Team:NTU Taida/Circuit3

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NTU-Taida

Corneum decomposition --- Keratinaes

Inspiration :

Exfoliation involves the removal of the oldest dead skin cells on the skin's outermost surface to help maintain healthy skin, softening the look of wrinkles and promoting collagen production. In additions to taking of skin with bioengineering designs, we also focus on producing keratinase in lab to explore the potential of cost reduction. Keratinase is enzyme derived from Streptomyces fradiac to degrade the disulfide (-S-S-) bond of the keratin substrate, and its effect on human skin has not been previously reported.

Our ultimate goalis to give E.coli the ability of producing an ideal amount of keratinase. In order to do so, we designed two circuits covering the functions of sensing, producing, and regulating. Having this goal established, we were faced with several precise sub-targets including obtaining keratinase gene sequence from the whole genome ofBacillus Licheniformis and ligating essential parts and biobricks. Eventually, we will test out the efficiency of keratinase production with practical methods.

Gene Ciruit :

Since the second part share the ideas with the first part, the principle mechanism is the same. The fad promoter is repressed by fadR protein. And the fadR protein can be repressed by acyl-coA, which is transformed from fatty acid. Therefore, the CI gene will be activated if there is fatty acid exists in the cell. Then, the CI protein will accumulate to a threshold concentration and repress the pCI promoter. At last, the expression of keratinase will be shut down after a period of time.

In addition, the time period of keratinase expression can be adjusted by two different ways. The first one is to enhance the expression of CI protein, and the second way is to use ribosome binding sites with different affinity. Since degrading too much keratin might cause our skin become vulnerable to allergy and infection, we can control the amount of keratinase by changing this time period.
Degrading too much keratin might cause our skin become vulnerable to allergy and infection. Also, we don’t want keratinase being produced before the bacteria live on human skin. So our gene circuit must function like this:
The model can be simulated by these equations, which is based on Hill functions. The simulating result shows the expression of target protein is repressed effectively after a specific time interval.

It sould be activated by sensing fatty acid in environment and it has to be shut down after a specific time interval. The transcriptional cascade model composes a repressing protein, a promoter and a target protein, which could be green fluorescence protein, keratinase or any other protein. The repressing protein CI can shut down the promoter preceding the target protein, as long as it has accumulated to a threshold concentration.
To secrete the enzyme outside the bacteria, a gene which functions as a signal sequence is inserted before the keratinase coding region. The signal peptide can guide the enzyme to the cell membrane. To further details, please check out the description in Skin Whitening --- PKEK page.

Testing Result :

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

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 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.

Exp 2: Function of J23119-RBS34-FadR-terminator-J23119-RBS34-FadL-terminator

Purpose:
Confirm that FadR protein and FadL protein assist the bacterial uptake of fatty acid.
Method:

1. We use modified LB medium for culturing E.coli strain. The amount of yeast extract is decreased.

tryptone 10g

yeast extract 1g

NaCl 10g /1L ddH2O

We added 20λ, 40λ, 60λ, 80λ, 100λ oleic acid in 2mL modified LB medium respectively. This fatty acid – composing broth is used for culturing E.coli.
2. The E.coli strain with FadR/FadL coding gene and the DH5α cell (without FadR/FadL coding gene) is cultured overnight in the modified LB medium, 2 c.c respectively, with different fatty acid ratio.

