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.
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
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).
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)
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)