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

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===C1a growth plot===
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----
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C1a is a k12-derivative strain. In order to know the behavior of this strain we did a growth curve. Every four hours two samples were taken from a C1a culture. We measured the optical density (OD) (550nm) of one of those samples, and the other was used to do dilutions and to plate them. The results are shown in the next table.<br>
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  tiempo (hrs) DO 550nm UFC/ml
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  0 0.0355 1207000
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  2 0.2533 25450000
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  4 0.944 160000000
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  6 1.2194 165000000
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A non-linear regression method was used to generate a logistic formula with the best adjustment. <br>
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[[Image:UFC_OD.png|800px]]<br>
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<span style="font-size:10px"> The unit time is hours, the y correspond to UFC and OD, respectively, and the x correspond to time </span><br>
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Both formulas were used to infer a correlation formula between OD and UFC, this formula was used to translate the OD measures to UFC.
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[[Image:FormulaeODvsUFC.png]]
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<br>
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===T3 and T7 infection plot===
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----
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====Without system====
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The experiment started when all the cultures had a OD around 0.8. Bacteriophages T3 and T7 were added to the culture, no phage was added to the control . Every twenty minutes an OD measure was taken. The results were plotted. In time 5 no cell was alive.
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[[Image:Growth_T3_t7.png|450px]]<br>
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====With system====
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We will follow the same procedure described in the last section. But in this case all the cells will be prepared with the whole system including the [[Team:LCG-UNAM-Mexico/Wet_Lab/Objectives#General objectives|defense, gossip and paranoia system]]. This is one of the last experiment we could do and is one of the most important. We will expect that most times the bacteria could contend with the infection and the population survives. So, if we will plate after two hours (it is the experiment's duration time) we would see viable colonies after eight hours.
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===Burst size determination===
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====Without system====
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====With system====
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===Test parts and devices===
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----
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====Multipromoter====
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Functionality was tested qualitatively using the strain [https://2009.igem.org/Team:LCG-UNAM-Mexico/Resources/Strains BL21(DE3)pLysS] that have an IPTG inducible T7 RNA polymerase; different conditions were tested Bl21/multipromoter IPTG+, Bl21/multipromoter IPTG- and Bl21/no-multipromoter IPTG+.
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[[Image:Gfp multi.JPG|300px]]
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In frame is BL21/multipromoter with IPTG inducer, unfortunately the microscope and camera was not suitable to check
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in full detail the BL21/non-multipromoter IPTG+ which gave no florescence and Bl21/multipromoter IPTG- which
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presented a diminished florescence.
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This result was only for T7 RNA polymerase but we are planning to implement a better assay system to include a WT T3 & T7
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RNA polymerase, a plasmid that have GFP and not the multipromoter and a strain that have our multipromoter and not T7 RNA polymerase. Also we need to clone the biobrick in a plasmid that only contain our multipromoter and no other in the same direction in order to avoid licking from near upstream promoters, this is important because we are using quite toxic bacterial proteins that will be induced under the presence of T3 or T7 so estimation of the basal transcription its important to test the viability of our death system.
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====Quorum sensing system====
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[[Image:Quorumdevice.jpg|500px]]
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<span style="font-size:10px"> The quorum sensing device </span>
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After assembling the quorum sensing device, we will turn on the system by either T3 or T7 RNA polymerases. We expect to see green florescence at the point of induction and red in the neighborhood. At this time the toxines won't be in the construction to avoid the noise caused by the bacteria death. AHL spatial dynamics are simulated using the [[Team:LCG-UNAM-Mexico:CA|Cellular Automata]]. The final goal of the Quorum sensing system is to decrease even more the burst size by delaying capsid assembly using the asRNA that is designed to interfere with the phage's replication machinery.<br>
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[[Image:Quorum.png|400px]]<br>
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<span style="font-size:10px"> An AHL difussion example when an infection occurs </span>
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====asRNA====
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We will induce the asRNA construction with IPTG, afterwards we expect the infection of T7 and T3 to be with less efficiency of plaquing and/or smaller plaques.
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We will also induce the expression of the asRNA in a liquid medium and perform a [[Team:LCG-UNAM-Mexico/Wet_Lab/Experiments#C1a growth curve|growth curve]]. We expect that the decay is going to be slower and/or to a higer OD (with more resistant bacteria).
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====Toxins====
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The colicines (E3 and E9) will be under the transcriptional regulation of the promoter induced by IPTG. We will do a growth curve following the same procedure used to do the [[Team:LCG-UNAM-Mexico/Wet_Lab/Experiments#C1a growth curve|c1a growth curve]] as performed above . We will feedback the model with these results. We expect this curve to decay before the wild type curve. We will do another curve using the same procedure described in the section [[Team:LCG-UNAM-Mexico/Wet_Lab/Experiments#phage infection curve|phage infection curve]] but without phages and with the induction with IPTG in the time zero. We expect the growth curve obtained to decay before than that of the phages infection curve. We hope the toxic activyty of the toxins will be faster enough to kill the bacteria before the phages.
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Latest revision as of 02:55, 22 October 2009

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