Team:LCG-UNAM-Mexico/Notebook/2008-September

WET LAB

Cultures

1. One of the colonies obtained after we transformed the ligations (part1_part2_part 3 normal) + PRK415 and (part1_part2-part3Mutated)+PRK415 was cultured.
2. Cultures of 10 colonies of part1+part2+PRK415 in liquid broth were prepared.
3. 1% Agarose Gel (PRK415+(parte1+parte2) repetition)
   

   1.-Molecular marker 2.5 μl
   2.-[1] part1_part2 in PRK415 extraction 5μl
   3.-[2] part1_part2 in PRK415 extraction 5μl
   4.-[3] part1_part2 in PRK415 extraction 5μl
   5.-[4] part1_part2 in PRK415 extraction 5μl
   6.-[5] part1_part2 in PRK415 extraction 3 μl
   7.-[6] part1_part2 in PRK415 extraction 3 μl
   8.-[8] part1_part2 in PRK415 extraction 3 μl
   9.-[9] part1_part2 in PRK415 extraction 3 μl
   10.-[13] part1_part2 in PRK415 extraction 3 μl
   11.-[15] part1_part2 in PRK415 extraction 3 μl
   

   The size of the plasmids does not correlate the size we are expecting.

4. Step 2 was repeated.
5. Cultures with (Part1_part2_part3N)+PRK415 were prepared for extraction.
   

   1 PRK415 part1+part2
   12 PRK415 part1+part2
   13 PRK415 part1+part2
   14 PRK415 part1+part2
   15 PRK415 part1+part2
   16 PRK415 part1+part2
   17 PRK415 part1+part2
   18 PRK415 part1+part2
   19 PRK415 part1+part2
   20 PRK415 part1+part2
   21 PRK415 part1+part2

Dirty plasmid, in theory, it should not affect the PCR because the genes occupied for this are not in the E.coli genome. We proceeded to do the PCR. (yo digo que no pongamos la foto del gel esta muy sucio)

PCR

1.-[17] PRK415 + p1_p2 oligos 1upper 2 lower
2.-[8] PRK415 + p1_p2_p3N oligos 2upper 3 lower
3.-[9] PRK415 + p1_p2_p3N oligos 2upper 3 lower
4.-CN1 H2O
5.-CN2 PRK415 DR EcoR1 y BamH1 oligos 1upper 2lower
6.-CN3 PRK415 DR EcoR1 y BamH1 oligos 2upper 3lower

MODELING:
Lac promoter synthesis rate:
The effect of stochasticity on the Lac Operon: An evolutionary perspective from van Hoek F et al (2007)
The article's objective, as you can infer from the title, is to evaluate the effect of stochasticity in the evolution of a Promoter. In order to do so they built a comprehensive model including every parameter involved in transcription and translation. They measure some parameters but they depend mostly on literature to define them.

They perform both a deterministic and a stochastic analysis. To generate a stochastic model they added one parameter, the average burst size of protein translation (protein translation occurs in bursts, after an mRNA is synthesized, several proteins can be translated from the same mRNA). This was possible because when an mRNA molecule is translated it can not be degraded. Therefore after each translation it can either be translated again (p) or be degraded (1-p). This suggests that protein production occurs in bursts with a burst size geometrically distributed. Afterwards, they compared the noise levels in their model with experimental noise measurements and found correlation.

To model transcription they used a two-dimensional Hill-function dependent on the cAMP and allolactose concentration. (repress the glucose and lactose operon via cAMP and activates the operon via allolactose).

They use 11 biochemical parameters, including three of special importance for us:

2% Agarose gel with PCR samples of 2/09/08

1.-Molecular marker
2.- [1c2]  [17] PRK415 + p1_p2 oligos 1upper 2 lower
3.- [2]  [8] PRK415 + p1_p2_p3N oligos 2upper 3 lower
4.- [3]  [9] PRK415 + p1_p2_p3N oligos 2upper 3 lower
5.- [4]  CN1 H2O
6.- [5]  CN2 PRK415 DR EcoR1 y BamH1 oligos 1upper 2lower
7.- [6]  CN3 PRK415 DR EcoR1 y BamH1 oligos 2upper 3lower
8.- R6 Restriction EcoR1 HindIII 1_2_3 (5 μl)
9.- R8 Restriction EcoR1 HindIII 1_2_3 (2 μl)
10.- R9 Restriction EcoR1 HindIII 1_2_3 (2 μl)
11.- R17 Restriction EcoR1 XbaI 1_2_3 (2 μl)

No results.

