Team:LCG-UNAM-Mexico/Wet Lab/Objectives

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='''Wet Lab!!'''=
='''Wet Lab!!'''=
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<br><br>
=='''General objectives'''==
=='''General objectives'''==
 +
<br>The final aim is to get and test the following device.
 +
 +
[[Image:P4_genome.jpg]]
 +
[[Image:Delivery_sistem.png|470px]]
 +
 +
==='''I. Biobrick Assembly of the Kamikaze system'''===
 +
<br>
 +
''Many cuts, pastes and clones for biobrick organization into the suicide system!!<br> In the hands of: [[Team:LCG-UNAM-Mexico/Wet_Lab/Objectives#Abraham Avelar|Abraham]] and [[Team:LCG-UNAM-Mexico/Wet_Lab/Objectives#Enrique Paz|Paz]]''
 +
<br>
 +
 +
==='''II. Construction of the standardized P4 vector'''===
 +
<br>
 +
''An incredible and challenging fight against PCR and logical thinking!!<br>In charge: [[Team:LCG-UNAM-Mexico/Wet_Lab/Objectives#Fernando Montaño|Nando]] and [[Team:LCG-UNAM-Mexico/Wet_Lab/Objectives#Enrique Paz|Paz]]''
 +
<br>
 +
==='''III. Construction of the P4-producing strain'''===
 +
<br>
 +
''A passionate struggle with natural selection and recombination!!<br>Leading:
 +
[[Team:LCG-UNAM-Mexico/Wet_Lab/Objectives#Uriel Urquiza|Uriel]], [[Team:LCG-UNAM-Mexico/Wet_Lab/Objectives#Laura Gómez|Laura]], and Miguel''
 +
<br >
 +
==='''IV. System testing and parameter obtention'''===
 +
<br>
 +
''The integrative side of the bite back<br> In the hands of: [[Team:LCG-UNAM-Mexico/Wet_Lab/Objectives#Laura Gomez|Laura]] (and Everyone else soon!)''
 +
<br>
=='''Personal Objectives'''==
=='''Personal Objectives'''==
 +
==== Uriel Urquiza ====     
 +
 +
[[Team:LCG-UNAM-Mexico:Journals:Uriel| Journal]]
 +
 +
** Construction of the phage production control system.
 +
 
 +
  In order to produce a grate amount of P4 phage particles that have our death system, we want to avoid the natural
 +
  early lysis that occur when WT P4 and P2 interact. This avoidance will be achieved by taking the control of the
 +
  two major regulators of P2 morphopoietic genes and deleting them form lysogenic P2; this will yield a P2 phage
 +
  which only has capsid and tail genes.  The control system that is going to be implemented is constituted by a
 +
  promoter inducible by IPTG in conjunction with transactivators cox and ogr from phage P2. All this will
 +
  allow us to grow bacteria in great quantities, induce lysis whenever we want and consequently a stock amount
 +
  of modified P2.
 +
 +
** Qualitative characterization of T7/T3 multipromoter.
 +
 +
  The multi-promoter that we have designed has the capacity to respond specifically to T7/T3 RNA polymerases
 +
  so if one or both polymerases are present in the cell the genes downstream of this promoter will be active.
 +
  A first characterization approach is the introduction of a plasmid carrying our promoter in the E. coli strain
 +
  BL21(DE3)pLysS that has a T7 RNA polymerase inducible by IPTG and then perform assays with and without this
 +
  inductor, finally microscope visualization with suitable filter and light will be performed to see a GFP that
 +
  is under control of the multipromoter.
 +
 +
====Laura Gomez====
 +
 +
 +
[[Team:LCG-UNAM-Mexico/LauraJournal|Journal]]
 +
 +
** To obtain all the relevant experimental information about T7 and T3.
 +
  Model validation needs some critical parameters like strain(C1a) growth rate and the T3 and T7 burst sizes. The
 +
  parameters found in the literature correspond to k12 strain however we used a k12 derivative strain, C1a, for this
 +
  reason it was indispensable to obtain experimentally a C1a growth plot. In the same way, simulations
 +
  showed us that the burst size is a critical parameter to determine the efficiency of our system.
 +
 +
** Generation of data to feedback the infection model.
 +
  Our [[Team:LCG-UNAM-Mexico:Modelling | Model]] Simulates both Molecular and Population Dynamics for the Defense System. Important  parameters must be estimated
 +
  experimentally. The most important parameter in the defense system is the[[Team:LCG-UNAM-Mexico:BSD| Burst Size]]. Experimental measures
 +
  of the Burst Size are of vital importance. The [[Team:LCG-UNAM-Mexico:Molecular model| Molecular Model]] will simulate the intracellular
 +
  dynamics and will generate a Burst Size Distribution. With my experimental results we will validate and improve the model.
 +
  At the population scale the [[Team:LCG-UNAM-Mexico:CA|Cellular Automaton]] will simulate the interaction between populations of 
 +
  bacteria and phages, T7 infection experiments will provide valuable information to the Population model.
 +
 +
====Abraham Avelar====
 +
 +
[[Team:LCG-UNAM-Mexico/AbrahamJurnal|Journal]]
 +
 +
** Ensamble the kamikaze construction
 +
 
