Team:LCG-UNAM-Mexico/Wet Lab/Objectives
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=='''General objectives'''== | =='''General objectives'''== | ||
- | + | <br>The final aim is to get and test the following device. | |
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- | 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: | + | ''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: | + | ''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> | + | ''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 | + | ''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> | ||
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=='''Personal Objectives'''== | =='''Personal Objectives'''== | ||
- | ==== Uriel Urquiza ==== | + | ==== Uriel Urquiza ==== |
+ | |||
+ | [[Team:LCG-UNAM-Mexico:Journals:Uriel| Journal]] | ||
** 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 | + | 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 |
- | major regulators of P2 morphopoietic genes. The control | + | two major regulators of P2 morphopoietic genes and deleting them form lysogenic P2; this will yield a P2 phage |
- | promoter inducible by IPTG | + | which only has capsid and tail genes. The control system that is going to be implemented is constituted by a |
- | us to grow bacteria in | + | 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 | + | ** 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. | ||
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** Generation of data to feedback the infection model. | ** Generation of data to feedback the infection model. | ||
- | The experimental | + | 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 | ||
- | Clone the colicines with the multipromoter | + | Clone the colicines with the multipromoter, design and Clone the asRNA and help in the ensamble of all the device. |
- | + | ||
- | + | ||
** 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 | + | 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. |
- | + | ||
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====Fernando Montaño==== | ====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==== | ====Enrique Paz==== | ||
- | ** | + | [http://openwetware.org/wiki/User:Paz_C._Enrique/Notebook/Paz_C._Enrique_-_Projects#Personal_objectives Journal] |
- | + | ||
- | to do it in order to construct a bacteriophage compatible with standards in synthetic biology. | + | ** Design of our modified version of bacteriophage P4 |
- | ** Construction and test of the | + | |
- | + | 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 | ** 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'''== |
Latest revision as of 03:51, 22 October 2009