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 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)

Figure 1.

Characterization of LacI/Plac

The dxs gene is placed under the control of the lactose promoter (which is IPTG inducible). Therefore, we assayed our capability of inducing the expression through addition of IPTG. As part of the experiment, it was tested if expression was suppressed 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 a device. The GFP fusion device with and without the lacI:LVA device was assayed to check for expression control. One triplicate of MG1655 and two triplicates of 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 (Fig. 1).

Strains not overexpressing LacI:LVA does not repress expression from the lactose promoter even without induction, whereas strains overexpressing LacI:LVA represses the promoter until induction. Approximately 90 min after induction of the strain overexpressing LacI:LVA the protein level is at its maximum. Furthermore, the maximum protein level is lower than that of the strain not overexpressing LacI:LVA, and not all of the cells expresses the the Dxs-GFP protein fusion.

Our obtained experience has been added to the experience of the part encoding the lacI:LVA basic part on parts registry

Characterization of AraC/Para

Figure 2. 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 minutes, 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 minutes after induction. (Fig. 2).

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 minutes (instead of the 60 minutes specified in the previous study) (Fig. 3). 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 3. 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 4. 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.

2 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 at 180 rpm. OD₆₀₀ measurements were done every half hour, and 1 of each triplicate was induced at time 2.5 hours. All strains grew at the same pace and induction didn’t impair growth rate (Fig. 4).

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 SOP0031 - Rubber purification which was made by us, based on a literature search and chemical evaluation of solubility of the polyisoprene. The cells were sonicated in ethanol suspension, washed in acetone, and extracted in n-hexane (both steps was ON).

We tried two different methods to evaluate the most efficient extraction method. In the first method we tried to wash with acetone and extract with n-hexane in a soxhlet extractor. As the second method, we tried to exclude the time consuming soxhlet steps and washed for a shorter duration with acetone in 50 mL falcon tubes (15 min shake at 37 deg) and extracting the rubber by adding hexane to the cell suspension and spinning down the sample to save the supernatant (hexane solution). We tested the rubber extractions on WT + polyisoprene on both soxhlet and non-soxhlet methods and evaluated the result on H¹-NMR. The result seemed similar, and therefore we chose to stay with the non-soxleth method as our SOP for purifying rubber, since the time required for that protocol was significantly less.

MALDI-ToF
We did a thorough literature study of polyisoprene on MALDI-ToF and found that the formation of adducts by adding AgNO^3- would make it possible to ionize the long alkene chain even though it has no 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 we never had time to test on anything but our standard polyisoprene (Mw 38 kDa,) which unfortunately was too large a molecule to be detected by the bruker MALDI-ToF machine. We have come to the conclusion that the machines’ hardware settings are not matching the requirements for these large molecules and therefore we might still be able to find our sample even though we cannot see our standard, since the sample is expected to be around 2-10 kDa.Unfortunately we have not had the time to test this, since the machine is frequently occupied by other research groups.