Part:BBa_K2599017

T7 Promoter+RBS+GS linker+αS1-casein

NCTU_Formosa 2018 designed a Biobrick contains αS1-casein [http://2014.igem.org/Team:SF_Bay_Area_DIYbio/Parts#Alpha-s1_casein_.28CSN1S1.29] and a GS linker (BBa_K1974030) ahead as a Curcumin biosensor.

Figure 1. Composite part of αS1-casein

The goal of our system is to regulate the soil microbiota in order to reach the maximum crop productivity. To accurately predict the curcumin content from NPK content in soil, we create a bio-sensor. This sensor can precisely detect the curcumin containment in turmeric. After the detection of curcumin, results can be fitted into our productivity model and utilize artificial intelligent to increase the accuracy. With the cooperation of productivity model and curcumin transformation model, we can perfectly predict the crop productivity and maintain balance soil microbiota.

Introduction

Curcumin

Curcumin is a natural lipid-soluble yellow compound from the plant tumeric. It is a potent antioxidant as well as antitumorigenic and anti- inflammatory molecule. Although curcumin has been proved its therapeutic efficacy against many human ailments, but the problem is it is hard to absorb by human cells. To solve this problem, a paper has discovered a curcumin carrier protein called αS1-casein, shows high binding affinity with curcumin. We then utilize this property of αS1-casein to create a curcumin bio-sensor.

αS1-casein

Casiens are proteins commonly found in mammalian milk and is a mixture of four phosphoprotein. One of the phosphoprotein is αS1-casein, which contains no disulfide bonds and relatively little tertiary structure. As their primary function is nutritional, binding large amounts of calcium, zinc and other biologically important metals, amino acid substitutions or deletions have little impact on function.

The Binding Between Curcumin and αS1-casein

According to the reference, we found how this two bind together.

Curcumin has a β-diketone moiety, flanked by two phenolic groups, that helps bind to proteins through hydrophobic interactions.

The carboxyl-terminal of αS1-casein (100−199 residues) predominantly contains hydrophobic amino acids, which may be involved in the binding process. Residues 14−24 in αS1-casein are hydrophobic in nature and form a surface “patch” of hydrophobicity. Curcumin may probably be binding at these two sites, with two different ranges of affinity through hydrophobic interaction. One with high affinity [(2.01 ± 0.6) × 106 M−1] and the other with low affinity [(6.3 ± 0.4) × 104 M−1].

Establishment of Curcumin Biosensor

Cloning of αS1-casein

WWe got the amino acid sequence of αS1-casein from NCBI, and adjust the DNA sequence to optimize its expression in E. coli. We also added a GS linker ahead to enhance the function of sensor and synthesized the gblock fragment from IDT. First of all of cloning process, we did PCR to acquire the product of GS Linker-αS1 casein DNA fragment. (Fig. 2) Next, we digested the fragment and ligated it to pet30a vector. Finally, we transformed the plasmid with GS Linker-αS1 casein to E. coli. BL21 DE3 and made protein expression.

Figure 1. PCR product of αS1-casein

Chip Production

αS1-casein Modification to Gold Chip

1. Dip the gold chips in 10mM Mua, RT for 4hrs.

2. Wash the chips with 95% EtOH three times and dry.

3. Add EDC+NHS mixture (100+100mM in DDW) on chips, RT for 1hrs.

4. DDW rinse the chips and dry.

5. Add αS1-casein on chips, RT for 1hrs.

6. Wash with PBS three times and dry.

6. Dip the chips in blocking solution, RT for 1.5hrs.

7. Wash with PBS three times and dry.

Detection Method of Curcumin Biosensor

Electrochemistry Introduction

After choosing αS1-casein as our biosensor, we should choose a method to detect curcumin. We choose the Differential Pulse Voltammetry (DPV) method.

Differential Pulse Voltammetry

DPV uses the difference between before and after the pulse application in order to solve the influence of background noise. This principle is difference from EIS. We hope we can find out which method is more sensitive to curcumin, and create more accurate formula.

Measurement protocol of Curcumin Biosensor

Add the diluted curcumin samples on our biosensor to react for 30min.

