Team:UTP-Panama/Week 17

Week 17: September 26 to 28

September 26

WET LAB

Transformation of competent cells with Bristol 2010 - BBa_K381001.

Electrophoresis to BBa_K672000 – RENBO (sent to the registry) + RENBO (Ligation Product).

During this procedure we couldn’t get a good picture of the dna in the transiluminator because there wasn’t enough dna in the samples. We are going to redo this procedure.

Experiment Design

We designed the experiments we are going to do with the three BioBricks used (Bristol 2010 - BBa_K381001, GaTech 2010 – BBa_K410000, UTP-Panama 2011 – BBa_K672000).

We chose 4 temperatures:
• 5-10°C
• 10-20°C
• 20-30°C
• 37°C

These temperatures were selected so we could evaluate the response to cold shock in various ranges of our BioBricks. Since the lab equipments we had available couldn’t reach temperatures between 11-20°C this temperature was not used during the experience. Finally, the lab equipment allowed us to make the experiments in: 37°C, 23°C and 8°C.

We also chose 3 different Growth Media:
• Saline Solution
• Liquid LB
• Minimal Media
These growth medias where selected to evaluate the response of our BioBricks in different metabolic pathways. According to the characterization made by Team Edinburgh 2009, the Pyear promoter (BBa_K216005) used by Bristol 2010, works best in minimal media and complex media with a nitrate concentration of 40mM. Using this reference, we added 40mM of KNO3 to every growth media in the different temperatures for Bristol 2010 - BBa_K381001 and UTP-Panama 2011 – BBa_K672000.

September 27

WET LAB

Transfer of a colony of RENBO into Liquid LB to grow overnight.

Preparation of Minimal Media and Saline Solutions for the experiment.

M9 Minimal Media Recipe (1000 ml

1. Make M9 salts

 To make M9 Salts, aliquot 800ml H2O and add:

 a) 64g Na2HPO4-7H2O

 b) 15g KH2PO4

 c) 2.5g NaCl

 d) 5.0g NH4Cl

 e) Stir until dissolved

 f) Adjust to 1000ml with distilled H2O

 g) Sterilize by autoclaving

2. Measure ~700ml of distilled H2O (sterile)

3. Add 200ml of M9 salts

4. Add 2ml of 1M MgSO4 (sterile)

5. Add 20 ml of 20% glucose (or other carbon source)

6. Add 100ul of 1M CaCl2 (sterile)

7. Adjust to 1000ml with distilled H2O

We set up the experience designed in a total of 27 15mL Falcon tubes.

September 28

WET LAB

Miniprep of The RENBO (BBa_K672000)

Electrophoresis of The RENBO BBa_K672000

The first column shows the reference. The other 4 columns show The RENBO.