Team:UST Beijing/Parts
Parts
We design a new standard BioBrick Part cantral to our project and submit this part to the iGEM registry and we experimentally validate this part works as expected. And more, we document a new application of a BioBrick part from a previous iGEM year.
New application of BBa_1450004
The cell wall of notoginseng contains a certain concentration of arabinose, so we expected to use arabinose as Inducer to express T7RNApol. When the concentration of T7RNApol is higher than LacI, T7RNApol would have priority to bind to pET28a plasmid, activate the expression of β-galactosidase. pSB1C3-BBa_1450004 and pET28a-β-galactosidase were transformed into E.coli. Psb1C3 contains T7 RNA Polymerase gene and can be regulated by pBAD. This double-plasmid system is expected to be regulated by pPAD, and expresses a large number of T7RNA polymerase to inhibit the effect of LacI repression, switch on the expression of β-galactosidase.
We added Lac, Ara, PNPG, and bacteria solution in each well of a 96-well palte. After a period of time, we measured the A620 and A450 using microplate reader.( A450 represents the concentration of brokendown PNPG, A620 represents the concentration of bacteria.) After the experiments, we found out that actual A450 could be calculated by this formula：A450real=A450-1.5A620
The catalyst efficiency of enzymes expressed by E.coli is represented by A450real/A620
The result displayed that the concentration of brokendown PNPG decreases when the concentration of lactose increases. Since the the similarities of structure between lactose and PNPG, high concentration lactose has a competitive effect on PNPG. There is 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
Because double-plasmid system containing ara operon and lac operon may be influenced by complex factors when it works, we connect 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 expressβ-galactosidase.
In this part，we use the orign sequence of araC and pBAD(pC) which copy from genome of E. coli. 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 are added at the end of coding sequence of araC and β-glucosidase. The prefix and suffix are necessary of parts’ both ends.
Plasmid verification
To validate the availability of plasmid, we test the abilities of decompositing pNPG of three kinds of E.coli strains.
We can know that the efficiency of E.coli containing BBa_K2072000 is higher, 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-galactosidase. We speculate that a small amount of enzyme background expression lead the E.coli which is not induced by arabinose to decompose the PNPG.
We added E.coli with BBa_K207200, without this part, ONPG and arabinose with varies concentration in different wells. After a period of time, absorbance was measured under 450nm light, the result shew that catalytic effect of arabinose reach peak when its concentration is 7mM, and then it went down while concentration increasing. It was even inhibited when concentration keep increasing.
