Team:UST Beijing/Parts
Parts
For our iGUT project, we designed a new composite BioBrick part (BBa_K2072000) (http://parts.igem.org/Part:BBa_K2072000:Design) and submitted it to iGEM registry for medal qualification. We did some preliminary characterization of this part. In addition, we used one BioBrick part from the toolbox of 2016 in our project iGUT.
New application of BBa_1450004
Since the cell wall of notoginseng root cells is composed of a high percentage of arabinose, we hope to use arabinose-inducable promoter to express our sugar degradation enzyme. One biobrick part, BBa_1450004, designed by Univ.British Columbia in 2014, controls T7RNApol expression through pBAD promoter. We co-transformed BL21(DE3) E.coli cells with pSB1C3-BBa_1450004 and pET28a-β-glucosidase. We measured the enzymatic activity of double-plasmid containing cells in different culture conditions, including variable concentrations of lactose, arabinose, IPTG, and kanamycin. For comparison we also measured the enzymatic activity of E.coli cells with single or no plasmids. The results indicated that our synthetic β-glucosidase gene was successfully expressed from the pET28a construct.
We added lactose, arabinose, PNPG, and diluted bacteria culture into each well of 96-well plates. After a period of time of culturing at 37C, we measured the A620 and A450 using a micro-plate reader (A450 reflects the concentration of PNP and cell density, A620 only reflects cell density). To calculate the PNP concentration, we used this formula：A450real=A450-1.5*A620. We validated this method using PNP solution in combination of bacteria culture over a wide range of concentrations (A620: 0.01-0.9).
The enzymatic efficiency in E.coli was standardized by calculating (A450-1.5*A620)/A620.
The result displayed that the concentration of PNP decreased when the concentration of lactose was increased. Since the similarities of structure between lactose and PNPG, high concentration lactose may pose a competitive inhibitory effect on PNPG. There was an optimum concentration of arabinose as inducer, so less enzymes would be expressed if the concentration of arabinose exceed optimum concentration or is lower than it. The process of bacterial metabolism uses sugar in each well, so that lactose would be less competitive, then enzymes would begin to decompose PNPG.
Plasmid design (BBa_K207200)
In the double-plasmid system, as a proof of principle, β-glucosidase was expressed under lactose and arabinose. However, T7 RNA polymerase inhibited the growth of E.coli. Next we designed an arabinose-inducing composite Biobrick by replacing the E.coli K12 araC operon B.A.D. genes with our synthetic β-glucosidase gene. We connected three gene fragments together on the basis of principle of ara operon. One of the gene fragments can produce araC protein, a type of ara binding protein—inhibiting the activity of pBAD gene. pBAD gene controls the expression of araC gene to one direction and regulates araB, araA, araD genes to another direction. The last gene fragment is a DNA sequence that can express β-glucosidase.
In this part，we borrowed the original sequence of araC and pBAD(pC) from the genome sequence of E. coli K12. Considering the direction of transcription on 5'→3', we replaced the sequences of araB、araA and araD with reverse complementary sequence of β-glucosidase. Terminal J61048 and B0015 were added at the end of coding sequence of araC and β-glucosidase. The prefix and suffix were necessary of parts for biobrick ends.
Plasmid verification
To test enzyme expression function of the new plasmid, we measured degradation of pNPG in three strains of E.coli culture.
We observed that the enzyme efficiency of E.coli containing BBa_K2072000 is greater, and the catalyze efficiency of E.coli with adding arabinose is faster than non-arabinose. So it prove that our part can be Induced by arabinose to express β-glucosidase. We speculated that a small amount of enzyme background expression led the E.coli which was not induced by arabinose to decompose PNPG.
We added E.coli with or without BBa_K2072000, ONPG and arabinose with varies concentration in different wells. After a period of time culturing, absorbance was measured under 450nm light, the result showed that catalytic activity of β-glucosidase reached peak when arabinose concentration in the culture medium is 7mM, and then it went down while concentration increasing. It was even inhibited when concentration kept increasing.
