Part:BBa_K750111
PtetGLT:GFP(lva) expression system controlled by (anhydro)tetracyclin
Description
This part includes TetR composition, promoter Ptet, RBS of 1.0 strength, a unstable GFP reporter and the double terminator. It is designed to work when (anhydro)tetracyclin is added. In the digital display part of our project E.Lumoli, we need different induction to activate the circuit and make GFP be produced or be degraded. The inspiration of this part comes from BBa_K145280(designed by iGEM08_KULeuven). We planed to use this biobrick in our project at the very start, but when we test this device by using 140 ng/ul atc(protocol from team KULeuven), the device could not work. So we changed the RBS from 0.3 strength to 1.0 strength. This time, the device worked.
Performance
In our project, we utilized tubes with different kinds of bacteria as seven segments. According to the theory of network design, genetic logic gates were assembled into circuits and then constructed using synthetic biology method. Based on the knowledge of bioinformatics and synthetic biology, there are only two results for a single logic gate: on or off. Here, instead of electric signals representing streams of binary ones and zeros, the chemical concentrations of specific DNA-binding proteins and inducer molecules act as the input and output signals of the genetic logic gates, i.e. present or absent of a specific signal molecule represents binary one or zero. The genetic circuits we constructed actually using Binary-Coded Decimal (or BCD, a device which converts the binary numerical value to decimal value) to compute signal molecules.To display two results (in this case, number one and zero) necessitates one kind of signal molecule, we called it a Single-input BCD Decoder (As shown in Table 1).
Table 2 presents the design of device displaying the number 0, 1, 2, which we called Dual-input BCD Decoder. Once the construction is completed, Engineered E.coli with different circuits can be immobilized into microcapsules and then be put in seven transparent tubes which acting as segment of display.
As can been seen in Figure 5, the result after inducing are both significant, which 70ng/mL shows better properties for the device: fast response as well as fast degradation. Therefore, 70ng/mL became the optimize concentration for the device, in spite of the higher peak value reached using 140ng/mL.
Another thing worth to be mentioned is that one part in the Distribution Kit seem failing to work as expected. The trouble circuit is Part BBa_K145280, consisting of a TetR generator and a GFP with LVA tag under control of a TetR promoter. It is supposed to be able to response to the addition of (anhydro)tetracycline. However, almost no fluorescence can be observed or determined after the addition of aTc. At first, we suspected that aTc we use might be the cause for the problem, for its unsuitable concentration (140ng/mL, as the designer used before). But further experiment showed that all gradients of concentration also failed (250, 25, 0.25, 0.025ng/mL). We even suspected that aTc itself might be ineffective due to inappropriate storage or preparation. Then we noticed that the strength of RBS in front of gfp is 0.3, a medium level, which may lead the low expression and finally counteracted by fast degradation. Consequently, we constructed a new circuit, changing the strength of RBS from 0.3 to 1.0 and successfully obtaining satisfying data as mentioned before.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1615
| control | R0040 |
| device_type | signaling |
| protein | GFP(lva) |