged, first we had to divide 1500 uL to each tube and then these épendor épendor if they are spinning.

4. The supernatant from the previous centrifugation was recovered and divided into tubes épendor. 5. They took these épendor centrifuge to 13000Rpm per minute. 6. Recover the supernatant and place it in épendor new tubes, add 300 L of each épendor P1 (in total 6 épendor). 7. We added 300 L of P2. 8. Tubes kept on ice for 5 minutes. 9. We added 300 L of P3 (the buffer had to be cold). 10. Tubes kept on ice for 5 minutes.

Note3: the above is worked out of the extraction chamber.

11. Centrifuged for 10 minutes to 13000Rpm. 12. Transfer supernatant to new tubes. 13. Add 1000 uL of phenol (working within the extraction chamber) Note 4: The phenol used was more dilute than that recommended protocol. 14. The tubes were shaken by inversion. 15. Centrifugation at 13000 rpm for 5 minutes. 16. We poured tubes and place upside down. 17. Were allowed to dry about 10 to 15 minutes, then re suspended in 30 ul and were maintained at 65.5 ° C.

Work Session #40 (afternoon)
OBJECTIVE: Electrophoresis 1. Preparation of 1% agarose. 2. Moisten the chamber walls. 3. In a 70 mL Erlenmeyer flask add the agar and put a minute in the microwave, then take it off shake it and put it in the microwave 20 seconds. 4. Ethidium bromide placed in the microwave and cooled with tap water to a temperature between 60 ° C and 70 º C. 5. Placed between 2.5 to 5 micolitros Ethidium bromide. We mix before pouring it into the camera. 6. Once the gel solidified cover it with TAE (1X). (A) 7. 35μL were added ultra filtered water plasmids were 65.5 ° C. 8. Droplets were eliminated on the walls with the sping-down applied to the tubes. 9. We put the tubes in the microwave 5 minutes. 10. Add loading buffer which was frozen thawed How? • Put dots of paper loading buffer. • Of the 35 uL of plasmid take 10 mL (B). • Re mix in the dots and then the wells. 11. A and B meet. Plasmid preparation and gel electrophoresis. 12. The plasmids with the loading buffer in the wells

Note: It is important to place the (-) side of the DNA

13. We ran the gel for 3 hours. 14. The plasmids containing épendor labeled with 1, 2, 3 are kept the cooler den -81 º C (they were 6 tubes).

08/10/2011 (afternoon)

OBJECTIVE: Preparing for bacteria to become competent. 1. 250ml of medium was pipetted 2.5 ml. 2. Bacteria that were taken were in the freezer and took a small aliquot of which was suspended in 250 mL. 3. We took a L of JM109 cells and inoculated into the medium which was incubated overnight at 27 ° C Celsius at 100 rpm. 4. Prepare one liter of LB media using solid and liquid: • 10g tryptone • 5g yeast extract • 10g of NaCl 5. Liquid agar was ready to be used 500mL 6. For solid agar were added 7.5 g of Agar 7. We measure and adjust pH to 7.0 with NaOH droplets

Finishing Miniprep, preparation of Plasmid for electrophoresis. Left running the Electrophoresis.

Human Practice: preparation of concepts and definitions to the outreach campaign.

August 11

WET LAB

Work Session

OBJECTIVE: Preparation of Competent Cells. 1. 250ml 100l take an aliquot of frozen cells at -80 º C. 2. Grow cells in a shaker at 37 °C

OBJECTIVE: Transforming Competent Cells using the BB of Bristol (BBa_K381001) 1. Antibiotic resistance of this BB is C11H12Cl2N2O5 Cloramphenicol Molar mass = 323.1g/mol.

Note = this resistance is given by the plasmid backbone that uses (pSB1C3). This backbone is the same project GaTech-UNAM and CINVESTAV. The final concentration in the dish of this antibiotic is 33 mg / mL.

2. To prepare 10 mL of stock: • We weighed powder 340mg Cloramphenicol • We add the above to 10 mL of ET-OH • Vortexiamos until completely dissolved • Filters with 0.2 mm filter • The final concentration is 34 mg STOCK / mL • We prepare 15 dishes with 10 ml of solid LB each

3. Concentration of antibiotic in each plate 33 mcg / mL For a plate: 10mL of LB (33 mg antibiotic / mL of LB) (mL/34mg antibiotic) = 0.0097 = 9.7 uL / ​​plate

4. Transformation Protocol, • Prepare 3 tubes of 50 ml falcon to make the total of 150 mL to fill the dishes. • To prevent the LB solidify place it in a water bath at 50 ° C. • We added 48.5 L of antibiotic to each tube to achieve falcon concentration of 33 mg / mL • The vertieron10 mL of LB plus antibiotic to each petri dish • Place the BB BBA-K381001 Bristol, was on the plate 4, position 1A. • Click on the plate and add 10 mL of purified water and then resuspended. • We stand for 5 min. • Remove everything that was in the booth 1 and place it in a épendorf tube, labeled with the name of Bio Brick. • In a épendorf control, Brick Bio 2μL added plus 50 uL of competent cells. • Leaving the tube in ice for 30 minutes. • The negative control hiso in an Eppendorf tube that contained only 50 uL of competent cells and also kept on ice for 30 minutes. • Heat shock was performed in a water bath at 42.5 º C for 1 minute and allowed 5 minutes on ice. • Add 200 L of SOC to each eppendorf tube (negative and positive control) and resuspended. • Incubate at 37 ° C with vigorous stirring (200rpm) for two hours. • Add 200 L of LB solid positive control and the same for the negative control. Labeled.

Note: SOC medium was cold, it is important to have fully resuspended proper concentration when you add it. When placed in eppendorf incubator should be sealed with a piece of parafilm.

Human Practice

Preparation of brochures for Outreach. Preparation of the slides regarding to the presentation for Outreach. Mindstorm about SynBio definitions & implications.

August 12

Human Practices
We were studying about the pros and cons of the SynBio and the safety regulations (for example the Synthetic Biology Report of the Presidential Commission for the Study of Bioethical Issues) for explain the risks and the safety to the people.

August 13

OBJECTIVE: Verify the results of past experience.