 +
  Clone the colicines with the multipromoter, design and Clone the asRNA and help in the ensamble of all the device.
 +
 +
** Test the construction
 +
 
 +
  Obtain the time that E3 takes to kill ''E. coli'', this parameter will be provided to the model. The preference <br> for E3 above E9 is due to a [https://2009.igem.org/Team:LCG-UNAM-Mexico:BSD#BSD_using_the_Kamikaze_System modeling suggestion] that shows the effectivity of our system when the translation<br> machinery is disrupted, test the efficience of plaquing with the asRNA by itself and the efficience of all the<br> device.
 +
 +
====Fernando Montaño====
 +
 +
[[Team:LCG-UNAM-Mexico:Journals:Nando's|Journal]]
 +
 +
**Assembly of the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K242051 P4 vector]
 +
 +
We want P4 to work as a [[Team:LCG-UNAM-Mexico/Description#Delivery|transduction vector for biobricks]]. Several things need to be done, which include <br>individual  amplification of P4 sid1 essential region, which we expect to be sufficient for a stable permanence <br>of the biobrick inside the cell. To test this, the first biobrick added will be a reporter gene, which is intended <br>to permanently stay with our P4 version. This is enough to further produce our P4 viral particles and assess<br> functionality of the delivery by transduction into several wild-type bacterial strains as reported in literature.
 +
 +
**Checking functionality for the [[Team:LCG-UNAM-Mexico/Description#Defense|Kamikaze system]]
 +
 +
Now we will be ready to ligate the entire device. Many things have to be assessed, as the complete packaging and <br>delivery of the vector into the host cells, and of course, the functionality of the system. This involves <br>checking for presence and reaction to AHL, production of antisense RNAs and many parameter calculations.
 +
 +
**P2 control system functionality
 +
 +
The [[Team:LCG-UNAM-Mexico/Description#P4sid1 standardized production|cox/ogr]] control system in which [[Team:LCG-UNAM-Mexico/Wet lab/Objectives#Uriel Urquiza|Uriel]] is working needs experimental validation of functionality.<br>I intend to transform P2 lysogenic strains and expect lysis through activation of such genes.<br>This will prove the construction works.
 +
 +
**P4 as an iGEM Plasmid
 +
 +
P4 will have to suffer many more modifications in order that it functions as an iGEM standard vector. The design <br>automatically eliminated forbidden restriction sites, but we also need transcription terminators and universal <br>primers. After addition of such sequences, functionality has to be assessed again.<br>
 +
 +
====Enrique Paz====
 +
 +
[http://openwetware.org/wiki/User:Paz_C._Enrique/Notebook/Paz_C._Enrique_-_Projects#Personal_objectives Journal]
 +
 +
** Design of our modified version of bacteriophage P4
 +
 +
We pretend to modify P4 to use it as an standard vehicle for synthetic constructions. My mission: Define what we
 +
need to do over P4 genome and how to do it in order to construct a bacteriophage compatible with standards in
 +
synthetic biology.
 +
 +
** Construction and test of the gossip device (quorum sensing)
 +
 +
Some -a lot- of restrictions, ligations, transformations, tests, etc. to assemble this device. The idea of this
 +
device is that infected bacteria will send and alarm to the surrounding bacteria. The alarm consist in a molecule
 +
of quorum sensing. Until the alarm will not save any bacteria, this molecule will activate transcription of an
 +
antisense to delay the cycle of the virus.
 +
 +
** Test the host range of our modified P4
 +
 +
According to litterature bacteriophage P4 has an unusual host-range. We would like to test it so we can transport
 +
easily synthetic construction to another interesting bacteria species! Some of this bacteria reported as hosts for
 +
P4 are: E.Coli, Klebsiella, Serratia and Rhizobium.
 +
 +
** Test the biobrick of P4 cos-site
 +
 +
P4 cos-site is a DNA fragmen of P4 genome that serves as a signal for packaging its DNA into the capsid. The idea
 +
is that with our sistem for P4 production and this biobrick you can encapsidate any!! DNA sequence and transduce it.
=='''Work Journals'''==
=='''Work Journals'''==
Line 27: Line 148:
*[[Team:LCG-UNAM-Mexico/LauraJournal|Laura's Lab Journal]]
*[[Team:LCG-UNAM-Mexico/LauraJournal|Laura's Lab Journal]]
-
* [[ Paz Paz]]
+
*[http://openwetware.org/wiki/User:Paz_C._Enrique/Notebook/Paz_C._Enrique_-_Projects#Personal_objectives Enrique's Lab Journal]
<!--Do not remove the first and last lines in this page!-->{{Template:LCG_bottom_Netscape}}
<!--Do not remove the first and last lines in this page!-->{{Template:LCG_bottom_Netscape}}

