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

=Wet Lab!!=

General objectives
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!)

Uriel Urquiza
Journal

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.
 * Construction of the phage production control system.


 * 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

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.
 * To obtain all the relevant experimental information about T7 and T3.

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.
 * Generation of data to feedback the infection model.

Abraham Avelar
Journal

Clone the colicines with the multipromoter, design and Clone the asRNA and help in the ensamble of all the device.
 * Ensamble the kamikaze 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.
 * Test the construction

Fernando Montaño
Journal


 * Assembly of the 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.


 * Checking functionality for the Kamikaze system

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
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

 * Nando's Lab Journal


 * Uriel's Lab Journal


 * Abraham's Lab Journal


 * Laura's Lab Journal


 * Enrique's Lab Journal