Team:SDU-Denmark/Tour52

Expression

Can we control it?

“And by what, O Socrates, is the soul nourished?” - Hippocrates
“By knowledge, of course, I said.” - Socrates

Characterization of our biobricks is a chance for us to prove that our design works as intended. These next two pages will slowly guide you through our results, but keep in mind that not everything is presented below. For all the details, consult our protocol page where you will find a comprehensive picture of our project.

The question of design and therefore of function is two-fold: How do we - and can we - control the expression of our system? And do the expressed proteins work as intended? These are the questions this page sets out to answer. Specifically, we have characterized our regulable biobricks (LacI/Plac and AraC/Para)

Characterization of LacI/Plac

The dxs gene is placed under the control of the lactose promoter (see Design). We assayed the inducible capabilities of our design and, as part of the experiment, we tested the ability to suppress expression prior to induction. The assay was carried out by measuring protein levels of Dxs fused to GFP using FACS. FACSFluorescence Activated Cell Sorting

A LacI:LVA basic part was available at Parts Registry, and we added a promotor and a terminator, producing a device. The GFP fusion devices with and without the LacI:LVA device were assayed to check for expression control. One triplicate of MG1655 and two triplicates of each MG1655 strains carrying either pSB1C3-Plac-dxs (B. subtilits)-GFP or pSB1C3-Pcon-lacI:LVA-term-Plac-dxs (B. subtilits)-GFP were grown from OD₆₀₀=0.005 to approximately OD₆₀₀=0.2. At this OD the MG1655 triplicate and one triplicate of each strain carrying constructs were induced with 1 mM IPTG at time 0 min. FACS measurements were done at times: -30, 0, 30, 60, 90, 120, and 150 min, thus showing the expression of GFP in each strain - both when induced and when not induced (Fig. 1).

The results show that strains lacking LacI:LVA do not repress expression from the lactose promoter. Even without induction, there is clear expression of GFP. Conversely, strains expressing LacI:LVA repress the promoter until induction. Approximately 90 min after induction of the strain expressing LacI:LVA, protein level is at its maximum. Still, maximum protein level is lower than that of the strain lacking LacI:LVA. Also, the fraction of flourescent cells is lower in the samples of pSB1C3-lacI:LVA-Plac-dxs (B. subtilits)-GFP compared to pSB1C3-Plac-dxs (B. subtilits)-GFP, indicating that expression of GFP is somewhat repressed, despite induction.

This experience has been added to the experience of the part encoding the LacI:LVA basic part on Parts Registry

BBa_K1088009) (+lacI:LVA) were grown from OD₆₀₀ 0.005 to approximately 0.2. At this OD the MG1655 triplicate and one triplicate of each strain carrying constructs were induced with 1 mM IPTG at time 0 min. FACS measurements were done at times: -30, 0, 30, 60, 90, 120, and 150 min. A) Growth curve shows that WT grows slightly faster than strains bearing plasmids. B) Per cent of population above fluorescence threshold. None of the WT cells were fluorescent, almost all of the -lacI:LVA cells were constitutively fluorescent, and only cells overexpressing lacI:LVA weren’t fluorescent when not induced. Upon induction increasingly per cent of +lacI:LVA became fluorescent and reaches a maximum of 70-75 per cent after 90 min. C) Mean GFP fluorescence of entire population. The -lacI:LVA cells became increasingly more fluorescent over time, both with and without induction. Though, the induced cells were slightly more fluorescent, which is probably because of the relief of repression from LacI naturally present in the cells. For the +lacI:LVA cells the results from B is reflected."> Figure 1.

Although LacI:LVA was shown to be functional, we found the prolonged and delayed response time together with lowered maximum protein level after induction to be less than optimal. In summary the performance of LacI:LVA as a repressor did not live up to our expectations when comparing it to the constitutively active strains, nor when comparing it to the wildtype. Therefore, we built a device of similar design to the LacI:LVA device with natural LacI: LacI(N). To test its qualities compared to LacI:LVA, a FACS was analysed. The experimental setup was similar to the previous. Two triplicates of MG1655 strains carrying either pSB1C3-Pcon-lacI(N)-term-Plac-dxs (B. subtilits)-GFP or pSB1C3-Pcon-lacI:LVA-term-Plac-dxs (B. subtilits)-GFP were used and an additional measurement at time 180 min was performed (Fig. 2).

Both strains repress expression when uninduced. The strain expressing LacI:LVA reaches its maximum percentage of fluorescent cells approximately 150 min after induction, whereas the strain overexpressing natural LacI reaches its maximum after a mere 30 to 60 min. The maximum protein levels in the strain overexpressing natural LacI reaches a level approximately 3 times higher than the strain expressing LacI:LVA. Clearly, the function of the natural LacI is superior to that of LacI:LVA.

Figure 2.

To test its qualities compared to LacI:LVA further, another FACS was performed. The experimental setup was similar to the previous. Dublicates of MG1655 strains carrying either pSB1C3-Pcon-lacI(N)-term-Plac-dxs (B. subtilis)-GFP or pSB1C3-Pcon-lacI:LVA-term-Plac-dxs (B. subtilis)-GFP were induced with IPTG concentrations rangingfrom 0.05-0.5mM (Fig. 3). After 180 min upon induction, the strains expressing LacI:LVA with the IPTG concentrations ranging from 0.05-0.5mM reached approximately 25-30 % cells above the fluorescent threshold. Comparing this observation to the strains overexpressing the natural LacI, even the least induced (0.05mM) reached a higher percentage of cells above the fluorescent threshold after 180 min. All of the cells in the culture overexpressing natural LacI induced with 0.125-0.5mM IPTG are above the fluorescent threshold within 180 min. In general, the response time is faster for higher concentrations of IPTG.

Figure 3.

According to Parts Registry, the LVA-tagged LacI part has been used 684 times despite of its poor function. The natural LacI parts that have been submitted to the Registry by previous teams are pending (and thus did not pass HQ quality control). We have therefore submitted the coding sequence of natural LacI to the Registry for future teams to use as an improvement to the LacI:LVA biobrick. Furthermore, our experience has been added to the experience of the part encoding the LacI:LVA basic part (BBa_C0012) on Parts Registry.

Characterization of AraC/Para

Figure 4. The HRT2 gene is under the control of the arabinose promoter (see Design). We assayed the inducible capabilities of our design and, as part of the experiment, we tested the ability to suppress expression prior to induction. The assay was carried out by measuring the mRNA levels of HRT2 using the Northern blotting technique.

To test whether overexpression of AraC improved expression control, devices with and without the araC device were assayed. Duplicates of MG1655 strains carrying either pSB1C3-Para-HRT2 or pSB1C3-Pcon-araC-term-Para-HRT2 were grown to late-exponential phase: OD₆₀₀=0.8. At this OD, the strains were induced with 0.2 % arabinose at time t=0 min, and samples were taken at times: -2 min, 15 min, and 30 min. Total RNA purified from the samples were run on a gel, blotted onto a membrane, and hybridized with probes specific for HRT2 mRNA and 5S rRNA (loading control), respectively.

The results prove that we are capable of inducing our HRT2 devices with arabinose. There is only little expression before induction and within the first 15 min, expression is at its maximum. Overexpression of AraC does not seem to have an effect on the expression levels after 15 min compared to natural levels of AraC. However, it is inconclusive whether AraC might contribute to an effect at times less than 15 min after induction. (Fig. 4).

This experience has been added to the experience of the part encoding the arabinose promoter on Parts Registry.