Team:LCG-UNAM-Mexico/Notebook/2008-August 2

WET LAB:

Ligation repeat

Performed in order to obtain at least part 1 and part 2 joined

Part 1	5 μl
Part 2	5 μl
Buffer 5x	4 μl
T4DNA Pol	1 μl
H2O	5 μl
Total	20 μl

Ligation PCR of P1_P2

H2O	33 μl
Buffer	5 μl
dNTPs	2.5 μl
Oligo 1 up	2.5 μl
Oligo 2 low	2.5 μl
Mg(Cl)2	2.5 μl
DNA	1 μl
Taq	1 μl
Total	50 μl

*The ligation and the PCR were done ___

Plasmid Extraction

Plasmid PBBIMCS_5 was extracted from the cultures of the last day.

PCR ligation Gel of part1_part2

WET LAB:

Ligation  part1+PRK415

The tube DR of PRK415 was used along with the following samples of part 1:

• DR Part1_9/pJET (130808)
• DR Part1_6/pJET (130808)
• DR Part1_3/pJET (130808)

Recipe for each ligation tube

Vector	4 μl
Part 1	8 μl
Buffer 5x	4 μl
Enzyme	1 μl
H2O	3 μl

Restriction

Linearization of pBB to use it as a control in RcnA+pBB Gel

Recipe for each restriction tube:

H20	10 μl
Buffer	3 μl
DNA	16 μl
EcoR1	1 μl
	30 μl

Plasmid Extraction Gel pBB+RcnA

1. Molecular Marker (2 μl)
2. [1] Cage2 /11_1
3. [2] Cage1 /1_2
4. [3] Cage2B /1_1
5. [4] Cage2a /10_1
6. [5] Cage2b /8_1
7. [6] Cage2b /6_1
8. [7] Cage1 /5_1
9. [8] Cage1 /2_1
10. [9] Cage2b /4_1

	1	2	3	4	5	6
Water	7 μl	7 μl	7 μl	5 μl	7 μl	7 μl
Buffer	2 μl	2 μl	2 μl	2 μl	2 μl	2 μl
DNA	10 μl	10 μl	10 μl	12 μl	10 μl	10 μl
Enzyme	1 μl	1 μl	1 μl	1 μl	1 μl	1 μl
	20 μl	20 μl	20 μl	20 μl	20 μl	20 μl

*In order to verify the size of the insert in pBB

Extraction plasmid restrictions (pBB+RcnA) with EcoR1

We used:

• [1] Cage2 /11_1
• [2] Cage1 /1_2
• [3] Cage2B /1_1
• [4] Cage2a /10_1
• [5] Cage2b /8_1
• [9] Cage2b /4_1

Gel

1. Molecular Marker (2.5 μl)
2. Restriction EcoR1 pBBIMCS-5 2b/4 (2 μl)
3. Restriction EcoR1 pBBIMCS-5 2a/10 (2 μl)
4. Restriction EcoR1 pBBIMCS-5 2b/5 (2 μl)
5. Restriction EcoR1 pBBIMCS-5 2a/14(2 μl)
6. Restriction EcoR1 pBBIMCS-5 2a/9(2 μl)
7. Restriction EcoR1 pBBIMCS-5 limpio con kit(2 μl)
8. Restriction EcoR1 BamH1 PRK415 R1(2 μl)
9. Restriction EcoR1 BamH1 PRK415 R2(2 μl
10. PCR control1 biopart 1 oligo 1up 2low (5 μl)
11. PCR control1 biopart 2 oligo 1up 2low (5 μl)
12. Negative Control PCR (5 μl)

Conclusions of the previous gel:

• The Oligos 1up and 2low don't join in unexpected sites
• RcnA wasn't inserted in pBB
• It's necessary to repeat the double restriction of PRK

PCR

1. Double ligation
2. Control without DNA
3. Control without oligos (¿Is there something contaminated?)

We run a gel of this three PCRs and we didn't obtain any product.

