Characterization of Experiments

First proof of concept: LacH+Mtase experiments

Upon finishing the insertion of our synthesized Methyltransferase under the control of the LacH promoter in the pSB1AT3 backbone, we were able to start testing our first biobrick and first proof of concept.

Read-out

Since there is one ScaI site residing in the pSB1AT3 backbone and we created one ScaI site via a scar inside our memory module there are different possible restriction profiles to expect. Knowing that the LacH promoter has some basal activity, it is in our line of expectation to observe either an off or intermediate state.

[picture]

In figure X the combination of bands indicate an intermediate state as there are bands visible that correspond to the ‘off’ and ‘on’ state.

Inducer concentration variation (different concentrations of IPTG)

The next step was to see if induction of MTase expression upon activation by IPTG will modify the methylation state of our biobrick thus changing the resulting restriction profile. In the presence of 1mM IPTG, the promoter must be activated and the MTase will be produced inside the cell. Since the [model] for this experiment suggests that methylation will occur at a fast rate we expect that after 16h of IPTG induction, the read-out will dramatically shift to the uncut profile.

[picture]

Figure X doesn’t display a fully ‘on’ restriction profile but a gradual shift towards the restriction profile that represent s more uncut plasmid. This indicates that induction leads to an increase of methylation of the plasmid. Although, we still observe a gradual shift of the restriction profile meaning that the read-out doesn’t leave the intermediate state.

Log vs stationary

Bacteria in different phases of growth are known to react differently to experiments. When using LacH and IPTG it is known that bacteria perform better in the stationary phase because then the lac-permease, needed for uptake of IPTG, can be quantitatively integrated into the membrane. The same bacterial batch is used in both experiments but let the stationary experiment grow first over a longer period of time(at least more then 24 hours), either with or without the induction.

[picture]

When comparing the restriction profiles from figure X, there is a clear difference visible between the different phases. The presence of induction over a longer period of time shows a different restriction profile which corresponds with a shift to uncut plasmid.

Growth curve digestion

During the [growth curve experiment] several samples were taken on different time points to investigate the occurrence of methylation over the progression of time.

Figure X shows first the samples without and then the samples with induction reveals an intermediate restriction profile over the progression of time with a gradual shift toward cut, without methylation, plasmid. But the two samples taken after 24 hours show a difference. The sample with signal shows only uncut, fully methylated, plasmid.

LacIq experiments

In an attempt to get rid of the intermediate restriction profile without induction and create an ‘off’ result, a LacIQ strain was used in the following experiments conducted on pSB1AT3+LacH+Mtase. Investigation of the LacH promoter revealed that a higher expression of LacI will show a better suppression and decrease of basal activity of the LacH promoter.

Basic read-out experiment

[picture]

The results of the read-out for the LacIQ strain are inconclusive. Higher expression of LacI doesn’t reveal a restriction profile that confirms an ‘off’ state nor creates a shift towards an ‘off’ state.

pBAD experiments

Further characterization of the memory module requires testing more than one signal. Thus a another signal was chosen in the form of the much less leaky pBAD promoter which is induced by arabinose. This is also a well characterized promoter and less leaky than the LacH.

Read-out

[picture]

After cloning the pBAD in front of the Mtase, in pSB1AT3, first a basic read-out was conducted as shown in figure X. This was performed on a culture after 16 hours which shows the same intermediate digestion profile as in the LacH experiments.

Inducer concentration variation

In the presence of arabinose the pBAD will be activated, a culture was grown for 16 hours in with x amount of 10% arabinose.

[picture]

The result of figure X shows a gradual shift towards the restriction profile fitting the ‘on’ state.

48 hour exposure/ Log vs Stationary

As with the LacH we suspect that the construct will act differently when taken from a stationary culture. So two experiments were performed on construct pSB1AT3+pBAD+Mtase where bacteria were grown for a minimum of 24 hours to reach stationary phase in the presence of arabinose.

Both figures X and Y show a dramatic shift from the intermediate digestion profile to the ‘on’ digestion profile. This is considered as a great success.

Growth curve digestion

As with the LacH a growth curve experiment was conducted in similar fashion to characterize the acquired construct of pSB1AT3+pBAD+Mtase.

[picture]

Over the course of time the methylation pattern found in the gel show a shift towards the ‘on’ digestion profile in the presence of arabinose. After 24 hours though the digestion profile shows a complete ‘off’ state again. This is probably due to the E. coli using arabinose when it reaches the stationary phase and needs to find a different energy source for its metabolism.