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

WET LAB:

We left PCR of:

• Part 1 of Biopart BBa_I79006
• Biopart cI BBa_C0051
• Biopart 3 (normal) ofBBa_I79006
• Biopart 3 (mut) of BBa_I79006
• Operator of rcnR + RcnA

MODELING:
Hill cooperativity 5th Reaction Reminder:

A + B <--> AB
Ka=Keq=[AB]/[A][B]=1/Kd
θ=[AB]/([AB]+[A])=[B]/([B]+Kd)

MWC Model (Cooperativity)
A + nB <--> ABn
Ka=Keq=[ABn]/[A][B]n=1/Kd
θ=[B]n/([B]n+Kd)
log(θ/(1- θ))=nlog(B)-log(kd) …Hill's equation

Suppression mediated by cI:
ρ + nCI <--> ρ:CIn (k+, k-)
Keq=Ka=[ρ:CIn]/[ρ][CI]n
Si ρ0=[ρ]+[ρ:CIn]
… ρ0=[ρ]+Keq[ρ][CI]n
=> ρ= (ρ0/Keq)/((1/keq)+[CI]n)

Flow= k+[ρ][CI]n = K+((ρ0/Keq)/((1/Keq)+[CI]n))[CI]n

Flow= k+([ρ0]/Keq) [CI]n / ((1/Keq)+[CI]n)

=> Vm= k+([ρ0]/Keq) & Kp=1/Keq=Kd

Therefore:
Keq = exp( -ΔG / R T )
k+ = (KB/h) T exp( -ΔG / R T ) = (KB/h) T Keq

WET LAB:

Gel

We run a gel with the PCR products obtained the day 01-08-08

Purification

From the PCR of the 01-08-08 we took 120 μl to purify DNA in a low fusion point agarose gel in line with the kit.

We run an agarose gel to verify the status of the purified PRC products.

1. Molecular Marker
2. Part 1 of biopart BBa_I79006
3. Biopart cI BBa_C0051
4. Biopart 3 (normal) of BBa_I79006
5. Biopart 3 (mut) ofBBa_I79006
6. Operator of rcnR + RcnA

Restrictions

We left restrictions over night(double and simple) of each biopart.

First Simple Restriction

The bioparts PCR product was cut with 2 simple consecutive restrictions. The reactives and volumes used in the first reaction were the following:

• Part 1; BamH1

  Buffer U.... 4 microlts
  BSA.......... 4 microlts
  BamH1...... 2 microlts
  DNA PCR... 15 microlts
  H2O......... 15 microlts   
  Total......... 40 microlts
  

• Part 2_cI; BamH1

  Buffer U.... 4 microlts
  BSA.......... 4 microlts
  BamH1...... 2 microlts
  DNA PCR... 15 microlts
  H2O......... 15 microlts   
  Total......... 40 microlts
  

• Part 3 Normal; Xba1

  Buffer U.... 7 microlts
  BSA.......... 7 microlts
  Xba1......... 3 microlts
  DNA PCR... 28 microlts
  H2O......... 25 microlts   
  Total......... 70 microlts

• Part 3 Mutated; Xba1

  Buffer U.... 7 microlts
  BSA.......... 7 microlts
  Xba1......... 3 microlts
  DNA PCR... 28 microlts
  H2O......... 25 microlts   
  Total......... 70 microlts

• Part 4 RcnA; Xba1

  Buffer U.... 7 microlts
  BSA.......... 7 microlts
  Xba1......... 3 microlts
  DNA PCR... 28 microlts
  H2O......... 25 microlts   
  Total......... 70 microlts
  

MODELING:
Hill Cooperativity

5th Reaction, conflict

If we consider that:

  - Keq = exp (-ΔG / R T)

  - k + = (KB / h) T exp (-ΔG / R T) = (KB / h) T Keq

and given that the flow is (k + / Keq) [ρ0] [CI] n / ((1/Keq) + [CI] n), the value of the maximum speed of the flow loses its meaning.

The speed limit is being determined by (k + / Keq) [ρ0], but k + / Keq = (KB / h) * T, and we know that [ρ0] is arbitrary, i.e., Vmax is no longer based on the reaction as such, which does not make sense.

For example: Take the same reaction that we are considering, the maximum speed of the flow of the reaction would be the same with the promoter that has the operators of CI, that if you used one with a random sequence, so, whether we repeated the experiment, with the same temperature and the same concentration of DNA and an equal number of copies of the sequence, the maximum speed reached by the flow would be the same for the real promoter as for for any sequence, without taking any consideration with their affinity for their substrates... That does not makes sense!

The proposed explanation is that the equation used to determine k + does not fit our model. We should explore other possibilities.

WET LAB:

Restrictions

Second simple restriction

Before the second simple restriction we cleaned the product oof the first restriction with the purification Kit.
Due to the purification protocol we knew that the DNA was clean and diluted in 40 μl of buffer, and in order to obtain an efficient restriction we try to dilute the less the DNA-Buffer mix, obtaining the following volumes.