Repetición PCR

We repeated the previous PCR reaction using the following recipe:

DNA	2 μl
H2O	32 μl
dNTP’s	2.5 μl
Mg(Cl)2	2.5 μl
Oligo 1up	2.5 μl
Oligo 2low	2.5 μl
Taq	1 μl
Buffer	5 μl

WET LAB

Restrictions gel pBB+RcnA

1. Molecular Marker (2.5 μl)
2. [1] Cage2 /11_1
3. [2] Cage1 /1_2
4. [3] Cage2B /1_1
5. [4] Cage2a /10_1
6. [5] Cage2b /8_1
7. [9] Cage2b /4_
8. pBB1
9. pBB2
10. pBB3
    

Cultured from a strain result of the transformation

We received an e-mail from the Mexico-UNAM-IPN with several questions in regards to our project, here is our reply:

Hello,
We apologize for the late reply, but we had to discuss carefully our answer.
First of all, we think you are confused about what our project really is. We want to make bacteria to modify the extracellular nickel concentration in response to an external signal (AHL in this case), and of course, be able to predict to what extent the concentration of the input signal will affect the amount of nickel in the medium. To achieve this, it is true we have to synchronize our cell population at the beginning. This is easy to do and doesn't represent any technical problems.
We are very conscious of the facts you tell us, first: we know the half-life of the lactones is relatively long (24 hrs as you say). That's why we are including AiiA under a constitutive promoter in our model, which degrades AHL very efficiently. This will ensure AHL does not saturate the medium. Second, we know AiiA does not diffuse freely through the cell membrane. However, we don't need that to happen, as each cell will degrade its own AHL (yes, we are assuming that all AHL will enter a cell within a window of time).
In other words, we do not need to synchronize the bacterial population more than in the first step. We are considering that some cells may respond earlier than others. However, we are assuming that, as we are not changing the physical nor chemical conditions, the proportion of cells responding "earlier" will remain constant, thus allowing us to draw some conclusions of the behaviour of the population as a whole. We hope you see why the synchrony is no longer important for our project.

To summarize what we plan to do, AHL will enter the cell and form a dimer with LuxR (which is under a constitutive promoter, so AHL is the only limiting step). This will start the transcription of cI*, which will repress the expression of RcnA. RcnA is the nickel efflux pump, and thus we are aiming to predict the amount of AHL necessary to get the desired extracellular nickel concentration.
We are doing small moves. At first, we only want to make one successful assay. We hope that in the near future we will be able to use the response time of the system to generate a succession of desired nickel concentrations, thus generating a song.
We hope this letter answers your questions,

LCG-UNAM-Mexico Team
Cuernavaca, Morelos

MODELING:
Reaction 3, AHL:LuxR
Conflict: k3 (ON) <k3 (OFF)?
Reference: Goryachev et al. (2006)

The references they cite for obtaining their parameters were not specific for that kind of parameters, in fact, one mentions the rate of RNA polymerase in HUMAN!
- Check whether the article mentions how they transformed the parameters.

They seem to explain why in their model, the k3(ON) is "so small" to start with:
<<common to all models considered here, is that the stability of the state "off" defined by the constitutive Transcription levels of I and R comes at a price of high value for the critical self Extracellular concentration.>>

This explains the criteria they used to determine their parameters:
<<For each layout we attempted to identify a set of parameters that optimize the functional fitness of the network. The search in the parameter space is constrained by requesting that the kinetic parameters must remain in the biologically realistic range and the resulting network should demonstrate the behavior compatible with our present understanding of the phenomenon quorum sensing.>>

WET LAB

Agarose Gel 1% low fusion point for band purification

1. Gel1
2. Molecular Weight marker
3. [1]
4. [2]
5. [3]

Ligation PCR parte1+parte2(35 ciclos)

1. 1_2 oligo 1up-2lower
2. 1_1 oligo 1up-1lower
3. 2_2 oligo 2up-2lower
4. 1_3 oligo 1up-3lower
5. 1_2 oligo 1up-2lower

Recipes:

DNA	2 μl
Water	32 μl
dNTP’s	2.5 μl
Mg(Cl)2	2.5 μl
Oligo 1up	2.5 μl
Oligo 2low	2.5 μl
Taq	1 μl
Buffer	5 μl

MODELING:

Reaction 4: Natural degradation of cI*

Half life of cI*:

The half-life of a modified cI is of 4 minutes, according to Elowitz & Leibler (2000). They analyze the LAA tail, and JB Andersen et al (1998) conclude that the LAA and LVA tails confer about the same time of life to GFP.

Reaction rate:

Once we get the half-life time of the protein, how do we calculate the rate of reaction and the flow?

