Part:BBa_K098988:Experience

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Experience by [http://2012.igem.org/Team:NTU-Taida 2012 iGEM NTU-Taida]

We tried to modify pCI, the well-known regulator from lambda phage. The original design of pCI in iGEM community is pR, which is composed of OR1 in -10 region, and OR2 in -35 regions. The transcriptional factor CI would preferentially bind to OR1 and then OR2 with cooperatively binding, via C terminal domain interaction. In the normal physiological condition, the binding of OR2 would further tighten the binding of RNA polymerase to pRM promoter and stimulate its own transcription. The binding site -10 upstream of starting point is critical to the function of the repressor. Under this notion, it seems more reasonable to pick a sequence unit with a higher affinity in -10 region. Thus we incorporate OL1, only one base pair different from OR1, and 10 times higher affinity, in our modified pCI repressor, with OR1 in -35 region.

Reference

1. Dodd, J.B. Shearwin; Egan, J.B.; Egan, JB (2005). "Revisited gene regulation in phage lambda". Curr Opin Genet Dev 15 (2): 145–152.
2. Direct demonstration and quantification of long-range DNA looping by the λ bacteriophage repressor, Nucleic Acids Res. 2009 May; 37(9): 2789–2795

Experience by 2011 iGEM NCTU_FORMOSA

In order to characterize this high temperature induced device BBa_K098995, the fluorescence intensity of BBa_K098988 is measured by the flow cytometry (Figure. 1).

Figure1. Part BBa_K098988 Design. The heat induced device BBa_K098995 uses gene BBa_K098997 coding for cI repressor to inhibit the cI promoter BBa_R0051. The activity of cI repressor is decreased by elevating temperature from 30 ℃ to 42 ℃.

Method
First day, we incubated E.coli which contains this circuit (BBa_K098988) included GFP (Green Fluorescence Protein, BBa_E0040) at 37℃ overnight. The next day, we transferred them to new three tubes with M9 medium. When the O.D.(optical density) reached 0.08, we incubated them to 25℃, 37℃, and 42℃, respectively. We collected the samples once an hour, diluting them 200x for accurate GFP (Green Fluorescence Protein, BBa_E0040) measurements. Here is our experiment original data (Table 1). The data after unit-conversion is shown in Table 2. We can use Table2 data to draw line chart, which is easy for us to compare the mean fluorescence at different temperatures, 25℃, 37℃, and 42℃. (Figure 2) The vertical axis is mean fluorescence, and the horizontal axis is time (unit: an hour).

Original Data (Related fluorescent unit)

	25℃	37℃	42℃
0	9.02	8.76	9.14
1	4.66	8.36	17.54
2	2.82	6.46	29.24
3	2.37	5.00	26.44
4	1.70	4.69	22.27
5	1.80	4.40	21.94
6	1.52	4.72	21.57
7	1.58	4.78	21.01
9	1.22	3.9	22.21

Table 1. Related fluorescent unit at 25℃,37℃,42℃ in 9 hours

Data after unit conversion

	25℃	37℃	42℃
0	2400.9500	2326.2350	2435.4930
1	1175.9040	2211.6410	4926.7290
2	683.2785	1673.7370	8559.5680
3	566.2294	1268.9040	7677.1730
4	395.3901	1184.0880	6377.2450
5	420.5876	1105.1540	6160.8630
6	350.3407	1192.2770	6160.8630
7	365.3116	1208.6670	5988.1660
9	276.2400	970.0604	6358.6760

Table 2. Molecules of equivalent fluorescence at 25℃,37℃,42℃ in 9 hours

"

Figure 2.
Line chart of molecules of equivalent fluorescence performance at 25℃,37℃,42℃ in 9 hours. The vertical axis is mean fluorescence, and the horizontal axis is time. Form the experimental data, we find that MEFL increases steeply at 42℃ in first two hours, which means the expression of GFP is high. Although after the first two hours its performance decreases and maintains at certain volume, it still expresses a lot more than the other two samples at 25℃,37℃.

