• In our software design, we follow the concept that to solve the difficulties in synthesize target products in complex

  • microorganism environment, we can craft a new bacterium community in our software, which means we regard

  • different combinations of chassis species and circuits as different bacterium group, and use artificial restriction

  • conditions, simulating natural selection pressure, to filter out the most appropriate one

  • Our software also uses the concept of “design and redesign” in synthetic biology, generating the protocol for

  • experiment with standard frame and filling it with provided messages in synthetic biological system.

  • Validation experiment used violacein as the target product, and we wanted to test if the software can give us the

  • appropriate combination of circuits and chassis species and if we can have acceptable results with protocol generated

  • from our software.

  • Design From

  • Software

Step 1: Input violacein as target product

  • We input violacein as our target product and set maximum concentration, time, medium and background bacterium.

Step 2: Derive circuit, plasmid and chassis species

  • Our software recommended us to use CDSs from VioA to VioE, constructing three expression vectors, and

  • transduce them into two strains of Escherichia coli with A, B, E in one and C, D in another. CRAFT also recommend us

  • use BBa_J23100 as promoter and BBa_B0034 as RBS.

Step 3: Generate protocol

  • Continue the progress and CRAFT will generate specific protocol correspounding to the synthetic biological system

  • previous chosen.

  • Validation

  • Experiment



  • In order to confirmed that the combination from CRAFT performed best, we also constructed other combinations with

  • promoter BBa_J23114 and RBS BBa_B0032, and combined them with Vio gene respectively (Table 1).

  • Also following the results from CRAFT, we transduced the circuits into corresponding strain (Table 2).


  • After the plasmids successfully finished, the strains will be several times screened by the medium with chloramphenicol and

  • kanamycin. Then they can be mixed into the co-culture system.

Determination of Intermediate products

  • Two strains of E. coli were cultivated in overnight separately and diluted bacterium were mixed togerther in LB broth with

  • chloramphenicol and kanamycin. 10ml of sample were taken every 3 hours for HPLC-MS analysis after 12-24h growth. Ethyl

  • acetate was used for extraction and extracts from bacterial cultures were subjected to HPLC-MS module coupled to a analyses

  • which was performed with an Alliance chromatographic module coupled to a 2996 photo-diode array detector and a ZQ4000

  • mass spectrometer (Waters, Mi- cromass, Milford, MA).


Combination Confirmation

  • The constructed plasmids were confirmed by enzymatic digestion and sequencing to make sure the results were not

  • influenced by the plasmids.

  • Plasmids containing VioA and VioB genes, VioC gene, VioD gene, VioE gene were used to running the gel after enzymatic

  • digestion (digest with EcoR1 and Pst). The picture showed that most plasmids had the right electrophoretic band of insert

  • genes and plasmids backbone except some plasmids that contain VioA1+VioB gene. We selected the constructs of VioA1+VioB gene

  • that had the right electrophoretic band for co-culture experiment. And all the plasmids used in co-cultured were also

  • confirmed by sequencing (Fig. 1).

Fig. 1 The enzymatic digestion results of plasmids containing Vio genes

  • Constructs containing GFP were also confirmed by enzymatic digestion and sequencing.

Fig. 2 The enzymatic digestion results of plasmids containing GFP gene

  • The results of fluorescence showed the recommended combination of promoter and RBS gave stronger expression of GFP

  • than other combinations, which means that they will have a higher expression level when expressing Vio genes. Its curve

  • also meet the prediction from CRAFT. These results showed that our “natural selection” concept had been successfully

  • implemented in CRAFT and filtered the most appropriate circuit out.

Fig. 3 The results of OD (left figure) and Fluorescence value (right figure)

HPLC-MS Confirmation

  • We measured expression of violacein by resersed-phase HPLC-MS. Before measuring the violacein quantificationally, we saw

  • the color of medium changed into purple. And longer the bacteria were co-cultured , deeper the color present. Because

  • none of the intermediate products in violacein synthesis pathway presents to be purple, we can predict that the medium

  • has produced the violacein.

Fig. 4 Extract of violacein co-culturing for different time

  • Compared with HPLC-MS map of sterling violacein, products from our experiment showed similar curve and the concentration

  • of violacein was in consistent with simulation of CRAFT, demonstrating that the design and protocol from CRAFT can

  • perform well and follow them the user can receive their predicted results.

Fig. 5 HPLC-MS results of sterling violacein

Fig. 6 HPLC-MS results of violacein group 1 (up figure) and 2 (down group)