Team:SDU-Denmark/Tour52

Characterization

“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. We invite you along on a journey through our attempts to obtain proof of concept; to show that Bacteriorganic Rubber is a true possibility. This page 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) and our central genes (dxs(B. subtilis) and HRT2)

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 Fluorescence Activated Cell Sorting (FACS).

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 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 result shows 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

Figure 1.

Although lacI:LVA was shown to be functional, we found the prolonged response time and lowered maximum protein level after induction to be less than optimal. That is to say that 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 taken.

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.

According to parts registry, the LVA-tagged lacI part has been used 684 times in spite 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 part of natural lacI to the registry for future teams to use, as an improvement upon 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.

Figure 2.

Characterization of AraC/Para

Figure 3. 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. 3).

This experience has been added to the experience of the part encoding the arabinose promoter on parts registry.

Characterization of dxs (B. subtilis)

Functionality assay
To optimize the flow through the MEP pathway, the dxs gene was overexpressed, the expectation being increased levels of IPP and DMAPP(see Specification). To examine if overexpression indeed resultats in an increase in substrate, we attempted to assay the levels of DMAPP using a headspace gas chromatography (GC)-technique.

DMAPP was hydrolyzed in acid to the volatile hydrocarbon gas isoprene. The gas was subsequently analyzed with headspace GC. A linear relationship between amount of detected isoprene and DMAPP concentration has previously been established. Source: Alison J. Fisher et. al; Nonradioactive Assay for Cellular Dimethyllyl Diphosphate We were capable of producing a standard curve by reacting DMAPP with acid for 2 min (instead of the 60 min specified in the previous study) (Fig. 4). At this time, we obtained optimal peak detection for standard solutions. We were, however, incapable of detecting isoprene, even in high concentrations of bacterial samples treated with acid. The test was expanded to include acid hydrolyzation for 2, 30, 60 or 90 min, yet we could not detect isoprene, and therefore not detect DMAPP.

Figure 4. Optimization of the procedure is needed before characterization of the dxs bricks can be completed using this approach. We suspect that the complexity of the bacterial samples is too high, and thus the reaction does not take place as fast as might be necessary for detection in our setup. Sonication of bacterial samples with and without addition of standard DMAPP, and subsequent measurements might shed some light on this hypothesis. However, it should be noted that the GC wasn’t fully functional during the test period, consequently leading to broader peaks and thus lowered the sensitivity of the instrument. On the 3rd of October, we received a mail from Professor Lars Porskjær Christensen, Department of Chemistry-, Bio- and Environmental Technology, University of Southern Denmark:

"...The GC has now been repaired and the sensitivity has been improved considerably. The GC-peaks should be very sharp now. This may be the reason that you have not observed any release of isoprene from your samples...MailTranslated from danish".

Unfortunately, with only 2 days left to wiki-freeze, there was no time for another round of testing.

Growth Experiment
Figure 5. Carrying the dxs devices and expressing the gene could impair the growth, and hence be important in production purpose. To test if the growth of MG1655 bacteria is impaired when carrying and expressing our dxs devices, we measured the growth rate with OD₆₀₀ measurements.

Two triplicates of MG1655 carrying no vector, empty pSB1C3 vector, pSB1C3-Plac-dxs (B. subtilis), pSB1C3-Pcon-lacI-Plac-dxs (B. subtilis), or pSB1C3-Pcon-lacI:LVA-Plac-dxs (B. subtilis) were started from ONC at time 0 hours, OD₆₀₀=0.005, and grown at 37ºC and 180 rpm. OD₆₀₀ measurements were done every half an hour, and 1 of each triplicates was induced at time 2.5 hours. All strains grew at the same pace and induction didn’t impair growth rate (Fig. 5).

Characterization of HRT2

Rubber purification
To discover rubber, it is useful to isolate the rubber from the rest of the bacterial cells. This will allow us to remove as many variables in the detection assays as possible. The rubber was purified according to self-written SOP0031 - Rubber purification. The method of purification was based on a literature search and chemical evaluation of solubility of the polyisoprene. Cells were sonicated in ethanol suspension, washed in acetone, and extracted in n-hexane (both steps were overnight).

Two different methods were tested to evaluate the most efficient extraction method. In the first method, we washed with acetone and extract with n-hexane in a soxhlet extractor. The second method excluded the time-consuming soxhlet steps and washed with acetone for a shorter duration of time (15 min shake at 37 deg). Rubber was extracted by adding hexane to the cell suspension, then centrifuging the sample. The supernatent was saved (hexane solution). We tested the methods of rubber extraction on a positive control made from MG1655, mixed with polyisoprene. Both the soxhlet and the non-soxhlet method were evaluated using H¹-NMR. We deemed the results to be similar, and since the required time for non-soxhlet extraction was significantly less, we chose to use the non-soxleth method.

The H¹ NMR test
We now seemed able to detect rubber that was extracted from a mixture of bacterial debris and polyisoprene, and felt ready for a test of our production system. H¹ NMR allowed us a certain specificity and although a significant amount of polyisoprene was needed to enable detection, it was preferred over C¹³NMR. All samples were run an a bruker 400 mHz NMR spectrometer. We also knew that we could control the expression of the genes in both of our plasmids. We were ready to put our abilities to extract rubber through the non-soxhlet method and detect it using H¹ NMR to the test. In other words, we were ready to find the rubber present in our strain!

During the first test of our CPS bacteria (MG1655 transformed with both plasmids - see Design), bacteria were grown to late exponential phase at 37 deg, then induced with 1mM IPTG, 0.2% Ara, and 1mM MgCl2. It was then moved to room temperature for a further 4 hours of growth. Non-soxhlet extraction was used to purify any rubber present in the sample, which was then compared to a positive control (WT mixed with polyisopren - also undergone non-soxhlet extraction) and a standard isoprene solution.

