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−	<p>The sequnence above is the amino acid sequenc of this part. Displayed here to make codon optimization and protein engineering easier. The sequence in deep green (html#003300) codes for the truncated major enzyme unit of the HRT2 and HRT2trunc. The sequence in pink is the 6xHis-tag introduced to this part (#ff99cc).</p><br>	+	<p>The sequnence above is the amino acid sequenc of this improved part HRT2trunc. Displayed here to make codon optimization and protein engineering easier. The sequence in deep green (html#003300) codes for the truncated major enzyme unit of the HRT2 and HRT2trunc. The sequence in pink is the 6xHis-tag introduced to this part (#ff99cc).</p><br>
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Revision as of 09:14, 1 October 2024

HRT2 prenyltransferase from Hevea Brasilianis (ara promoter without 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.

Using Northern blot technique 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 promoter regulator 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 "-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 C-terminal to the HRT2 prenyltransferase, but ins't part of the translated protein (HRT2 has it's natural stop-codon).

The rubber producing capabilities of HRT2 were assessed 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 precense 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.

     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 HRT2+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. 

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 +HRT2 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 HRT2+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, 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, comparing WT and HRT2+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 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 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.

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. 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. 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.

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.

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. 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=" H¹-NMR reference: Donald L. Pavia and Gary M. Lampman. Introduction to Spectroscopy, International Edition 4e (Book) ISBN-13: 9780538734189 / ISBN-10: 0538734183 [http://edu.cengage.co.uk/catalogue/product.aspx?isbn=0538734183 (Link)] </html>

Uses and Improvements

This part is re-designed by Imperial_College 2024, where the truncated version of HRT2 (HRT2trunc, [[Part:BBa_K5416000|BBa_K5416000]]) now can work in composite parts to form artifical rubber-producing organelles.

HRT2trunc (Rubber cis-1,4-polyprenyltransferase HRT2, truncated) This part encodes the truncated version of rubber producing prenyltransferase HRT2 from the rubber tree H brasiliensis (K1088024). HRT2 uses isopentenyl diphosphate (IPP) as substrates to produce cis-1,4-polyisoprene natural rubber^[1][2]. This enzyme has a unique unit consisting of a hydrophobic channel, where allows the polymerization of the IPP precursors to occur at the entrance and eject the polymerized natural rubber chain through the exit.

Design

Protein Engineering

Inspired by HRT2 parts designed by SDU_Denmark (K1088024), we applied several major modifications on protein structure of HRT2. These generate with new HRT2 with higher solubility; and prevent the formation of HRT2 dimer during expression. This modified HRT2 is coupled with multiple strategies (detailed below) during our project for higher rubber yield.

The protein engineering process to design this part begins with identify the function of each protein domains according to the literature and protein structure database. Whereby we have identified the several domains from the original amino acid sequence of this HRT2 ^[2][3].

In the structure of HRT2 (wt) predicted via using the ColabFold (Alphafold 2) ^[4]. The domains previously identified by the literatures are highlighted in different colors as follows: the N terminus and C terminus domain (coloured in blue and teal); dimerizing helix (white); major enzyme catalytic unit (green); substrate binding and catalytic residues (red); residues near the existing channel of polyisoprene chain (yellow).

A protein structural alignment is hence conducted using Swiss-Model to identify the structural homologies with its homologous protein from Homo sapiens (HsCPT) ^[5]. In which the dimerizing helices, N terminus and C terminus domain (indicated with the arrows) appears to be less conserved after aligning the structures. Inspired by this finding, we hence truncated these domains entirely form the HRT2. Where on the scar that was left after removing the dimerizing domain, a 6xHis tag is inserted to provide the possibility of future protein purification ^[6].

The amino acid sequence of the engineered HRT2 – now called HRT2trunc, was used to predict its final structure again with Colab Fold (alphafold2) ^[4]. The structure is analysed with Pymol where the hydrophobic channel is retained after the modification. Indicating a general success in this process of in silico protein engineering.

Expression

Burden

The burden of this part is studied in the growth assay of its E. coli BL21 transformant, where no significant burden in cell growth is identified.

Variants of This Part

Through the path of modular engineering, Imperial-College 2024 have also designed the variants of this parts for the formation of artifical organelles.

We report proteins HRT2trunc-NT-ASIP (BBa_K5416070); HRT2-SP (BBa_K5416001); PhaC_HRT2trunc(BBa_K5416030); to be variants of this part. For a comparison, the same domain has been colored in green.

Reference:

1. Asawatreratanakul, K., Zhang, Y.-W., Wititsuwannakul, D., Wititsuwannakul, R., Takahashi, S., Rattanapittayaporn, A. and Koyama, T. (2003). Molecular cloning, expression and characterization of cDNA encoding cis-prenyltransferases from Hevea brasiliensis. European Journal of Biochemistry, 270(23), pp.4671–4680. doi: https://doi.org/10.1046/j.1432-1033.2003.03863.x.
2. Yamashita, S. and Takahashi, S. (2020). Molecular Mechanisms of Natural Rubber Biosynthesis. Annual Review of Biochemistry, 89(1), pp.821–851. doi: https://doi.org/10.1146/annurev-biochem-013118-111107.
3. Takahashi S, Koyama T. Structure and function of cis‐prenyl chain elongating enzymes. The Chemical Record. 2006;6(4):194-205.
4. Mirdita M, Schütze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M. ColabFold: making protein folding accessible to all. Nature methods. 2022 Jun;19(6):679-82.
5. Schwede T, Kopp J, Guex N, Peitsch MC. SWISS-MODEL: an automated protein homology-modeling server. Nucleic acids research. 2003 Jul 1;31(13):3381-5.
6. Maree HJ, van der Walt E, Tiedt FA, Hanzlik TN, Appel M. Surface display of an internal His-tag on virus-like particles of Nudaurelia capensis ω virus (NωV) produced in a baculovirus expression system. Journal of virological methods. 2006 Sep 1;136(1-2):283-8.

Index

MIPTHIAFIL DGNGRFAKKH KLPEGGGHKA GFLALLNVLT YCYELGVKYA TIYAFSIDNF RRKPHEVQYV MNLMLEKIEG MIMEESIINA YDICVRFVGN LKLLDEPLKT AADKIMRATA KNSKFVLLLA VCYTSTDEPH HHHHHPYINP YPDVLIRTSG ETRLSNYLLW QTTNCILYSP HALWPEIGLR HVVWAVQ

The sequnence above is the amino acid sequenc of this improved part HRT2trunc. Displayed here to make codon optimization and protein engineering easier. The sequence in deep green (html#003300) codes for the truncated major enzyme unit of the HRT2 and HRT2trunc. The sequence in pink is the 6xHis-tag introduced to this part (#ff99cc).

Sequence and Features