Cultures in 2ml of LbTc10 of PRK415 +part1+part2 and of DH5alfa 123 (3normal) ligation were prepared.

1% Agarose gel with extractions
2.-[3] PRK415 part1_2_3N Ligation
3.-[5] PRK415 part1_2_3N Ligation
4.-[6] PRK415 part1_2_3N Ligation
5.-[8] PRK415 part1_2_3N Ligation
6.-[9]PRK415 part1_2_3N Ligation
7.-[10] PRK415 part1_2_3N Ligation
8.-[11] PRK415 part1_2_3N Ligation
9.-[12] PRK415 part1_2_3N Ligation
10.-[13] PRK415 part1_2_3N Ligation
11.-[1.2] PRK415 part1_2_3N Ligation
12.-[3.2] PRK415 part1_2_3N Ligation
13.-[5.2] PRK415 part1_2_3N Ligation
14.-[6.2] PRK415 part1_2_3N Ligation
15.-[8.2] PRK415 part1_2_3N Ligation
16.-[9.2] PRK415 part1_2_3N Ligation
17.-[10.2] PRK415 part1_2_3N Ligation
18.-[11.2] PRK415 part1_2_3N Ligation
19.-[12.2] PRK415 part1_2_3N Ligation
20.-[13.2] PRK415 part1_2_3N Ligation

PCR ligation p1_p2_p3 in PRK415
No positive results.

Electrophoresis ligation p1+p2 PRK415

1.-Molecular marker
2.-[1] p1+p2 PRK415
3.-[2] p1+p2 PRK415
4.-[3] p1+p2 PRK415
5.-[4] p1+p2 PRK415
6.-[5] p1+p2 PRK415
7.-[17] p1+p2 PRK415
8.-[18] p1+p2 PRK415
9.-[19] p1+p2 PRK415
10.-[20] p1+p2 PRK415
11.-[21] p1+p2 PRK415

4 cultures of PRK415 were left.

Restrictions

part1_part2  (PCR)
part 3 mutated (PCR)

Part1_part2 ligation sep-09

PCR ligation part1 + part2 with RttH.

Ligation and transformation of part1_part2 in pJET. (check kit)

PBB + RcnA was streaked again. (1,2,5 and 9)

PBB + RcnA restriction
DNA 10 μl
H2O 12 μl
BSA 3 μl
XbaI 1 ml
HindIII 1 ml
30 μl

1% Agarose gel

1 .- molecular marker
2.-DR1 pBB+RcnA
3.-DR2 pBB+RcnA
4.-DR5 pBB+RcnA
5.-DR9 pBB+RcnA
6.-DR PRK415 2
7.-DR PRK415 3

Plasmids were purified from the samples:
1.-pBB+Rcna[2]
2.-pBB+Rcna[5]
3.-pBB+Rcna[9]
4.-part1_part2 PRK415[6]
5.-part1_part2 PRK415[8]
6.-part1_part2 PRK415[9]
7.-part1_part2+part3N PRK415[5]
8.-part1_part2+part3N PRK415[8]
9.-part1_part2+part3N PRK415[9]

1%  Agarose gel
Samples were run through an agarose gel at 1%

1.-molecular-weight marker
2.-[1]pBB+Rcna[2]
3.-[2]pBB+Rcna[5]
4.-[3]pBB+Rcna[9]
5.-[4]part1_part2 PRK415[6]
6.-[5]part1_part2 PRK415[8]
7.-[6]part1_part2 PRK415[9]
8.-[7]part1_part2+parteN PRK415[5]
9.-[8]part1_part2+parteN PRK415[8]
10.-[9]part1_part2+part3N PRK415[9]
11.-PCR rTth part1_part2
12.-molecular marker

Restrictions were made of the previous samples.

