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

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=='''General objectives'''==
=='''General objectives'''==
-
[[Team:LCG-UNAM-Mexico/Wet_Lab/Objectives#Uriel Urquiza|Uriel]]
+
<br>The final aim is to get and test the following device.
-
[[Team:LCG-UNAM-Mexico/Wet_Lab/Objectives#Laura Gómez|Laura]]
+
-
[[Team:LCG-UNAM-Mexico/Wet_Lab/Objectives#Fernando Montaño|Nando]]
+
-
[[Team:LCG-UNAM-Mexico/Wet_Lab/Objectives#Abraham Avelar|Abraham]]
+
-
[[Team:LCG-UNAM-Mexico/Wet_Lab/Objectives#Enrique Paz|Paz]]
+
-
 
+
-
The final aim is to get and test the following device.
+
[[Image:P4_genome.jpg]]
[[Image:P4_genome.jpg]]
 +
[[Image:Delivery_sistem.png|470px]]
==='''I. Biobrick Assembly of the Kamikaze system'''===
==='''I. Biobrick Assembly of the Kamikaze system'''===
<br>
<br>
-
''Many cuts, pastes and clones for biobrick organization into the suicide system!!<br> In the hands of: <br> [[Team:LCG-UNAM-Mexico/Wet_Lab/Objectives#Abraham Avelar|Abraham]] and [[Team:LCG-UNAM-Mexico/Wet_Lab/Objectives#Enrique Paz|Paz]]''
+
''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>
<br>
==='''II. Construction of the standardized P4 vector'''===
==='''II. Construction of the standardized P4 vector'''===
<br>
<br>
-
''An incredible and challenging fight against PCR and logical thinking!!In charge: <br> [[Team:LCG-UNAM-Mexico/Wet_Lab/Objectives#Fernando Montaño|Nando]] and [[Team:LCG-UNAM-Mexico/Wet_Lab/Objectives#Enrique Paz|Paz]]<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>
<br>
==='''III. Construction of the P4-producing strain'''===
==='''III. Construction of the P4-producing strain'''===
<br>
<br>
-
''A passionate struggle with natural selection and recombination!!<br>leading:<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''
[[Team:LCG-UNAM-Mexico/Wet_Lab/Objectives#Uriel Urquiza|Uriel]], [[Team:LCG-UNAM-Mexico/Wet_Lab/Objectives#Laura Gómez|Laura]], and Miguel''
-
<br>
+
<br >
==='''IV. System testing and parameter obtention'''===  
==='''IV. System testing and parameter obtention'''===  
<br>
<br>
-
''The integrative side of the bite back<br> In the hands of: [[Team:LCG-UNAM-Mexico/Wet_Lab/Objectives#Laura Gómez|Laura]] (and Everyone else soon!)''
+
''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>
<br>
-
 
-
 
-
'''Are you ready 2 rumble??'''
 
=='''Personal Objectives'''==
=='''Personal Objectives'''==
Line 49: Line 42:
** Construction of the phage production control system.
** Construction of the phage production control system.
    
    
-
   In order to produce a grate amount of P4 phage particles that has our death system, we want to avoid the natural
+
   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  
   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  
+
   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 systems that is going to be implemented is constituted by a  
+
   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  
   promoter inducible by IPTG in conjunction with transactivators cox and ogr from phage P2. All this will  
-
   allow us to grow bacteria in grate quantities and induce lysis when ever we want and as a consequence a grate
+
   allow us to grow bacteria in great quantities, induce lysis whenever we want and consequently a stock amount
-
   production of modified P2.  
+
   of modified P2.  
-
** Qualitative characterization of multipromter.
+
** 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====
====Laura Gomez====
 +
 +
 +
[[Team:LCG-UNAM-Mexico/LauraJournal|Journal]]
** To obtain all the relevant experimental information about T7 and T3.
** To obtain all the relevant experimental information about T7 and T3.
Line 68: Line 71:
** Generation of data to feedback the infection model.
** Generation of data to feedback the infection model.
-
   The experimental data would be used to feedback the model in order to obtain the most accurate model as possible.
+
   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====
====Abraham Avelar====
 +
[[Team:LCG-UNAM-Mexico/AbrahamJurnal|Journal]]
** Ensamble the kamikaze construction
** Ensamble the kamikaze construction
Line 79: Line 88:
** Test the construction
** 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 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.
+
   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====
====Fernando Montaño====
-
<br>
 
-
====Assembly of the P4 vector.====<br>
 
-
* I must create an eternal bacterial source of P4 sid1. Therefore I must Infect C1a with it.
 
-
*I need to develop a good protocol for P4 sid1 lysate production.
 
-
* I must obtain the essential region of P4 sid1 by PCR. This will add the preffix and suffix to the region.<br>
 
-
3) I need to ligate the "biobricked" P4 region to a reporter; better off RFP or a hard-resistance antibiotic.<br> Then, I have to transform to produce P4 (preferably with our stock producing bacterium) and infect wild-type strains to look for reporter-associated colonies. <br>(Also try in many bacterial strains)<br>
 
-
4) When the kamikaze system is ready, I need to ligate the P4sid1 region to it and transform an E.coli C1a strain with it. <br>
 
-
5) then we can start testing the system!<br>
 
-
6) ultimate goal: standardize P4 as an iGEM vector.
 
-
====Testing for activity of the control system====
+
[[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.
-
Uriel's cox and ogr construction needs to be tested experimentally to assureThe procedure will be:
+
**P4 as an iGEM Plasmid
-
1) Transform a P2 lysogenic strain with the construction.
+
  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>
-
2)assess IPTG induced lysis activation
+
====Enrique Paz====
====Enrique Paz====
-
** Desingn of our modified version of bacteriophage P4
+
[http://openwetware.org/wiki/User:Paz_C._Enrique/Notebook/Paz_C._Enrique_-_Projects#Personal_objectives Journal]
-
  What we need to do over P4 genome and how to do it in order to construct a bacteriophage compatible with standards
+
** Design of our modified version of bacteriophage P4
-
  in synthetic biology.  
+
 
-
+
  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)
** Construction and test of the gossip device (quorum sensing)
-
  Some -a lot- restrictions, ligations, transformations, tests, etc. To achieve a high quality device for quorum
+
  Some -a lot- of restrictions, ligations, transformations, tests, etc. to assemble this device. The idea of this
-
  sensing that we will use for construction of our "alarm" system.
+
  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
** 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
  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!
+
  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'''==

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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