Part:BBa_K598019

B0015+pBAD+vioABD+B0015+pBAD+TPP Down-regulated Hammerhead Ribozyme 2.5+vioE

This part was constructed to demonstrate the applicability of RNA toolkit developed by 2011 iGEM Peking_R team. Upon adding TPP, the amount of byproducts of violacein biosynthetic pathway would decrease and the purity of the desired product would increase.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 262
  Illegal NheI site found at 1296
  Illegal NheI site found at 5973
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 202
  Illegal BamHI site found at 5913
  Illegal BamHI site found at 6105
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 4081
  Illegal AgeI site found at 4277
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 5506
  Illegal SapI.rc site found at 5581
  

Introduction

To testify our platform for genetic softcoding, we chose to apply it to a segment of violacein biosynthetic pathway, because violacein and its precursors have notable antibacterial, antiviral, antiparasite and many other biological activities, rendering them having great potential in medical applications. Besides, this biosynthetic pathway is more characterized, including the intermediates, the shunt products, the mechanism of each reaction and so on. Moreover, the products in violacein biosynthetic pathway are often colored, so it’s relatively easier to pick positive colonies and to verify the experiment primarily by naked eyes. And most importantly, the enzymes involved in this pathway are readily available in the Registry of Standard Biological Parts.

Violacein originates from a soil bacterium Chromobacterium violaceum, and genes for violacein biosynthesis are arranged in an operon consisting of vioA, vioB, vioC, vioD and vioE. These genes have been successfully transformed to E. coli to produce violacein . In the segment of the pathway investigated in our project, four enzymes are involved (Fig. 1). VioA is an FAD dependent L-tryptophan oxidase, which transforms L-tryptophan to an IPA imine. VioB would further convert the IPA imine into a dimer. VioEis responsible for the indole shift that converts the IPA imine dimer to prodeoxyviolacein, which can be taken over by VioD to form proviolacein. In E.coli an additional side reaction occurs, producing a green pigment called deoxychromoviridans, which is produced by condensing two prodeoxyviolacein molecules.

Figure 1 Scheme of a segment of violacein biosynthetic pathway. VioA, VioB, VioE function sequentially to convert L-tryptophan to prodeoxyviolacein, which would either form deoxychromoviridans due to intrinsic reactions in E. coli, or be converted to proviolacein by VioD.

Design of Constructs to Fine-tune Violacein Biosynthetic Pathway

After analyzing the metabolic flux in violacein biosynthetic pathway, we decided to fine-tune the translation strength of enzymes VioE and VioD, which have direct links to the side products, as shown in Fig.1. Therefore, we substituted the RBS of vioE or vioD with thiamine pyrophosphate (TPP)-responsive hammerhead ribozyme variants, which were previously characterized in our project. Decreasing the amount of VioE may lower the pool of prodeoxyviolacein, hence lowering the production of deoxychromoviridans, thus we inserted a TPP down-regulated hammerhead ribozyme (TPP ribozyme 2.5) in front of vioE (Fig.2A), so that the purity of the product proviolacein may increase as TPP is added to the culture(Fig.2B). Similarly, increasing the amount of VioD may increase the yield of proviolacein, thus we inserted a TPP up-regulated hammerhead ribozyme (TPP ribozyme 1.20) upstream of vioD (Fig.2A), so that upon adding TPP into the culture, the purity of the products may increase(Fig.2C)..

Figure 2 (A) Design of constructs to fine-tune violacein biosynthetic pathway. Hexagon: Stem-loop terminator (Part:BBa_B0015); Bent arrow: pBAD promoter (Part: BBa_I13453); Oval: Ribosomal binding site; Straight arrow: Coding sequence originated from Part: BBa_K274003; ribbon shape: TPP-responsive ribozyme. (B) Decreasing the amount of VioE may lower the pool of prodeoxyviolacein, hence lowering the production of deoxychromoviridans, thus we inserted a TPP down-regulated hammerhead ribozyme (TPP ribozyme 2.5) in front of vioE, and the metabolic flux would favor proviolacein. Dash arrow: down-regulated. (C) Increasing the amount of VioD may increase the yield of proviolacein, thus we inserted a TPP up-regulated hammerhead ribozyme (TPP ribozyme 1.20) upstream of vioD, and the metabolic flux would favor proviolacein. Dash arrow: down-regulated; Filled arrow: up-regulated.

Results

We have successfully constructed a plasmid that would enable us to fine-tune the translation strength of vioE by TPP ribozyme 2.5 (Part: BBa_K598019). The plasmid was transformed to E. coli, and after induction by arabinose and culturing in different concentrations of TPP, the bacteria produced some pigments that were visible by naked eyes (Fig.3). After lysating the bacteria with 10% SDS and extracting the pigments with ethylacetate, the extraction samples were analyzed by HPLC (Agilent systems 2000 series, Spursil C18 5m column, mobile phase 50% methanol, 50% water, monitor wavelength 650nm). The two peaks eluted at 0.7-1.0 min were confirmed as the products of the bacteria that were extracted into ethylacetate(Fig.4), and we tentatively assumed that the two peaks corresponded to proviolacein (left) and deoxychromoviridans (right) (Fig.4).

Figure 3 E. coli producing pigments. When induced by arabinose, the engineered E. coli produced dark-green pigments (second tube comparing to first one). Upon addition of different concentration of thiamine pyrophosphate (TPP), the color of the bacteria gradually shifted from dark-green to dark-brown (from second tube to sixth one).

To see if adding TPP could affect the purity of the products extracted, we induced the bacteria under different concentrations of TPP. The samples were prepared as described above. The pelleted cells produced some pigments, and the color changed as the concentration of TPP increased (Fig. 3). The HPLC results turned out that upon adding TPP into the cultures, the amount of deoxychromoviridans decreased, and the ratio between proviolacein and deoxychromoviridans increased upon increasing TPP concentration (Fig.5), until deoxychromoviridans was not detectable when TPP concentration reached to 10M. These results proved that our RNA controller had the ability to fine-tune the biosynthetic pathway and increased the purity of the products as we desired.

Figure 4 HPLC results of the bacterial extraction when different concentrations of TPP were presented in the cultures. When induced with arabinose, the bacterial extraction produced two peaks eluted at 0.7-1.0 min ((b) comparing to (a)), and we tentatively assumed that the two peaks corresponded to proviolacein (left) and deoxychromoviridans (right). Upon adding TPP into the cultures, the amount of deoxychromoviridans decreased, and the ratio between proviolacein and deoxychromoviridans increased upon increasing TPP concentration ((b) to (f)), until deoxychromoviridans was not detectable (f).

Figure 5 The area ratio between peaks of proviolacein and deoxychromoviridans on HPLC when different concentrations of TPP were presented in the cultures. Note that deoxychromoviridans was not detectable when TPP concentration reached to 10 uM, so the ratio was not presented in this figure.

Due to time limit, we have not performed calculated RBS sequence substitution yet, but further experiment that would fix the RBS sequence of vioE designed by our RBS calculator will enable us to settle down the optimized biosynthetic pathway. This brand-new approach greatly saved us from time-consuming library construction and screening, which facilitated our experiment progress to a large extent. The versatile and extensible platform for softcoding of genetic program has proved its applicability in fine-tuning biosynthetic pathway, and it will prove its robustness in more bioengineering fields.

Reference

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