RcnA

Part1+part2 in PRK415

Part1+part2+part3N in PRK415

1%  Agarose gel
Gel with the bioparts restrictions

1.-molecular-weight marker
2 .- [1] PBB + Rcna [2] double restriction with XbaI and HindIII
3 .- [2] PBB + Rcna [5] double restriction with XbaI and HindIII
4 .- [3] PBB + Rcna [9] double restriction with XbaI and HindIII
5 .- [4] part1_part2 PRK415 [6] double restriction with EcoRI and Xba I
6 .- [5] part1_part2 PRK415 [8] double restriction with EcoRI and Xba I
7 .- [6] part1_part2 PRK415 [9] double restriction with EcoRI and Xba I
8 .- [7] part1_part2 + parteN PRK415 [5] double restriction with EcoRI and PstI
9 .- [8] part1_part2 + parteN PRK415 [8] double restriction with EcoRI and PstI
10 .- [9] part1_part2 + part3N PRK415 [9] double restriction with EcoRI and PstI
11.-Molecular Marker

Plating
Striated colonies resulting from cloning in PJet
Cloning in PJet of ligation Part1 + Part2
Transformation according to the manual

Restrictions with XbaI of:

1.-Part 1 + part2
2.-Normal Part 3

The restrictions were left alone for 2:30 hrs.

Ligation
Ligations were made of [part1 + part2] + parte3N and [part1 + part2] + parte3M in a final volume of 50μl

10 colonies from every LB Petri dish Amp with Part1_Part2_part3mutated and Part1_Part2_Part3normal were scratched.

RcnA [2] Big flask alkaline lysis
RcnA[1] Purification tuve with kit

Plasmid extraction gel

1.-Molecular marker
2.-part1 + part2 pJet 2
3.-part1 + part2 pJet 34
4.-part1 + part2 pJet 21
5.-part1 + part2 pJet 1
6.-part1 + part2 pJet 8
7.-part1 + part2 pJet 29
8.-part1 + part2 pJet 36
9.-part1 + part2 + part3M  pJet 6
10.-part1 + part2 + part3M  pJet 4
11.-part1 + part2 + part3M  pJet 3
12.-part1 + part2 + part3N  pJet 3
13.-part1 + part2 + part3N  pJet 11
14.-part1 + part2 + part3N  Petri dish2 pJet 5
15.-part1 + part2 + part3N  Petri dish2 pJet 12
16.-part1 + part2 + part3M  Petri dish2 pJet 2
17.-part1 + part2 + part3M  Petri dish2 pJet 5
18.-part1 + part2 + part3M  6
19.-part1 + part2 10 Petri dish 2
20.-part1 + part2 21

There is no need to make a gel. Nothing definite yet.

4 tubes of part3 normal were restricted, and 2 of ligation part1 + part2.

MODELING:
Exploring Sensibility Analysis:
Normalizing sensitivity
- None: dx(t)/dt
- Medium: 1/x(t)*dx(t)/dt
- Complete: k/x(t)*dx(t)/dt (Allows you to compare dimensionless.)

How is it measured? Are the k values moved in a range? Or is it a property of the system?

Wilkinson (1978).
The parameters are systematically perturbed from their given values…
… change from the given value… (although it is recommended to define them in each system).

  Ingalls & Sauro (2002)
- Before the analysis, it is recommended that you detect the 'preserved structures' (linear units, eg moieties).

How to reduce the system:

- The response coefficient, defined above, provides a measure of the difference between this ‘‘perturbed trajectory’’ and the ‘‘nominal’’ (unperturbed) trajectory at each time t: As time tends to infinity, each trajectory will converge to its steady state, and so the response coefficient will converge to the steady-state response of MCA.

- At steady state, these coefficients reduce to their standard MCA counterparts—flux responses.

NOTE: The sensitivity analysis is sensitive to the initial concentrations of metabolites.

There are, broadly speaking, four options to choose from:

1) The faculty of physiology at UNAM has a sensor for variations of voltages of orders that could be useful (Question: If it is not specific for nickel, is there a way to filter the noise?).
Among the benefits versus the other possibilities: sensitivity appears to be very good and we know this because similar experiments have been done previously. This in turn gives us the assurance that the sensor has already been tested in other biological systems in line with the results expected. Plus, a member of our team already knows how to use this system. On the other hand, the fact that the Insitute is part of the UNAM has the advantage of working with people from the same team, not counting the enormous advantage of being physically close.

2) At the University of Guadalajara, there is a device that measures the medium resistivity in the orders of 10^-9 Molar. We have not checked with sufficient detail the operation of this system, but at least the sensitivity offered is very promising. Furthermore, we believe that the metal used in a phase of measurement reacts specifically with nickel also producing a easily measurable and identifiable optical effect.
Among the advantages this option provides are: that it is sensitive and that the software used to process and record each measurement is very comprehensive and drops the noise reliably. The main disadvantage, is that the apparatus is in a laboratory of the UdG, which means that we would have to carry biological material.