The following diagrams, Figure3-1.~Figure3-3., are from the Flow cytometer experiment we did. The green fluorescence intensity of E.coli incubated at 25℃ (Figure3-1) and 37℃(Figure3-2) for 9 hours is lower than incubated at 42℃(Figure3-3),which also can refer to the data from Table 1. In Figure3-2., the peak of green fluorescence intensity of E.coli incubated at 37℃ for 9 hours moves right a little bit than incubated at 25℃ for 9 hours. Last, in Figure3-3., incubated at 42℃ for 9 hours, the peak of green fluorescence intensity is significantly stronger than incubated at 25℃ and 37℃ for 9 hours.

Figure 3-1. Fluorescence of sample at 25℃, 0hr &9 hour. X-axis is related green fluorescence intensity, and Y-axis is cells number.

Figure 3-2. Fluorescence of sample at 37℃, 0hr &9 hour. X-axis is related green fluorescence intensity, and Y-axis is cells number.



Figure 3-3. Fluorescence of sample at 42℃, 0hr & 9 hour. X-axis is related green fluorescence intensity, and Y-axis is cells number.

Comment

Form the experimental data, we find that MEFL increases steeply at 42℃ in first two hours, which means the expression of GFP is high. Although after the first two hours its performance decreases and maintains at certain volume, it still expresses a lot more than the other two samples in different temperatures. We can regard GFP as our target protein. This means that our hypothesis has been confirmed that any protein placed after this circuit BBa_K098995can be regulated by elevating or lowering temperature. At higher temperature(42℃), our target protein will perform significantly. On the contrary, at lower temperature(25℃), it will express slightly.

We also build the modeling of Part:BBa_K098988

Modeling and simulations of high temperature induced device BBa_K098988 – cI promoter & cI repressor

A differential equation is used to calculate protein expression activity of BBa_K098995 as follows.

This equation describes the concentration of GFP in BBa_K098988 change with time (Figure. 1). Alpha-Temp is the protein expression rates corresponding to BBa_K098995 which is a temperature sensitive expression device. To describe transition during log phase and stationary phase, the alpha-Temp is assumed to zero in stationary phase. Gamma-GFP are decay rates of the GFP proteins. When bacteria divide, the molecular in a bacterium will be dilute. Because bacteria grow faster, the dilution rate d(t) is included in this model and can be calculated from OD ratio of medium (Figure. 4). The values of the kinetic parameters used in the simulation were initially obtained from the literature and experimental data. Data computations were performed with Matlab software. A program was written and used as a subroutine in Matlab for parameter optimization using nonlinear regression (Figure. 5).</p>

Figure 4. The OD ratio is increased faster in log phase than it in stationary phase. The dilution rate d(t) can be calculated from OD ratio and used in our model.

Figure 5. The behavior of high temperature induced device BBa_K098988 at 25°C, 37 °C and 42°C. Experimental data (dot) and simulated results (line) of the model suggest this temperature-dependent device can control the expression level of the target protein by the host cell’s incubation. The fitting results indicate our dynamic model can quantitatively assess the protein expression activity of BBa_K098988 during log phase and stationary phase.

Using least squares estimation from experimental data, the relative the protein expression activity of BBa_K098988 at 25°C, 37 °C and 42°C were estimated (Figure 6).

Figure 6. The relative the protein expression activity of BBa_K098988 at 25°C, 37 °C and 42°C estimated using least squares estimation from experimental data. The protein expression activity at 42°C is higher than 25°C, 37 °C

According to the fitting results (Figure. 5), the dynamic model successfully approximated the behavior of our high-temperature induced system. The model equation presents interesting mathematical properties that can be used to explore how qualitative features of the genetic circuit depend on reaction parameters. This method of dynamic modeling can be used to guide the choice of genetic ‘parts’ for implementation in circuit design in the future.

References

Alon, U. (2007) An Introduction to Systems Biology: Design Principles of Biological Circuits. Chapman & Hall/CRC.

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