Latest revision as of 03:51, 22 October 2009




Contents

Wet Lab!!



General objectives


The final aim is to get and test the following device.

P4 genome.jpg Delivery sistem.png

I. Biobrick Assembly of the Kamikaze system


Many cuts, pastes and clones for biobrick organization into the suicide system!!
In the hands of: Abraham and Paz

II. Construction of the standardized P4 vector


An incredible and challenging fight against PCR and logical thinking!!
In charge: Nando and Paz

III. Construction of the P4-producing strain


A passionate struggle with natural selection and recombination!!
Leading: Uriel, Laura, and Miguel

IV. System testing and parameter obtention


The integrative side of the bite back
In the hands of: Laura (and Everyone else soon!)

Personal Objectives

Uriel Urquiza

Journal

    • Construction of the phage production control system. 
  In order to produce a grate amount of P4 phage particles that have our death system, we want to avoid the natural
  early lysis that occur when WT P4 and P2 interact. This avoidance will be achieved by taking the control of the 
  two major regulators of P2 morphopoietic genes and deleting them form lysogenic P2; this will yield a P2 phage 
  which only has capsid and tail genes.  The control system that is going to be implemented is constituted by a 
  promoter inducible by IPTG in conjunction with transactivators cox and ogr from phage P2. All this will 
  allow us to grow bacteria in great quantities, induce lysis whenever we want and consequently a stock amount
  of modified P2.
    • Qualitative characterization of T7/T3 multipromoter. 
  The multi-promoter that we have designed has the capacity to respond specifically to T7/T3 RNA polymerases
  so if one or both polymerases are present in the cell the genes downstream of this promoter will be active. 
  A first characterization approach is the introduction of a plasmid carrying our promoter in the E. coli strain
  BL21(DE3)pLysS that has a T7 RNA polymerase inducible by IPTG and then perform assays with and without this 
  inductor, finally microscope visualization with suitable filter and light will be performed to see a GFP that 
  is under control of the multipromoter.