3) Someone offered to buy the specific sensor for nickel and lend it to us during the measuring stage. We need to contact him and describe the project and what we need at the time of sensing. His only requirement is that he appears as a collaborator in the experimental publication resulting from this research.

4) Finally, most certainly not least, Trejo is still building up the sensor as we had planned initially. He has progressed well and in about two weeks it will be ready.

- We decided to wait for Option 4 and, as a backup, the support of Dr. Pena (1), but this does not rule out the option 3, which will be investigated for further details such as shipping time and specificity of the device.
- We need to define the requirements for the bioparts and make the oligos. The oligos are being designed and probably by Friday they will be sent.
- Design of experiments to estimate parameters (probable date September 10-12).
They are still working on the buildings, but there will be a first meeting on Tuesday, Sept. 23, at 4:00 pm.

MODELING:
Converting units:

ΔG ° =- 23.81 kcal / mol
Keq = exp (-ΔG º / RT)

*Units supposedly do not affect this formula's usage

Correction of the synthesis reaction of cI:
Units of the k3ON, estimated in reference 3 are 1/molecules*seconds, which means that the reaction is of second order.
In that same article, they suggest that the sole presence of the dimer ensures the production of cI with k3ON rate (that is, that bonding is efficient). Since the estimated values do not consider the intermediate step of the promoter's union and the complex, we should not consider it.

3.1 ρcI + (AHL: LuxR): (AHL: LuxR) -> CI + ρcI + (AHL: LuxR): (AHL: LuxR)
k3ON

Unknown parameters:

MODELING:
Dimerization of cI
k4.1 & k-4.1?
2 cI <-> cI: cI

¿Quasi-equilibrium?
The initial concentration of cI varies over time, but the proportion is preserved.
...avoid kinetic analysis of the fast reactions, ie to take into consideration only their equilibrium constants instead of considering their rates (Kholodenko et al. 1998).

Keq = [cI:cI]/[cI][cI] -> [cI:cI] = Keq [cI] 2

d(cI:cI 2cI)/dt = v+ - v-
d(Keq[cI]^2+2cI)/dt = v+ - v-

MODELING:

We will also design experiments to obtain the missing parameters through the experimental measurements (viable).

- Sensitivity analysis: Although we don't yet understand the particular units in which SimBiology returns the results, the first graphics that display the basic parameters of the model are ready and they are the ones involved in the degradation of AHL and its dimerization with LuxR (the beginning of the cascade) and the ones regarding the entry and exit of Nickel. We must do this analysis later, as we have seen that this is sensitive to the initial concentrations of metabolites, which are not yet fully defined.

- Stationary States and Jacobian: They were stopped briefly because we need the parameters for further analysis.

WET LAB:

MODELING:
Correcting reaction 5

The Keq is dimensionless, but for formality in the reaction, it is sometimes necessary to add units.

In the case of the reaction 5, the only information we have is the ΔGº of the reaction, so the Keq is calculated as exp (-ΔG º / RT), which returns a value without units.

As the definition of Keq is given in concentration, we interpret this value in terms of molarity. We know that once defined, they are completely interconvertibles: Molar <-> moles <-> molecules and since we are working on molecules for our model, we make the relevant adjustments.

We purified the PCR products of RcnA and the promoter region of RcnA.
4 reactions of RcnA
We prepared a 1% low melting point agarose gel, let it cool down for about 20min at the freezer. We use 2.5 μl of loading dye Buffer.
We run the gel at 4°C, 130 Volts for about 40min.
Total volume of the loaded samples:
45μ per sample of RcnA (1,2,3,4
We put the gel in a recipient with 100ml of distilled water and 120μl of Ethidium Bromide   for 10min.
We cut from the gel the 900bp band using a sterilized scalpel.
We purified the gel band using the QIAquick Gel Extraction Kit.
We ran a  1% agarose gel to verify if we have the purified PCR product.

September 29 2008
WET LAB

We Cloned the RcnA PCR fragment according to the ColoneJet protocol of the  CloneJET PCR cloning Kit by Fermentas, and let the cells grow all the night.

We Cloned the RcnA PCR fragment according to the ColoneJet protocol of the  CloneJET PCR cloning Kit by Fermentas, and let the cells grow all the night.

We streak 18 colonies on an LB Am100 agar plate.