Difference between revisions of "Part:BBa K1088016"

Revision as of 13:12, 26 October 2013

HRT2 prenyltransferase from Hevea Brasilianis (ara promoter with araC: arabinose inducible)

This part encodes the rubber producing prenyltransferase HRT2 from the rubber tree Hevea brasiliensis. It uses isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) as substrates to produce cis-1,4-polyisoprene natural rubber.

The part is designed to express the enzyme when induced with arabinose under glucose scarce conditions. Arabinose binds the ara promoter regulator, AraC, and arabinose bound AraC then activates the transcription from the ara promoter.

The coding region of AraC with a constitutively active promoter, a strong RBS and a efficient terminator was added to this part to see if the expression from the ara promoter would be elavated compared to a similar construct (BBa_K1088024) not bearing the AraC device.

Using Northern blot analysis it was proved that the construct in E. coli K-12 MG1655 doesn't express the prenyltransferase when grown without arabinose. Upon addition of arabinose to the media prenyltranferase mRNA was detected. The addition of the AraC device did not prove to elevate the expression level. See BBa_K1088017 for more details.

A) Normalized intensity of HRT2 mRNA using intensity of 5S rRNA as reference. The normalized intensity of sample -araC -2min were set to 1 and the other samples are relative to this. Within 15 min of induction the expression levels are at its maximum in both strains, and overexpression of AraC does not seem to be necessary for expression control of the arabinose promoter. B) Northern blot result reflecting diagram.

In conclusion we proved that we can induce the expression by addition of arabinose.

A 3xFLAG tag is behind the HRT2 prenyltransferase, but ins't part of the protein (HRT2 has it's natural stop-codon).

The rubber producing capabilities of HRT2 were assesed by purifying the rubber content of the bacteria expressing the HRT2 gene under control of the arabinose promoter. The purification was done according to the SOP developed by the SDU-Denmark 2013 team and can be found at our wiki or by clicking here:[[1]]The purification was based on acetone and hexane. The purified rubber was investigated by H-NMR and the results indicated the presence of rubber. However, due to technical difficulties, the experiments must be repeated to ensure scientific certainty.

• • • Abreviations that might occur throughout the following test***

HRT2=Hevea Rubber Transferase 2

PT=Prenyltransferase (in this case PT=HRT2)

DXS=1-deoxy-D-xylulose-5-phosphate Synthase

NMR=Nuclear Magnetic Resonance

• • • Abreviations that might occur throughout the following test***

The following information is available at our wiki as well: 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.

     Spectrum illustrating the pure polyisoprene standard (Mw = 38 kDa) H NMR spectrum. 
     The peaks at 5.12, 2.04 and 1.68 indicate the (A), (B) and (C) protons of the isoprene monomer, respectively.
     This sample was not dried in vacuum oven, and therefore we have a large peak at approximately 1.55 corresponding to water.

     Spectrum illustrating the WT + polyisoprene standard added to it, rubber purified, H NMR spectrum.

The peaks at 5.12, 2.04 and 1.68 indicate the (A), (B) and (C) protons of the isoprene monomer, respectively, in this spectrum as well as the aforementioned pure polyisoprene H NMR spectrum. The different solvents from the rubber purification procedure can be seen as well, however, these are not interfering with isoprene proton shifts.

     Spectrum illustrating our PT+DXS part. In this spectrum it is important to notice the same peaks as before,           
     5.12, 2.04, and 1.68 which indicate the presence of polyisoprene (rubber). The same solvents as in the previous spectrum are also present, but the focus should be put towards the isoprene presence, proving the function of HRT2/PT. 

First round: In the spectrums seen above, you can see from the first figure 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 during the rubber purification). The peak at 0.00 ppm is the defining peak of the ppm axis and represents TMS which is a calibrating standard.

Our rubber purification (SOP0031 - Rubber purification) of WT + polyisoprene give the same peak positions as the pure polyisoprene ((A), (B) and (C)), however, the integration of the 3 peaks show a relationship of approximately 1:5:4. This can be explained by the impurities in the area 0-2.5 ppm which might add additional integration value to the peaks assigned to polyisoprene (B) and (C) causing a disruption of the true relationship. Peaks originating from the solvents used to purify the rubber (acetone, ethanol and hexane) and a small amount of water (1.56 ppm) can be seen as well.

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

       Spectrum illustrating WT bacteria which have undergone rubber purification.
       Notice that none of the peaks for the isoprene units are present in this spectrum.
       However it should be noted that the vacuum oven was malfunctioning, and therefore this result might be due   
       to insensitivity of the spectrometer since the noise from the solvents are much greater than before.

       Spectrum illustrating PT+DXS which have undergone rubber purification.
       The peak at 5.12 is vaguely present, and the rest of the peaks assigned to isoprene are hidden in the noise    
       from the solvents and impurities.
       The peak at 5.12 is a triplet which corresponds to the previously observed shape of the 5.12 peak, and we   
       expect that it is a indicator of the presence of polyisoprene production

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, comparing WT and PT+DXS. From the WT spectrum you can see that there are 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 spectrum we observe only a very weak peak at 5.12 indicating the (A) hydrogens. This peak has the same splitting pattern as in the first round of H1-NMR we performed 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.

Sequence and Features