B0015+pBAD+vioAB+B0015+pBAD+TPP Up-regulated Hammerhead Ribozyme 1.20+vioD+B0015+pBAD+vioE
B0015+pBAD+vioAB+B0015+pBAD+TPP ribozyme 1.20+vioD+B0015+pBAD+vioE
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 262
Illegal NheI site found at 1296
Illegal NheI site found at 4826
Illegal NheI site found at 6466
- 21Illegal BamHI site found at 202
Illegal BamHI site found at 4766
Illegal BamHI site found at 6406
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 4081
Illegal AgeI site found at 4277
- 1000Illegal SapI.rc site found at 5915
Illegal SapI.rc site found at 5990
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.
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)..
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 5m 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).
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 10M. 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.
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.
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|chassis||E. coli DH5α|
|ligands||Thiamine Pyrophosphate (TPP)|
|n/a||B0015+pBAD+vioAB+B0015+pBAD+TPP Up-regulated Hammerhead Ribozyme 1.20+vioD+B0015+pBAD+vioE|