The half-life of a reaction (t1/2) is the time it takes for half of the reagents to become products. In a first order reaction, t1/2 is a constant and can be calculated from the rate constant, as follows:
t1/2 =-ln(0.5)/k=0.693/k
This reciprocal relationship between the half life time and the rate constant is very useful when making an estimate of the time a reaction will take to occur. Thus, for k = 0.01/s, the half life time would be about 70 s. For k = 10/s, the half life would be of about 0.07 s or 70 milliseconds. The average life of the reactions of first order is also independent of the initial concentration. If the first half of the molecules react in aprox 20 s, half of the remaining molecules will also take 20 s to react, and so on. The fact that the average lifetime in an unimolecular reaction is a constant means that, at any time of the reaction, a constant fraction of reactive molecules have enough energy to overcome the kinetic barrier and become molecules of product. This makes sense because the energy of a set of molecules is distributed randomly according to a Boltzmann distribution.
* With information from RT Sauer (1999).
NOTE: A first order reaction is the type A → B.

Today we sent an e-mail to Dr. Peter Chivers, an expert in RcnR, to ask him for information in regards to RcnA.

Our mail to Dr. Peter Chivers:
Dr. Peter Chivers,

Hi! My name is Carlos and I'm a student of the Undergraduate Program in Genomic Sciences of the National Autonomous University of Mexico and currently I'm on the fourth year.

The main reason of this e-mail is to get in contact with you, I'll explain you why as briefly as I can. Several students of the program decided to participate in a systems biology competition known as iGEM, and our project focuses on regulating nickel efflux in e. coli. We have to both build the circuit and design a mathematical model to accurately predict the behaviour of our system.

Taking several issues in consideration we decided to regulate RcnA.
Here is the page of our team in case you're interested in reading more about our project:

https://2008.igem.org/Team:LCG-UNAM-Mexico

However we've been having some trouble in finding parameters for our model (such as RcnA's half-life, efficiency, synthesis rate among others). Therefore, having exhausted (or so we believe) the literary resources and having found your name in many of the papers regarding RcnA we decided to contact you. I'll try to explain with more detail the parameters we still lack in case you're interested in providing us with information.

I greatly thank you for taking the time to read this e-mail and await your answer.

Carlos Vargas


Dr. Peter Chivers' reply

Dear Carlos,

Thank you for your e-mail. From what I read on your website, it looks like your team is tackling an interesting project. Unfortunately, there are very few papers on RcnA itself, and none of them have examined the parameters that you need for your model. As far as I know, the groups that have published in the RcnR/RcnA area don't typically do those sorts of measurements so they are unlikely that have the data you need. We have no plans to do these experiments as my lab focuses on the nickel responsive regulators, RcnR and NikR.

I'm sorry that I can't be of more assistance. Good luck with your project, I hope it is well received at the iGEM competition.

Best regards,

Peter

WET LAB

Agarose Gel 1% low fusion point for band purification

1. Molecular Weight Marker
2. [1] Cage2 /11_1 pBB+RcnA
3. [2] Cage1 /1_2 pBB+RcnA
4. [5] Cage2b /8_1 pBB+RcnA
5. [9] Cage2b /4_1 pBB+RcnA
   

Gel Purification PCR of pBB+rcnA

1. Purification 1
2. Purification 2
3. Purification 3
4. Purification 4 clean PRK
5. Positive control
6. Negative control

The cells seem to carry the plasmid with RcnA, However one of the negative controls gave product.

All of them were positive, however there are some spurious products.(no photo was added because the low melting gels are quite fragile and we couldn't carry it to the transiluminator with camera.)

Recipe:

DNA	2 μl
Water	32 μl
dNTP’s	2.5 μl
Mg(Cl)2	2.5 μl
Oligo 1up	2.5 μl
Oligo 2low	2.5 μl
Taq	1 μl
Buffer	5 μl

A gel with the extraction of the plasmid PRK415+part_1 was run.

