Coding
HRT2

Part:BBa_K1088003

Designed by: Andreas Kjr, Patrick Rosendahl Andreassen   Group: iGEM13_SDU-Denmark   (2013-07-10)

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 (Standard Operating Procedure) 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.

SDU2013_Characterization_NMR_1.png

     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. The peak at 0.00 ppm is the defining peak of the ppm axis and represents TMS which is a calibrating
     standard.

SDU2013_Characterization_NMR_2.png

     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.

SDU2013_Characterization_NMR_3.png

     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.

SDU2013_Characterization_NMR_4.png

       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.

SDU2013_Characterization_NMR_5.png

       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. Above 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. (Link)



We wanted to verify that our previous experiments where replicable as well as investigate the quantity and yield of rubber. To assess this we did a third round of H1-NMR on MG1655 (WT), and MG1655 carrying pSB1C3-Pcon-araC-term-Para-HRT2 (HRT2), or pSB1C3-Pcon-lacI(N)-term-dxs (B. subtilis) and pSB1K3-Pcon-araC-term-Para-HRT2 (Dxs + HRT2). 1 L LB media was inoculated with the strains, starting from OD600=0.05. The cultures were grown at 37ºC with shaking for 5 hours; the temperature was then lowered to 20ºC and cultures were induced with 1mM IPTG, 0,2% arabinose, and 1mM MgCl2 ON. Rubber purification was commenced the following day, and the final yield was solubilized in 2 mL d-chloroform and analyzed by H1-NMR.


From the spectrum reflecting the WT strain, none of the peaks originating from polyisoprene were observed. From the spectrum reflecting the Dxs + HRT2 strain, all three peaks originating from polyisoprene were observed. Lastly, from the spectrum reflecting the HRT2 strain, the peaks potentially originating from polyisoprene could not be conclusively identified indicating the decreased rubber production performance of this strain compared to the Dxs + HRT2 strain. This emphasizes the importance of excess IPP and DMAPP (rubber precursors) available as a consequence of Dxs overexpression.


Rubber purified MG1655 (WT) H1-NMR spectrum. The three characteristic polyisoprene peaks (5.12, 2.04, and 1.68 ppm) are all absent. SDU2013_Rubber_WT2.png Rubber purified MG1655 carrying pSB1C3-Pcon-lacI(N)-term-dxs and pSB1K3-Pcon-araC-term-Para-HRT2 (Dxs + HRT2) H1-NMR spectrum. The three characteristic polyisoprene peaks (5.12, 2.04, and 1.68 ppm) are all present. SDU2013_Rubber_CPS2.png Rubber purified MG1655 carrying pSB1C3-Pcon-lacI(N)-term-dxs (HRT2) H1-NMR spectrum. The three characteristic polyisoprene peaks (5.12, 2.04, and 1.68 ppm) cannot be concluded to be present. SDU2013_Rubber_HRT2.png


In order to determine rubber yield, we compared the yield from 1L of our Dxs + HRT2 strain with 5 rubber purifications on WT + a selected amount of standard polyisoprene. Yields from all 5 purifications were solubilized in equal volumes of d-chloroform (2 mL), and the resulting TMS peaks could consequently be used for normalization of the polyisoprene peaks at 5.12 ppm. This peak was selected for quantification due to its localization in a region without background noise. From these 5 purifications, we calculated a standard curve to which we compared the integral value of the 5.12 ppm peak originating from our Dxs + HRT2 strain.



From the sample with the lowest amount of polyisoprene added (0.2 mg), no peaks could be identified, indicating the detection limit of the H1-NMR setup. Together with normalized peak integrals from the other 4 samples, we calculated the standard curve depicted in the bottom. Based on the standard curve and the integral originating from the sample containing 3.2 mg polyisoprene (see below), we roughly estimate our polyisoprene yields to be in the range of 0.5-5 mg polyisoprene/L fig. 11). We realize that this yield estimation is far from optimal, however, only the sensitivity of H1-NMR allowed us to give a rough estimate of our yield.



Integral of the 5.12 ppm peak originating from the rubber purified MG1655 carrying pSB1C3-Pcon-lacI(N)-term-dxs and pSB1K3-Pcon-araC-term-Para-HRT2 (Dxs + HRT2) relative to the TMS peak. The relative integral value equals 0.16. SDU2013_Rubber_IntCPS2.png

Integral of the 5.12 ppm peak originating from the rubber purified MG1655 (WT) with addition of 3.2 mg polyisoprene standard relative to the TMS peak. The relative integral value equals 0.09. SDU2013_Rubber_Int32.png Standard curve of normalized integrals for known amounts of polyisoprene purified from MG1655 (WT) ONC. The standard curve displays a linear relationship between the normalized 5.12 ppm peak integrals (relative to the TMS peak) and amount of polyisoprene. <img src="SDU2013_Rubber_Standardcurve.png H1-NMR reference: Donald L. Pavia and Gary M. Lampman. Introduction to Spectroscopy, International Edition 4e (Book) ISBN-13: 9780538734189 / ISBN-10: 0538734183 (Link) Sequence and Features

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