Laura Gomez

Journal

    • To obtain all the relevant experimental information about T7 and T3. 
 Model validation needs some critical parameters like strain(C1a) growth rate and the T3 and T7 burst sizes. The 
 parameters found in the literature correspond to k12 strain however we used a k12 derivative strain, C1a, for this 
 reason it was indispensable to obtain experimentally a C1a growth plot. In the same way, simulations
 showed us that the burst size is a critical parameter to determine the efficiency of our system.
    • Generation of data to feedback the infection model. 
 Our  Model Simulates both Molecular and Population Dynamics for the Defense System. Important  parameters must be estimated
 experimentally. The most important parameter in the defense system is the Burst Size. Experimental measures
 of the Burst Size are of vital importance. The  Molecular Model will simulate the intracellular
 dynamics and will generate a Burst Size Distribution. With my experimental results we will validate and improve the model. 
 At the population scale the Cellular Automaton will simulate the interaction between populations of   
 bacteria and phages, T7 infection experiments will provide valuable information to the Population model.

Abraham Avelar

Journal

    • Ensamble the kamikaze construction 
 Clone the colicines with the multipromoter, design and Clone the asRNA and help in the ensamble of all the device.
    • Test the construction 
 Obtain the time that E3 takes to kill E. coli, this parameter will be provided to the model. The preference 
 for E3 above E9 is due to a modeling suggestion that shows the effectivity of our system when the translation
 machinery is disrupted, test the efficience of plaquing with the asRNA by itself and the efficience of all the
 device.

Fernando Montaño

Journal

    • Assembly of the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K242051 P4 vector] 
We want P4 to work as a transduction vector for biobricks. Several things need to be done, which include 
individual  amplification of P4 sid1 essential region, which we expect to be sufficient for a stable permanence 
of the biobrick inside the cell. To test this, the first biobrick added will be a reporter gene, which is intended 
to permanently stay with our P4 version. This is enough to further produce our P4 viral particles and assess
 functionality of the delivery by transduction into several wild-type bacterial strains as reported in literature.

Now we will be ready to ligate the entire device. Many things have to be assessed, as the complete packaging and 
delivery of the vector into the host cells, and of course, the functionality of the system. This involves 
checking for presence and reaction to AHL, production of antisense RNAs and many parameter calculations.
    • P2 control system functionality 
The cox/ogr control system in which Uriel is working needs experimental validation of functionality.
I intend to transform P2 lysogenic strains and expect lysis through activation of such genes.
This will prove the construction works.
    • P4 as an iGEM Plasmid 
P4 will have to suffer many more modifications in order that it functions as an iGEM standard vector. The design 
automatically eliminated forbidden restriction sites, but we also need transcription terminators and universal 
primers. After addition of such sequences, functionality has to be assessed again.

Enrique Paz

[http://openwetware.org/wiki/User:Paz_C._Enrique/Notebook/Paz_C._Enrique_-_Projects#Personal_objectives Journal]

    • Design of our modified version of bacteriophage P4 
We pretend to modify P4 to use it as an standard vehicle for synthetic constructions. My mission: Define what we
need to do over P4 genome and how to do it in order to construct a bacteriophage compatible with standards in 
synthetic biology.
    • Construction and test of the gossip device (quorum sensing) 
Some -a lot- of restrictions, ligations, transformations, tests, etc. to assemble this device. The idea of this
device is that infected bacteria will send and alarm to the surrounding bacteria. The alarm consist in a molecule 
of quorum sensing. Until the alarm will not save any bacteria, this molecule will activate transcription of an
antisense to delay the cycle of the virus.
    • Test the host range of our modified P4 
According to litterature bacteriophage P4 has an unusual host-range. We would like to test it so we can transport
easily synthetic construction to another interesting bacteria species! Some of this bacteria reported as hosts for
P4 are: E.Coli, Klebsiella, Serratia and Rhizobium.
    • Test the biobrick of P4 cos-site 
P4 cos-site is a DNA fragmen of P4 genome that serves as a signal for packaging its DNA into the capsid. The idea
is that with our sistem for P4 production and this biobrick you can encapsidate any!! DNA sequence and transduce it.

Work Journals

  • [http://openwetware.org/wiki/User:Paz_C._Enrique/Notebook/Paz_C._Enrique_-_Projects#Personal_objectives Enrique's Lab Journal]


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