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'''== | ||
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of modified P2. | of modified P2. | ||
- | ** Qualitative characterization of T7/T3 | + | ** Qualitative characterization of T7/T3 multipromoter. |
The multi-promoter that we have designed has the capacity to respond specifically to T7/T3 RNA polymerases | The multi-promoter that we have designed has the capacity to respond specifically to T7/T3 RNA polymerases | ||
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====Laura Gomez==== | ====Laura Gomez==== | ||
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+ | [[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 | ||
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** 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==== | ||
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- | + | [[Team:LCG-UNAM-Mexico:Journals:Nando's|Journal]] | |
- | ** | + | **Assembly of the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K242051 P4 vector] |
- | 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. | + | 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. |
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+ | **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 | **P2 control system functionality | ||
- | The cox/ogr control system in which [[Team:LCG-UNAM-Mexico/Wet lab/Objectives#Uriel Urquiza|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. | + | 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 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. | + | 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. | |
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** 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. | + | 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 | 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. | ||
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+ | ** Test the biobrick of P4 cos-site | ||
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+ | 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