Part:BBa_K2027039:Design

Rubber Transferases + Small Rubber Particle Protein Cassette

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 25
  Illegal SapI.rc site found at 80
  

Design Notes

This assembly consists of HRT1, HRT2, SRPP proteins optimized for expression in E. coli. Each protein also has a FLAG tag and a Lumio/His tag for visualization and purification.

The purpose of this construct is to introduce xenogeneic components of H. brasiliensis into E. coli to enable synthesis of isoprene polymers. We identified well characterized and isolated cDNAs from H. Brasiliensis for prenyltransferases participating in natural rubber biosynthesis. Two enzymes were selected, HRT1 and HRT2, which were two cis-prenyl chain elongating enzymes isolated from Hevea latex. These proteins are responsible for the synthesis of new rubber molecules and are also found expressed predominantly in fresh Hevea latex. Because either HRT1 or HRT2 could be used for rubber synthesis, we incorporated both genes into a plasmid construct under a IPTG inducible constitutive promoter to allow for maximum expression of enzyme.

Since prenyltransferase requires an SRPP and various chemical cofactors for functionality, we identified the most commonly expressed SRPP in Hevea latex as a possible cofactor for HRT1 or HRT2 (Accession No. AB061234.2 and AB064661.2). However, because SRPPs can play a variety of roles in Hevea latex production, we targeted SRPPs that had also been identified as rubber elongation factors. Our criterion for SRPP selection were that it had to be (1) a rubber elongation factor, (2) highly expressed in Hevea latex, and (3) of the small variety, since large rubber particle proteins would be difficult to produce in E. coli due to their size. The gene of our SRPP (AF051317) was subsequently incorporated into the prenyltransferase cassette. Since prenyltransferase activity depends on SRPP presence and gene order in an operon can influence expression level, we inserted the SRPP gene before the two prenyltransferases to account for the possibility the first protein in the cassette would be expressed the most. This cassette was the linked to a IPTG inducible T7 Elowitz high copy promoters to allow for regulated expression. All proteins were also tagged with a FLAG, tetracysteine, and hexahistidine tag to allow for protein purification assays . A double terminator, (BBa_B0010 and BBa_B0012) was also used to prevent RNA polymerase leak through.

Note: To enhance production of polyisoprene, part BBa_K2027040 can also be co-transfected to increase the amount of IPP substrate available for polymerization.

The cis-prenyltransferases and SRPP, have been biobricked into the following parts:

• H. Brasiliensis SRPP cDNA (https://parts.igem.org/Part:BBa_K2027053)
• H. Brasiliensis HRT1 cDNA (https://parts.igem.org/Part:BBa_K2027054)
• H. Brasiliensis HRT2 cDNA (https://parts.igem.org/Part:BBa_K2027055)

Additionally, our construct also includes the following ribosome binding sites, terminators, tags, and promoters:

• T7 Strong promoter (https://parts.igem.org/Part:BBa_Z0251)
• Elowitz RBS (https://parts.igem.org/Part:BBa_B0034)
• FLAG-Lumio His Tag (https://parts.igem.org/Part:BBa_K2027052)
• (pMS-AFa-DEa) pAKP444 RBS 1 extraction (https://parts.igem.org/Part:BBa_K2027073)
• (pMS-AFa-DEa) pAKP444 RBS 2 extraction (https://parts.igem.org/Part:BBa_K2027074)
• Terminator from E. coli rrnB (https://parts.igem.org/Part:BBa_B0010)
• Terminator from coliphage T7 (https://parts.igem.org/Part:BBa_B0012)

Sequence Verification

Because the construct was too large to sequence in its entirety, we sequenced it in ~800-1000 bp stretches. Front end of construct sequencing indicates that Hevea SRPP is present in the construct. Middle part of sequencing is spotty but suggests that the 5' and 3' end of HRT1 is present in the construct. Because the 5' and '3 ends sequenced extend beyond the gibson assembly junction, HRT1 is likely to be in the construct. Rear end of construct sequencing indicates that Hevea HRT2 is present in the construct.

Characterization

The following proteins are encoded by this construct and have the corresponding mass:

• SRPP - 24.97 kilodaltons
• HRT1 - 72.3 kilodaltons
• HRT2 - 70.47 kilodaltons

Because our Cellytic solution contains trypsin that cleaves between arginine and lysine residues. Consequently, we observe a darker band at >20kDa and at >10kDa that we suspect may be the HRT1, HRT2 fractions cleaved in two.

Source

1. http://www.ncbi.nlm.nih.gov/nuccore/AF051317
2. http://www.ncbi.nlm.nih.gov/nuccore/AB061234?report=GenBank
3. http://www.ncbi.nlm.nih.gov/nuccore/AB064661?report=GenBank

References

1. Asawatreratanakul K, Zhang YW, Wititsuwannakul D, et al. 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;270(23):4671-80.
2. Oh SK, Kang H, Shin DH, et al. Isolation, characterization, and functional analysis of a novel cDNA clone encoding a small rubber particle protein from Hevea brasiliensis. J Biol Chem. 1999;274(24):17132-8.