Team:LCG-UNAM-Mexico/Notebook/2008-July 02

MODELING:
We checked the dynamics of our equations further developing the beforementioned document
Reminder:
According to Michaelis-Menten: V0=[kcat*Et*S]/[km+S] where Vmax=kcat*Et (as long as the enzyme is saturated).

TO-DO LIST:
- Find missing parameters and start evaluating the model
- Add the dimerization of cI* and of the complex AHL:LuxR to the model
- Add the regulation by RcnR to define the initial intracellular nickel concentration
- Include the constitutive synthesis of cI* in the model.

MODELING:
Analyzed Hill Coefficient.

TO-DO LIST:
- Understand Hill Cooperativity
- Find missing parameters
- Determine initial Concentrations
- Include Dimerization

WET LAB:

Cultures

We cultured the cells with the plasmids

WET LAB:

Transformación

We transformed the biopart from Chiba(BBa_I729006) using two controls:

*Control 1: only DH5 alfa cells
*Control 2: without cells

PCR

1. Part 1 from Chiba's Biopart
2. Part 3 from Chiba's Biopart (normal)
3. Part 3 from Chiba's Biopart (mutated promotor )
4. RcnA with RcnR operator
5. RcnA control with RcnR operator

Recipe:

WET LAB:
The cell colonies transformed with the biopart from Chiba(BBA_I729006), grew successfully.
Three colonies from each cage were plated(concentred and no concentred), in 5 mL of liquid LB.

We run a with the PCR products.

Each track contains:

1. Molecular Marker
2. Product from part 1 in biopart BBa_I729006
3. Product from part 3 in biopart BBa_I729006
4. Product from part 3 in biopart BBa_I729006 Making a mutation
5. Product from RcnA
6. Product from RcnA in strain RnA-(control)

In the first and second tracks there's no product, we will repeat this reactions.

*Repeat PCR for Part1 and 2 in bioart BBa_I729006

PCR

We performed the PCR of the missing tubes

Purification of PCR Product

WET LAB:

We run a gel in order to observe the PCR products of the 25th.

Restrictions(cuts)

EcoR1-Up/Bam-Lowe           Parte1  (10 μl of PCR sample)
Xba1-Up/Pst1-Low               Parte3-N  (8 μl of PCR sample)
Xba1-Up/Pst1-Low               Parte3-M (5 μl of PCR sample)
Xba1-Up/HindIII-Lower        RcnA (5 μl of PCR sample) 
   
The overnight reaction was at 37ºC.

*BSA(Bovine Serum Albumin)

WET LAB:

From each one of the samples cultured on 28th,we chose 5 colonies and we plated in a 1 mL LB tube and we incubate it for 6 hrs. Our neative control was just LB.

Bioparts ligation in pRK415 and pBB

Transformation

We transformed the product of the ligation and plated it in cages with X-gal. With he purpose of save X-gal we chose to add it not in the LB-agar, but joint to the plated culture. In order to do this, we dilute 20 mL of X-Gal in 1mL of N-N-dimethylformamide. And from this mix we plated 25μl in each cage.

For the transformation we used two controls( one without plasmid and one without cell). When plating we excluded the control without cell and plated the DH5 cells without transformation in a Tc Cage and another of Gm.

Plasmid Extraction

We extract plasmid from te plated colonies. All the tubes pass the night with 1mL of ethanol at 100%.

Biopart cI

The missing biopart(cI), was cultured in 5mL of LB with 2.5 μl of ampicillin(50%). It was also plated in 5mL of LB without antibiotic and was plated in agar with ampicillin at 100%

WET LAB:

We finished the extraction of the plasmid, started on 29th.
We extracted the plasmid holding the biopart cI.

Gel

We run a Gel with the 15 samples of the plasmid pHET containing RcnA, part 3(normal) or part 3 (mut) and the three extraction plasmid samples containing the biopart cI+LVA