• Part 1; EcoR1

  Buffer U....... 5 μl
  BamH1........ 2.5 μl
  Clean DNA ... 40 μl
  H2O............2.5 μl
  Total........... 50 μl
  

• Part 2_cI; Xba1

  Buffer 2....  5 μl
  BSA.......... 2.5 μl
  BamH1...... 2.5 μl
  DNA PCR... 40 μl 
  Total......... 50 μl

• Part 3 (Normal); Pst1

Buffer 3.... 5 μl
Pst1......... 2.5 μl
DNA PCR... 40 μl
H2O......... 2.5 μl   
Total......... 50 μl

• Part 3 Mutated; Pst1

Buffer 3.... 5 μl
Xba1......... 2.5 μl
DNA PCR... 40 μl
H2O......... 2.5 μl   
Total......... 50 μl

• Part 4 RcnA; HindIII

  Buffer 2.... 5 μl
  HindIII..... 2.5 μl
  DNA PCR... 40 μl
  H2O......... 2.5 μl   
  Total......... 50 μl

Extraction

Plasmids pRK415 and pBBR1MCS-5 were extracted with the Roche kit(see Techniques).

Cultures

We cultured DH5alfa cells tranfromed with pJet+biopart

MODELING:
Hill Cooperativity:
5th Reaction, solving the problem:

The error in the previous approach was that we were considering ΔG to be the same for both equations (for Keq & k+).

We know that the equilibrium depends solely on the difference in the free energy of Gibbs between the substrate and the product (ΔG 'th). The one with less energy will be favored in the balance, while the rate of reaction depends on the activation energy needed for the conversion (ΔG ‡). A reaction reaches equilibrium faster or slower depending on the rate of reaction (depending on the magnitude of its ΔG ‡), but the balance itself does not change.

Thus:
Keq = exp (- ΔG 'º / R T)
k + = (KB / h) T exp (- ΔG ‡ / RT) ≠ (KB / h) T Keq

GROUP MEETING
Wet Lab Statusk

Objectives:
- Build the bioparts.
- Transform the bacteria with the construction that we have.
- Design the experiments to test our construction.
- Build the system.
- Collaborate with the modeling group.

To do:
- Extract DNA from the wild type strain to obtain RcnA.
- Get the bioparts catalog.
- Obtain a large amount of plasmid that we can use, and amplify the bioparts.
- Transformation of the bacteria with bioparts.

Currently:
- There are plasmids.
- There are parts already amplified and in a plasmid.

Problems:
- The DNA that we needed was not in the registry.
- The oligos were delayed 2 weeks and a half.
- Issues to extract the plasmid from the colonies.
- Make a PCR ligation with the three parts and amplify with the ends (it did not work).
- With the enzyme used the frequency of spontaneous mutation was increased to about an error every thousand base pairs.
- There is a problem with tetracycline. You get false positives.

What can be done:
- The biopart with RcnA can already be linked to the plasmid.
- For the other construction we will have to link two parts and digest them, then link them with the third part and digest once more, then insert into the final plasmid.
- HindIII can be used with the large biopart to verify the sequence.

Electrodes:
- Will they be specific for Nickel?

WET LAB:

Cultures

We left cultures of Biopart 1 in pJEt

Plasmid Extraction

Plasmid RcnA was extracted by alkaline lysis

Gels

We run a 2% Agarose gel with the following samples:

1. Molecular Marker (2.5 μl)
2. Restriction of part 1_3 (5 μl)
3. Double Restriction of RcnA_3 (5 μl) no purified to verify.
4. RcnA (purified 5 μl)
5. CI (5 μl)
6. Part 3 Normal (5 μl)
7. Part 3 Mutated (5 μl)

With this gel the parts were verified

PCR

We performed a PCR reaction for RcnA and part 1 using Taq pol.

Transformation y ligation

We took cut RcnA and then it was ligated at vector PBBIMCS_5

The transformation was performed by the previously mentioned technique and the strain was cultured in two Gentamycine cages(Gm 20)

PCR Cleaning

We cleaned the PCR and it was filled up to 40 μl

WET LAB:

4μl of each sample were charged in the following order:

1. Molecular Marker
2. RcnA 1
3. RcnA 3
4. RcnA 4
5. RcnA 5
6. RcnA 6
7. Part 1_1
8. Part 1_3
9. Part 1_6
10. Part 1_7
11. Part 1_9

<Falta pegar gel segun liber...>

Part 1_1 restriction (Double digestion)

After double digestion of part 1 we left massive ligation of part 1,2 and 3.

Ligation Recipe:

Restriction of the other bioparts was performed

WET LAB:

GEL

2% Agarose gel was run with the product of the massive ligation of the three parts

(we didn't obtain the desired product)

PCR

We pick up a PCR product with the folowing oligos:

a) 1up and 2low
b) 2up and 2low
c) control

The PCR reaction was prepared in the following way:

GEL

We run for 1 hr, an 2% Agarose Gel

1. Molecular Marker (2.5 μl)
2. P1_P2 (oligo 1 up y 2 low) ligation with part 3 (normal) (5μl)
3. P2_P3 normal (oligo 2 up y 3 low) (5 μl)
4. Negative Control(oligo 1 up y 2 low) (5 μl)
5. P1_P2 (oligo 1 up y 2 low) ligation with part 3 (mutated) (5 μl)
6. P2_P3 mutated (oligo 2 up y 3 low) (5μl)
7. Negative Control (oligo 1 up y 2 low) (5μl)

*The results suggest inspecific primer join.

Cultures

We left cultures of the RcnA + PBBRIMCS_5 ligation