Gel Electrophoresis of PCR_Ligation part1+part2

1. [5] 1_2 oligo 1up-2lower
2. [4] 1_2 oligo 1up-2lower
3. [3] 1_2 oligo 1up-2lower
4. Molecular marker
5. [2] 1_2 oligo 1up-2lower
6. [1] 1_2 oligo 1up-2lower

We have got part1+part2 ligation

WET LAB

PRK+part1 PCR
Recipe:

DNA	1 μl	11 μl
H2O	33 μl	363 μl
dNTP’s	2.5 μl	27.5 μl
Mg(Cl)2	2.5 μl	27.5 μl
Oligo 1up	2.5 μl	27.5 μl
Oligo 2low	2.5 μl	27.5 μl
taq	1 μl	11 μl
Buffer	5 μl	55 μl
	50 μl	550 μl

PCR gel of RcnA (Obtained of the purification in gel of pBB + RcnA)

1.-Molecular-weight marker
2.-[1] RcnA
3.-[2] RcnA
4.-[5] RcnA
5.-[9] RcnA
6.-Control +
7.-Control -

Ligation part1+part2 (rttH)  PCR

Reaction 1
DNA	2 μl
H2O	10 μl
dNTP’s	4 μl
Mg(Ac)2	3 μl
Oligo 1up	2.5 μl
Oligo 2low	2.5 μl
Buffer	6 μl

Reaction 2
H2O	9 μl
Buffer	9 μl
RttH	11 μl

Ligation part1_part2 Gel PCR

1.-Molecular-weight marker
2.-PCR Lp1+P2 1.1
3.- PCR Lp1+P2 1.2
4.- PCR Lp1+P2 5.1
5.- PCR Lp1+P2 5.2
6.-Negative control
7.-PRK + part 1_1
8.- PRK + part1_2

PCR gel of RcnA (Obtained of the purification in gel of pBB + RcnA) Repetition

1.- Molecular-weight marker
2.-[1] RcnA
3.-[2] RcnA
4.-[5] RcnA
5.-[9] RcnA
6.-Control +
7.-Control –

Ligation part1+part2 Restriction with BamH1  

	For1_1	For1_2
H2O	10 μl	5 μl
Buffer2	2 μl	2 μl
BSA	2 μl	2 μl
DNA	5 μl	10 μl
Enzime(BamH1)	1 μl	1 μl
	20 μl	20 μl

PRK+part1  Gel PCR

1.- Molecular-weight marker
2.-[3]
3.-[4]
4.-[5]
5.-[6]
6.-[7]
7.-[9]
8.-[10]
9.-c-

PCRs
With oligos 4 up, 4 lower

1.- pBB+RcnA 1 band purification
2.- pBB+RcnA 1 band purification
5.- pBB+RcnA 1 band purification
9.- pBB+RcnA 1 band purification
-1.-Negative control 1(prk)
-2.-Negative control 2(pBB)
+ Positive control (PCR August 22)

With oligos 1 up, 3 lower
L1 Ligation part1+part2 1_2
L- Negative control

DNA	1 μl
H2O	33 μl
dNTP’s	2.5 μl
Mg(Cl)2	2.5 μl
Oligo 1up	2.5 μl
Oligo 2low	2.5 μl
Taq	1 μl
Buffer	5 μl

WET LAB

2% Agarose gel with the next samples:

1.- Molecular-weight marker
2.-[1] pBB+RcnA 1 band purification
3.-[2] pBB+RcnA 1 band purification
4.-[5] pBB+RcnA 1 band purification
5.-[9] pBB+RcnA 1 band purification
6.-[-1]Negative control 1(prk)
7.-[-2]Negative control 2(pBB)
8.-[+ ]Positive control (PCR August 22)
9.- L1 Ligation part1+part2 1_2
10.- - Negative control
11.-1_1 Restriction BamH 220808 part1+part2
12.-1_2 Restriction BamH 220808 part1+part2
13.- Molecular marker
  * 5 μl of each sample.

Ligation part1+part2 restrictions

H2O	4 μl
Buffer EcoR1	5 μl
BSA	5 μl
DNA	30 μl
Xba1 enzime	3 μl
EcoR1 enzime	3 μl

PRK415 Restriction

H2O	5 μl
Buffer EcoR1	3 μl
BSA	3 μl
DNA	15 μl
Xba1 Enzime	2 μl
EcoR1 Enzime	2 μl

(part1+part2)+PRK415 Ligation
A quick ligation kit was used.

Vector (PRK415)	15 μl
Part1+Part2	7 μl
Buffer 5X	8 μl
Enzime	2 μl
H2O	8 μl
	40 μl

 (part1+part2)+ part3  Ligation
A quick ligation kit was used.