2. Rinse with wash buffer and dry the chips.

3. Wash the reference and counter electrodes with DDW, and dry them.

4. Set up the three electrodes system within electrochemical cell. (Fig. 3, left)

5. Use the prototype of electrochemical machine to measure the DPV method. (Fig.3, right)

Data Analysis

1. Electrochemical Impedance Spectroscopy (EIS)

First of all, we used EIS to check whether our biosensor can detect curcumin. As we mentioned above, EIS method tested the impedance change when charges transfered. Therefore, we compared the two samples, the red line was connected to general chip, and the blue line was connected with αS1-casein (Fig. 4). As long as our biosensor connected to curcumin , its impedance value would become larger. We can easily observed that our biosensor with αS1-casein produced more fierce Redox reaction than another. Figure 4 also represented that the biosensor connected with αS1-casein have more effect of detecting curcumin than none.

Figure 4. The sensitivity test of curcumin biosensor in EIS

2. Differential Pulse Voltammetry (DPV)

After proving that adding αS1-casein can detect curcumin, we hoped to use more accurate method to show our data. We chose DPV method to replicate the experiment above, and determined which method was more sensitive. Comparing with Fig 4 and Fig 5, we found that chips with αS1-caseinis showed almost no changes in EIS method, but changed a lot in DPV method. In this way, we can explain DPV method was more sensitive for detection and showed better signal than EIS.

Figure 5.The sensitivity test of curcumin biosensor in Dpv. (Reduction)

3. Improvement (DPV)

After knowing DPV is more sensitive, we want to know in which condition is more suitable to detect curcumin. We tried two buffers, PB and PBS, as curcumin solvent. In Fig 6, blue line was detection in PB buffer, and red line was in PBS buffer. In the result, LOD (limit of detection) of curcumin in PB buffer was 10pM and in PBS buffer was 1nM. This meaned that the sensitivity by using PB buffer was one hundred times more than using PBS buffer. We also confirmed that NaCl would increase background noise in our method. In the conclusion, we would chose the PB buffer as our solvent rather than the PBS buffer.

Figure 6.The improvement test of curcumin biosensor in Dpv. (Reduction)

Application

1. Determine Standard Curve from Detection of Curcumin in PB Buffer

We knew that connecting our biosensor with αS1-casein and detecting curcumin in PB buffer is the best condition to determine curcumin concentration by DPV. We used the data in this condition to fit the standard curve. Since it was the logarithmic function, we put the curcumin concentration into the natural logarithm, and did the polynomial curve fitting. We obtained the result in Figure 7, R² =0.9995. This represented the prediction from the following formula was really close to real value.

<p style="padding-top: 10px; font-size: 10px; text-align: center;"X = Curcumin Concentration; Y = DPV Peak Current</p>

Figure 7.The relationship between current and curcumin concentration.

2. The Detection Result of Real Samples from Turmeric

Our final goal is to predict the concentration of curcumin by the bio-sensor. Therefore, we milled turmeric and divided into two groups to detect. One of them was nonextracted, and the other was extracted, both of them were added with water. The sample which didn’t be extracted was unable to be detected, which is because curcumin uaually existed in cell. Furthermore, this result represented our sensor had strong specificity to curcumin. That is, our sensor would not be disturbed even if taking the whole turmeric to detect.

Figure 8.Detection of real samples from Turmeric

Conclusion

In our conclusion, we use EIS and DVP, two different kinds of electrochemistry method, to prove that we can detect curcumin if we use chips to connect with alphaS1-casein as bio-sensor. We also find the better condition to detect curcumin and create accurate formula to support our bio-sensor. Finally we certify the specificity is perfect. According to these experiments, we successfully create a new curcumin bio-sensor. Because of it, we can detect curcumin in time and feedback to our model.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Reference

1. Gupta, S. C., et al. (2012). "Discovery of curcumin, a component of golden spice, and its miraculous biological activities." Clin Exp Pharmacol Physiol 39(3): 283-299.

2. Le Parc, A., et al. (2010). "α(S1)-casein, which is essential for efficient ER-to-Golgi casein transport, is also present in a tightly membrane-associated form." BMC Cell Biology 11: 65-65.

3. Sneharani, A. H., et al. (2009). "Interaction of αS1-Casein with Curcumin and Its Biological Implications." Journal of Agricultural and Food Chemistry 57(21): 10386-10391.

4. Teresa Treweek (September 12th 2012). Alpha-Casein as a Molecular Chaperone, Milk Protein Walter L. Hurley, IntechOpen, DOI: 10.5772/48348.

5.Palazon, F.; Montenegro Benavides, C.; Léonard, D.; Souteyrand, É.; Chevolot, Y.; Cloarec, J. P. Carbodiimide/NHS derivatization of COOH-terminated SAMs: activation or byproduct formation?. Langmuir, 2014, 30, 4545-4550.