Wet Lab!!

General objectives

Uriel Laura Nando Abraham Paz

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

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!)

Are you ready 2 rumble??

Personal Objectives

Uriel Urquiza

- 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
  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. The control systems 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 grate quantities and induce lysis when ever we want and as a consequence obtation of grate
  amounts of P4 phage.

- Qualitative characterization of multipromter.

Laura Gomez

- 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.

 The experimental data would be used to feedback the model in order to obtain the most accurate model as possible.

Abraham Avelar

Ensamble and prove the functionality of the kamikaze device.
Get the time in wich a colicin, preferentially E3, kills E. coli c1-alpha.
Clone the bioparts received from Gene Art into any iGEM plasmid vector. Send them to the Registry of standard biological parts.
Design and test the asRNA

Partial goals:

Get the time in wich a colicin, preferentially E3, kills Escherichia coli c1-alpha. This parameter is going to be used to model the system. The election of E3 as a preffered toxin is because of the model's advice wich suggest that affecting traduction will be useful.
- Clone both colicines into any iGEM vector.
- Clone both colicines at the suffix of the biobrick BBa_R0010 in order to get an IPTG inducible device.
- Measure the time of death after the induction with IPTG.
Clone the MultiPromoter_GFP with all the death device
- Insert the MP_GFP biobrick into any iGEM vector.
- Insert the 2 colicines after the MP_GFP.
- Join this product with all the other biobricks.
Clone the asRNA and ligate it with all the device.

Fernando Montaño

1) I'm in charge of the assembly of the P4 vector. 2) I need to obtain the essential region of P4 sid 1 by pcr 3) I need to check for the characterization of p4 as an iGEM plasmid. 4) I need to ligate the P4sid1 plasmid to the BiteBack system and transform an E.coli C1-a strain with it. 5) then we can start checking for transduction of our system.

Enrique Paz

- Desingn of our modified version of bacteriophage P4

 What we need to do over P4 genome and how to do it in order to construct a bacteriophage compatible with iGEM standards

- Construction and test of the alarm device (quorum sensing)

 Some -a lot- restrictions, ligations, transformations, tests, etc. To achieve a high quality device for quorum sensing that we will use for construction of our "alarm" system.

- 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!