Part3	10 μl
Part1+Part2	10 μl
Buffer 5X	8 μl
Enzime	2 μl
H2O	10 μl
	40 μl

MODELING:
Checking parameters:

Reaction 6
The value that had been found before for k6 (Pl) = 0.20mM / h is in reality the flow of the reaction (which is why the units are mM/h). That is, how many mRNA molecules are being produced per unit of time. The system in which they measured this parameter is a derivative of the plasmid pBR322, pTrc99A. It has the same origin of replication and the number of copies is estimated at 15-20 but under certain conditions where replication is limited it appears to be between 3-5 copies.

Using the previous information we can calculate the value of k+ of the reaction in our system if we consider that each promoter acts independently and we multiply by the ratio between the number of copies of our plasmid and those of their plasmid.

WET LAB

PCR, ligation (1_2)_3

1.-PCR (1_2)_3 oligo 1up 3 low
2.-PCR (1_2)_3M oligo 1up 3 low
3.-c- negative control (no DNA)
4.- pcr (1_2)_3N oligo 2up 3 low control
*Independently added to each tube.
*The alignment temperature was raised 5ºC.
*Problem in oligo 3 low.

	One reaction	Four
DNA	1 μl	4μl
H2O	33 μl	132μl
dNTP’s	2.5 μl	10μl
Mg(Cl)2	2.5 μl	10μl
Oligo 1up	2.5 μl	10μl
Oligo 2low	2.5 μl	10μl
taq	1 μl	4 μl
Buffer	5 μl	20 μl
	50 μl	200μl

WET LAB

 1% Agarose Gel

1.- Molecular-weight marker
2.-[1] part (1_2)_3 oligo  1up_3low
3.-[2] part(1_2)_3M oligo 1up 3low
4.-[3] Negative control (no DNA)
5.-[4] Negative control  part(1_2)_3N

Vector PRK415 restriction to ligate (p1+p2)

	Simple restriction  Pst1	Double restriction  EcoR1/Pst1
H2O	3 μl	6 μl
Buffer 10X	4 μl	2 μl
DNA	30 μl	10 μl
Pst1	3 μl	1 μl
EcoR1	0 μl	1 μl
	40 μl	20 μl

Ligation [p1_p2]-p3 restriction
Final volume 70 μl

H2O	9 μl
Buffer 10X	7 μl
DNA	50 μl
Pst1	2 μl
EcoR1	2 μl
	70 μl

Electrophoresis, double restriction, band purification pBB+RcnA

1.- Molecular-weight marker
2.-[1]
3.-[2]
4.-[5]
5.-[9]

MODELING:

To-do List 1. Find Missing Parameters.
- cI Dimerization.
- Nickel efflux . (Estimate?).
- RcnA degradation.
- Nickel Internalization (Experimentally,?).
- Initial concentrations (aiiA, LuxR? (Define arbitrary medium), ? (by number of copies of the plasmid; change to molar), Ni-ext (experimental)).
2. Review tools in SimBiology (sensitivity analysis, parameter estimation, moiety conservation).
3. Stationary states of the system (whether there is multistationarity).
4. Jacobian.
5. Analysis of the stechiometric matrix (also analyze the null space and its transpose).
6. Electrochemical theory (the relationship between electric potential and the concentration of nickel).
7. Electrodes.
->We need to check for advances in the construction of the measurement device we will use.
->Take into account the possibility of asking for support to Dr. Peña.

WET LAB

Streak
Strains streak of PRK415+(part1_part2) in tube for plasmid extraction (verification)

Transformation
Transformation on ligartions part1_pat2_part3 in DH5α

Restrictions

	Bioparts	Plasmid
H2O	11μl	6 μl
Buffer 10X	5 μl	2 μl
DNA	30 μl	20 μl
Pst1	2 μl	1 μl
EcoR1	2 μl	1 μl
	50μl	30 μl

Ligation
Two ligations of every version of the ligation part1+part2+part3 were made. 25 μl of insert and 25 μl of plasmid were added to each one.

DNA insert	25 μl
PRK415	25 μl
Buffer T4 ligase 10X	6 μl
H2O	3 μl
T4 ligase	1 μl
	60 μl

WET LAB

Plasmid ligation p1+p2+PRK415 gel.