Part:BBa_K1088003
Cis-1,4-prenyltransferase obtained from Hevea brasiliensis cDNA
This part encodes the rubber producing prenyltransferase 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 sequence has been codon optimized for E. coli and is made from the cDNA sequence "HRT2" produced by Asawatreratanakul K et al.
The rubber producing capabilities of HRT2 were assesed by purifying the rubber content of 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 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
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.
Figure 1: Spectrum illustrating the pure polyisoprene standard (Mw = 38 kDa) H NMR spectrum.
The peaks at 5.12, 2.04 and 1.68 indicates 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 aprox. 1.55 corresponding to
water.
Figure 2: Spectrum illustrating the WT + polyisoprene standard, rubber purified, H NMR spectrum.
The peaks at 5.12, 2.04 and 1.68 indicates 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 of the rubber purification procedure is seen as well,
however theese are not interfering with out isoprene proton shifts.
Figure 3: Spectrum illustrating our HRT2+DXS part. In this spectrum it is important to notice the same peaks as
before,5.12, 2.04, and 1.68 which reveals the prescense of our polyisoprene. The same solvents as the previous
spectrum is present, but the focus should be put towards the isoprene presence, proving the effect of HRT2.
First round: On 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 as 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 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, Ethanol and Hexane) as well as a small amount of water (1.56 ppm) is seen as well.
DXS + HRT2 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.
Figure 4: 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 noticed that the vacuum oven was malfunctioning, and therefore this result might be due
to insensitivity of the spectrometer, since the noice from the solvents are far greater than before.
Figure 5: Spectrum illustrating HRT2+DXS which have undergone rubber purification.
The peak at 5.12 is vaguely present, and the rest of the peaks assigned to isoprene is hidden in the noice
from the solvent 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, HRT2+DXS and HRT2. The three samples where unfortunately not dried properly in the vacuum oven due to apparatus malfunction. Below you can observe 2 spectrums, matching WT, and HRT2+DXS. From the WT spectrum 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 HRT2+DXS spectrum 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.
References:
Asawatreratanakul K, Zhang YW, Wititsuwannakul D, Wititsuwannakul R, Takahashi S, Rattanapittayaporn A, Koyama T. Molecular cloning, expression and characterization of cDNA encoding cis-prenyltransferases from Hevea brasiliensis. A key factor participating in natural rubber biosynthesis. Eur J Biochem. 2003 Dec;270(23):4671-80. [http://www.ncbi.nlm.nih.gov/pubmed/14622254 (Link)]
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |