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 and 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 performed. 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.

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. 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 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 AgNO₃^- 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.

An introduction to Proton Nuclear Magnetic Resonance (H¹ NMR)
H¹ NMR is based on the absorption and re-emitting of electromagnetic radiation. The resonance frequency at which an atom absorbs, depends on the properties of the magnetic field as well as the isotope which is affected. since atoms with an equal number of protons and/or neutrons has a total spin of 0, it is only possible to detect chemical shifts from atoms with an unequal number. The most common types of NMR is C¹³ and H¹. It is sometimes useful to check with both methods to produce a 2D diagram, using different sets of information to produce stronger evidence for a hypothesis. However it should be noted that the C¹³ NMR is much less sensitive since the natural abundance of C¹³ atoms is 1.109 % whereas the natural abundance for H¹ is 99.98% and therefore this method is more sensitive. There are also more Hydrogen atom’s than carbon atoms in our rubber chain, so the sensitivity of H¹ NMR would be our best option for rubber detection. We tried both C¹³ and H¹ but as you will see from our data below, only the H¹ NMR is shown since the C¹³ NMR simply was too insensitive to detect anything of use to us.

First round
On the spectrums seen above (fig 6-8) you can see from fig 5, that the pure polyisoprene gives peaks at 5.12 A), 2.04(B) and 1.68(C) in the ratio 1(A):4(B):3(C). Additionally we see a peak at 1.56 indicating water (the standard was not dried in a vacuum oven ON as the rubber purification). The peak at 0.00 ppm is the defining peak of the ppm axis and represents TMSTMSTetramethylsilane which is a calibrating standard.

Our rubber purification (SOP0031 - Rubber purification) of WT + polyisoprene give the same peak placement as the pure polyisoprene ((A), (B) and (C)) however the integration of the 3 peaks shows a relationship of approx. 1:5:4. This can be explained by the impurities in the area 0-2.5 ppm that might add additional integration value to the peaks assigned to polyisoprene (B) and (C) causing a disruption of the true relationship. Some peaks from the solvents used to purify the rubber with (Acetone (ppm), Ethanol (ppm) and Hexane (ppm)) as well as a small amount of water (1.56 ppm) is seen as well.

DXS + PT shows the same peaks (A), (B) and (C) as both the WT + polyisoprene and pure polyisoprene tests indicating the presence of our rubber. We see the same distortion of the spectrum by solvents, as the rubber purification from WT + polyisoprene.

Second round To validate the first experiments we wanted to include a negative test (WT) as well, in order to exclude the possibility of a naturally occurring polyisoprenoid compound in E. coli. We performed rubber purification on WT, PT+DXS and PT. The three samples where unfortunately not dried properly in the vacuum oven due to apparatus malfunction.

Below you can observe 2 spectrums (Fig 9, and 10), matching WT, and PT+DXS respectively. From spectrum in fig 9 you can see that there is no peaks at all in 5.12, 2.04 and 1.68 proving the fact that we do not have any rubber present in our WT bacteria (or any other compound that might have the same chemical shift values). In the PT+DXS (fig 10) we observe only a very weak peak at 5.12 indicating the (A) hydrogens. 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 the machinery to have decreased sensitivity towards our isoprene peaks due to the high amount of solvent seen from the assigned peaks.

The future of our characterization
Our next move on the subject of H¹ NMR will be to properly dry the rubber purified samples from the second round. Then we will run the H¹ NMR again to verify the rubber presence in PT+DXS and also check that the WT is still lacking any sign of peak at 5.12 (since this area is less affected by noise, and therefore is a valid point of analysis). The rubber purification must be done again as well to produce more replicates of our H¹ NMR tests and investigate the yield in weight to further evaluate the two purification methods mentioned above.

All samples where run on a Bruker 400 mHz

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.

MG1655 strains carrying either no plasmid (WT), pSB1C3-Pcon-araC-term-Para-HRT2 (BBa_1088016) (HRT2), pSB1C3-Pcon-lacI(N)-term-Plac-dxs(B.subtilis) (BBa_1088024) (Dxs), or pSB1K3-Pcon-araC-term-Para-HRT2 and pSB1C3-Pcon-lacI(N)-term-Plac-dxs(B.subtilis) (BBa_1088024) (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 protein clouding. 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