Difference between revisions of "Part:BBa K1507014"

Latest revision as of 12:26, 17 October 2014

Fatty acid induced expression auto regulator (pfadBA-B0030-C0051-R0051-B0034-E0040-B0015)

A down-counting timer system. The expression of GFP (green fluorescence protein) will be shut down after a period of time (long) whenever the bacteria uptakes fatty acid.

Exp 1: Function of transcriptional cascade

Purpose:

Testing the properties of three transcriptional cascade circuit, which composed of different ribosome binding site and duplication of CI coding region respectively.

[Notice]Before reading further details, please notice that we have named these circuits with three symbols:

II*2: pfadBA-RBS34-CI-pfadBA-RBS34-CI-pCI-RBS34-GFP40-term

II 34: pfadBA-RBS34-CI-pCI-RBS34-GFP40-term

II 30: pfadBA-RBS30-CI-pCI-RBS34-GFP40-term

Method:

1. Three E.coli strains is cultured overnight in 200mL LB medium respectively in Erlenmeyer flask, under 37℃ environment.

2. 10mL oleic acid is added to the flasks respectively and mixed by hand-shake for 1 minute.

3. 30ml LB-bacteria mixture is moved from the flasks every 30 minutes. Then the lysis buffer is added to the mixture.

100 c.c Lysis buffer:

Na3PO4．12H2O 1.9006 g

NaCl 0.5844 g

EDTA(0.5M) 20λ

Triton X 100 200λ

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Add 1/1000 V β-mercaptoethanol & 120λ protein inhibitor/c.c(Roche)

4. E.coli in LB medium with lysis buffer is destructed by sonicator.

5. Centrifuge by 9000 rpm, 15 minutes.

6. Move the supernatant liquid into eppendorfs.

7. Records the absorbance of green fluorescence protein with fluorophotometer.

Result:

Hour*	0	0.5	1	1.5	2	2.5
II*2	169.4	275	205.0	152.1	105.8	101
II 34	137.6	435	125.5	78.91	105.5	121.5
II 30	62.44	353.2	139.4	123.8	78.55	182.8

Hour* : Time after adding oleic acid into the medium.

This is the raw data of Experiment 1. Since bacteria is keep growing in this 2.5 hour-long period, the amount of bacteria in each time point should be taken onto consideration. These data should be adjusted by dividing the theoretical amount of bacteria.

Adjusted value = log(O.D/Nbacteria)

The theoretical amount of bacteria is calculated by this equation, which is developed by Hiroshi Fujikawa in 2004 (A new logistic model for Escherichia coli growth at constant and dynamic temperatures):

dN/dt=rN(1-N/Nmax)(1-Nmin/N)^c

Parameters are proposed in the article:

T (K) 300

r 1.262802

Nmax 7.94E+08

c 0.74

Nmin 999.99

dt 0.01

N0 1000

These are the theoretical amount of E.coli in LB medium and GFP amount’s adjusted value:

Hour*	0	0.5	1	1.5	2	2.5
N bacteria	6.20E+03	1.09E+04	1.99E+04	3.65E+04	6.78E+04	1.26E+05
II 34 adjusted	-1.65E+00	-1.40E+00	-2.20E+00	-2.67E+00	-2.81E+00	-3.02E+00
II 30 adjusted	-2.00E+00	-1.49E+00	-2.15E+00	-2.47E+00	-2.94E+00	-2.84E+00

The expression of GFP protein decreases significantly after the bacteria senses fatty acid. At the beginning, bacteria still utilize nutrient component in LB medium (mainly yeast extract) as carbon source. Therefore, the metabolism of fatty acid has not started yet and repression of CI protein keeps going. This is the main reason that causes the increasing of GFP amount at the beginning of the curve. After the carbon containing material in LB medium being exhausted, the fatty acid metabolism started and the CI protein begins to be expressed. As a result, the amount of GFP keep decreases at the later part of the curve.

Although the decreasing rate of II 34 group is slightly greater than II30, the slope of two curves is quite similar with each other, suggesting that using different ribosome binding site in gene circuit seems to have limited influence on changing the decreasing rate of GFP protein in E.coli cells. To confirm the properties of the gene circuits, we need to conduct more experiment and lengthen the observation interval. The exact time point that the expression of GFP protein is completely shut down should be confirmed. In addition, the experiment of pfadBA-RBS34-CI-pfadBA-RBS34- CI-pCI-RBS34-GFP40-terminator plasmid will be conducted in future.