3. Record the absorbance of the suspension culture by spectrophotometer.

Since the amount of yeast extract is decreased significantly in LB medium, the bacteria is forced to metabolizes oleic acid for carbon source (yeast extract is the main carbon source in origin LB medium). Therefore, the E.coli strain that contain FadR/FadL coding gene will be more adjustable to the critical environment and grow better in the medium. We compared the growing condition of FadR/FadL (+) and FadR/FadL (-) strain in modified medium.
Result:

% of oleic acid plasmid (-) plasmid(+)

1% 2.542 2.661

2% 2.390 2.623

3% 2.523 2.357

4% 2.610 2.616

5% 2.351 2.653

Conclusion:

FadR/FadL(+) E.coli strain grows better in the critical environment, compared to the control group. The phenomenon suggests FadR and FadL protein play important roles in the uptake and metabolism of fatty acid in E.coli. Fatty acid sensing system can be designed on the basis of the dynamic interaction between FadR-acyl-coA complex , fatty acid molecules and pfadBA promoter

Background Knowledge :

The outermost layer of human skin is called keratin layer (F.g1), which acts as a protective barrier against outside environment. Keratin is the key structural material making up this outer layer. Although keratin layer is an essential structure of healthy skin, excessive accumulation of keratin can cause pigmentation and dryness of epidermis. Therefore, degrading keratin moderately is helpful . It can cause hydration of skin and increase skin elasticity. This property is proved by studies and degrading keratin has also come to cosmetic uses already.
Keratinase is an enzyme composes degrading capability. It hydrolyzes keratin by attacking disulfide bond of the substrate . It exists in a kind of bacteria, Bacillus licheniformis. And we got gene sequence of the enzyme by extracting whole genome of the bacteria. In addition, the keratinase exhibits mold dehairing capability.

Reference :

Food Microbiology 21 (2004) 501–509 A new logistic model for Escherichia coli growth at constant and dynamic temperatures Hiroshi Fujikawa*, Akemi Kai, Satoshi Morozumi Department of Microbiology, Tokyo Metropolitan Research Laboratory of Public Health, 3-24-1, Hyakunin-cho, Shinjuku, Tokyo 169-0073, Japan Received 15 November 2003; accepted 6 January 2004

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     <strong>Degrading too much keratin</strong> might cause our skin become vulnerable to allergy and infection. Also, we don’t want keratinase being produced before the bacteria live on human skin. So our gene circuit must function like this:</p><p><img src="https://static.igem.org/mediawiki/2014/c/cc/NTU_Taida_Keratinase_model.jpg" style="width:360px;height:160px"></p><p><strong>The model</strong> can be simulated by these equations, which is based on Hill functions. The simulating result shows the expression of target protein is repressed effectively after a specific time interval.</p><p><img src="https://static.igem.org/mediawiki/2014/2/24/NTU_Taida_Keratinase_model_2.jpg" style="width:360px;height:160px">
 </p><p><strong>It sould be</strong> activated by sensing fatty acid in environment and it has to be shut down after a specific time interval. The transcriptional cascade model composes a repressing protein, a promoter and a target protein, which could be green fluorescence protein, keratinase or any other protein. The repressing protein CI can shut down the promoter preceding the target protein, as long as it has accumulated to a threshold concentration.</p><p><img src="https://static.igem.org/mediawiki/2014/f/f0/NTU_Taida_Keratinase_model_3.jpg" style="width:360px;height:300px" ></p><div style="float:right"></p>
-	To secrete the enzyme outside the bacteria, a gene which functions as a signal sequence is inserted before the keratinase coding region. The signal peptide can guide the enzyme to the cell membrane. To further details, please check out the description above.</div></h5>
+	To secrete the enzyme outside the bacteria, a gene which functions as a signal sequence is inserted before the keratinase coding region. The signal peptide can guide the enzyme to the cell membrane. To further details, please check out the description in Skin Whitening --- PKEK page.</div></h5>
 <div class="container" style="width:1200px;line-height:4em;padding:200px;padding-top:10px;padding-right:100px;padding-bottom:10px;" float="left" id="test">
    <li class="boxG"style="line-height:4em" >

Hour*	0	0.5	1	1.5	2	2.5
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*	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

% of oleic acid	plasmid (-)	plasmid(+)
1%	2.542	2.661
2%	2.390	2.623
3%	2.523	2.357
4%	2.610	2.616
5%	2.351	2.653