Figure 6 illustrates the peaks given by pure polyisoprene are found at 5.12 (A), 2.04(B) and 1.68(C)in the ratio 1(A):4(B):3(C). Additionally, a peak is visible at 1.56 indicating water (the standard was not dried in a vacuum oven ON like the rubber extraction was). The peak at 0.00 ppm is the defining peak of the ppm axis and represents TMSTMSTetramethylsilane which is a calibrating standard.

Our rubber extraction of WT + polyisoprene gives the same peak placement as the pure polyisoprene ((A), (B) and (C)). However, the integration of the 3 peaks shows a relationship of approximately 1:5:4. The discrepancy can be explained by the impurities in the area between 0-2.5 ppm. Such impurities might add additional integration value to the peaks assigned to the polyisoprene peaks (B) and (C) - hereby causing a disruption of the true relationship. Some peaks from the solvents used to purify the rubber (Acetone, Ethanol, and Hexane), as well as a small amount of water (1.56 ppm) are seen, too.

The conclusion arrives in figure 8, named DXS+PT, which is our double plasmid CPS bacteria, displays the same peaks (A), (B) and (C) as both the WT + polyisoprene and pure polyisoprene. This is a strong indication of the presence of rubber - specifically, this is an indication that our CPS bacteria produces rubber: Bacteriorganic Rubber. Peak distortion of the spectrum due to solvents are the same as seen in the rubber purification from WT + polyisoprene.

A second round of testing was done to validate the first experiment. We wanted to include a negative test (WT), in order to exclude the possibility of a naturally occurring polyisoprenoid compounds in E. coli. We performed rubber purification on WT, our CPS bacteria as well as a strain containing only the pSB1K3-AraC-Para-PT device. Bacteria were grown and rubber extracted as described above. The three samples where unfortunately not dried properly in the vacuum oven due to apparatus malfunction.

The test provided the following results: Fig 9 lacks the characteristic peaks of polyisoprene at 5.12, 2.04 and 1.68. It can be concluded from their absence that there is not rubber (nor any other compound that might provide a similar chemical shift values) in the wildtype. In the CPS (fig. 10), we observe very a slight peak at 5.12, indicating the presence of (A) hydrogen. This peak has the same splitting pattern as the first round of H¹-NMR, but it has a very low intensity. The (B) and (C) peaks are hidden in the background noise, which is most likely due to cell debris and solvents, which did not evaporate appropriately. We suspect that the machinery has a decreased sensitivity towards the isoprene peaks due to the high amount of solvent seen from the assigned peaks, but the peak at 5.12 (with the recognizable splitting pattern) is a strong indication that rubber is once again present in our CPS bacteria (containing both PT and dxs plasmids).

The future of our H¹ NMR test rubber characterization experiments will see us replicate our results. Specifically, we must redo the second rubber extraction and rerun the H¹ NMR to verify the rubber presence in our CPS bacteria. Also, this particular rerun allows us to check that the WT is indeed lacking any sign of a peak at 5.12. In addition, we must investigate the rubber yield in terms of weight to further evaluate the two purification methods mentioned above.

MALDI-ToF
We thoroughly studied the literature concerning polyisoprene on MALDI-ToF and found that the formation of adducts by adding AgNO₃^- would make it possible to ionize the long alkene chain, despite its lack of functional groups that can be ionized.

We tried several matrixes including MBTMBT2-mercaptobenzothiazole, DHBDHB2,5-dihydroxybenzoic acid, CHCACHCAalpha-cyano-4-hydroxycinnamic acid, SASAsinapinic acid and DTDTdithranol, but didn't have the time to perform the tests on anything but our standard polyisoprene (Mw 38 kDa). Unfortunately, it appeared to be too large a molecule to be detected by the bruker MALDI-ToF machine. It was concluded that the machine's hardware settings could not match the requirements for such large molecules. However, the expected length that we expect HRT2 produces is in the 2-10 kDa range. This is within the limits of the machine, though we have yet to test this hypothesis as the machine is frequently occupied by other research groups.

Composite production system

Growth experiments
Carrying the HRT2, dxs and both (CPS) devices and expressing the genes could impair the growth, and hence be important in production purpose. To test if the growth of MG1655 bacteria is impaired when carrying and expressing our devices, we measured the growth rate with OD₆₀₀ measurements.

Four different MG1655 strains carrying either no plasmid (WT), pSB1C3-Pcon-araC-term-Para-HRT2 (HRT2), pSB1C3-Pcon-lacI(N)-term-Plac-dxs(B.subtilis) (Dxs), or pSB1K3-Pcon-araC-term-Para-HRT2 and pSB1C3-Pcon-lacI(N)-term-Plac-dxs(B.subtilis) (CPS), were grown from OD₆₀₀=0.05 and induced with 1 mM IPTG and/or 0.2% arabinose during growth. The first experiment showed impaired growth of the bacteria only carrying construct expressing HRT2. We thought that the reason why the strains expressing HRT2 as well as Dxs didn’t show impaired growth rate could be due to a phenomenon called inclusion bodies. When overexpressing too many proteins, there is a risk of misfolding and hence loss of function. A way to lower this risk is to lower the temperature. Therefore we set up a growth experiment where the temperature was lowered from 37 to 20ºC at time of induction. This did not prove to make a difference, and CPS strains with and without IPTG and/or arabinose induction grew at same pace as WT (fig 11)

Figure 11