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

C1a growth plot

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.

 tiempo (hrs)	DO 550nm	UFC/ml
 0		0.0355		1207000
 2		0.2533		25450000
 4		0.944		160000000
 6		1.2194		165000000

A non-linear regression method was used to generate a logistic formula with the best adjustment.

The unit time is hours, the y correspond to UFC and OD, respectively, and the x correspond to time

Both formulas were used to infer a correlation formula between OD and UFC, this formula was used to translate the OD measures to UFC.

Burst size determination

Without system

We already tried an experimient in wich we assumed a couple of things: all the phages are infecting a bacterium, all the cells were infected and almost all the bacteria died in the first round of infection. We know the number of bacteria due to the correlation obtained betwen UFC and OD.The burst size was calculated as the total number of phages produced divided by the total number of bacteria. The data from the first purification (1BS stock)indicate a burst size of 952 phages per bacterium. However the data from the 2BS stock indicate a burst size of 160,000 phages per bacterium and surprisingly the data from the 3BS stock indicate a bigger number.
We know that this number is not possible because phage production is limited by the amount of nucleotides available.

For this reason, we designed another experiment to measure the burst size. We will use the previous titrations to perform infections with just 1 or 0 phages per cell culture. We will let time to one lytic cycle, finally we are going to make plaques. We expect to see either the number of plaques representing the burst size or zero plaques, representing that we didn't take any phage.

With system

Test parts and devices

Multipromoter

Functionality was tested qualitatively using the strain BL21(DE3)pLysS which has an IPTG inducible T7 RNA polymerase. Different conditions were tested: Bl21/multipromoter IPTG+, Bl21/multipromoter IPTG- and Bl21/no-multipromoter IPTG+.

In frame is BL21/multipromoter with IPTG inducer at 100X. Unfortunately the microscope and camera were not suitable to check in full detail BL21/non-multipromoter IPTG+, which gave no fluorescence, and Bl21/multipromoter IPTG-, which presented a diminished fluorescence.

This result was only for T7 RNA polymerase, but we are planning to implement a better assay system to include a WT T3 & T7 RNA polymerase, a plasmid that has GFP and not the multipromoter to see basal transcription, a strain that has our multipromoter and not T7 RNA polymerase. We also need to clone the biobrick in a plasmid that only contains our multipromoter and no other in the same direction in order to avoid leaking 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 basal transcription is important to test the viability of our death system.

Quorum sensing system

The quorum sensing device

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 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.

An AHL difussion example when an infection occurs. The AHL gradient is shown, red corresponds to the more concentrated parts, in our system it would be green

asRNA

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. We will also induce the expression of the asRNA in a liquid medium and perform a phage infection curve. We expect that the decay is going to be slower and/or to a higer OD (with more resistant bacteria) in the final stage.

Toxins and Kamikaze system

The colicines (E3 and E9) will be under the transcriptional regulation of the promoter induced by IPTG. We will do another curve using the same procedure described in the section 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 because we want the toxic activity of the toxins will be faster enough to kill the bacteria before the phages. We will feedback the model with these results.

This phenomenon also has been studied in the theoretical molecular model, KZM and WTM. KZM has generated Burst size Distribution.

T3 and T7 infection plot

Without system

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 we did plates and no cell was alive.

With system

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 defense, gossip and paranoia system. This is the last experiment is one of the most importants because we will be able to conclude that all our design works as expected. We predict that most of the times bacteria will beat phage's infection and the population will survive. So, if we will plate after two hours (it is the experiment's duration time) we would see viable colonies after eight hours.

Helper P2 strain Development

Removal of Native P2 control genes

We need to remove the native Cox and Ogr gene. For this purpose we design special primers bordering the region in which these are coded. These primers are special because they are designed to do a PCR for an antibiotic resistance and to recombine this PCR product with the native P2 control region. The final phage won't have the cox an ogr gene allow us to control its lytic cycle and will have an antibotic resistance marker.

Functionality of Control Construction

The control construction built is IPTG-inducible under a lac promoter. The experiment intends to qualitatively assess the induction of such construction. When P2 lysogenic strains are transformed with this plasmid, one coud expect an effect on P2 lysis due to equilibrium disruption of the mutual negative regulation of native COX and C proteins in favor of COX. Such disruption would result in P2 late gene activation, and hence lysis will appear. The assay will check the quality of bacterial lawns by addition of different concentrations of IPTG stocks to part of the plate surface, so internal negative controls can be contrasted. Semi-quantitative Northern blot analysis is planned once the P2 helper lacking native cox and ogr genes is finished. Controls would be: non-induced construction (basal transcription), induced construction (induced transcription), native gene transcription, no construction.