Coding

Part:BBa_K1582000

Designed by: Dongqi Bao   Group: iGEM15_Tianjin   (2015-08-07)

inJanus from Grifola frondosa


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 121


Improvements

Group: TJUSLS_China 2016

Author: Zhuozhi Chen

Summary:inJanus-linker-GCW61 is a construction we designed, which aims to change the hydrophobicity of the outer surface of the cell wall. GCW61 is an anchoring protein derived from Pichia GS115. When fused with a certain protein on its C-terminus, such as inJanus, this protein can be displayed on to the cell wall. Thus, inJanus, a class I hydrophobin, can eventually manage to increase the hydrophobicity of the cell wall, having displayed on cell surface, and ultimately help in targeting hydrophobic substrates when the host cell is a whole cell biocatalyst. Another usage of this construct is to protect the host cell from severe or extreme environments, since hydrophobins are considered to be very stable and protective.
We co-displayed PETase-linker-GCW21/51/61 along with inJanus-linker-GCW61, in attempt to make our whole cell biocatalyst more efficient and tolerant. Previous studies on co-displaying hydrophobin with lipases have already shown positive results on the promotion of efficiency. It is confirmed that most lipases are more active when entering a more hydrophobic environment. Since PETase is also classified as lipase, we decided to build this construction and make this attempt.

New parts: BBa_K1921026.

Co-display HPLC Results

ProofTJU19.jpg
Figure 1. The activity of the first group of ppic9-PETase-linker-GCW51 & ppiczaA-inJanus-linker-GCW61 co-display transformants in different hours and amount of yeast.

ProofTJU20.jpg
Figure 2. The activity of the second group of ppic9-PETase-linker-GCW51 & ppiczaA-inJanus-linker-GCW61 co-display transformants in different hours and amount of yeast.

</html> Concluding, we successfully improved this part by constructing and characterizing a new BioBricks based on this part. More information on these parts can be found here: BBa_K1921026.

References

[1] Shosuke Y, Kazumi H, Toshihiko T, Ikuo Taniguchi, Hironao Y, Yasuhito M, Kiyotsuna T, Kenji M, Yoshiharu K, Kohei O. 2016. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science 351, 1196-1199. DOI: 10.1126/science.aad6359.
[2] Pan W, Jie H, Yufei S, Matthew R, Li Z, Shuangyan H, Shuli L, Haixin S, Ying L. 2016. Display of fungal hydrophobin on the Pichia pastoris cell surface and its influence on Candida antarctica lipase B. Appl Microbiol Biotechnol DOI 10.1007/s00253-016-7431-x
[3] Brzozowski A, Derewenda U, Derewenda Z, Dodson G, Lawson D, Turkenburg J, Bjorkling F, Huge-Jensen B, Patkar S, Thim L . 1991. A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex. Nature 351(6326):491–494. doi:10.1038/351491a0

Usage

Janus is a kind of amphipathic protein which could self-assembly spontaneously. Due to its special properties, we could make many new applications. We use them as substrate to fix antibodies on a high-flux tumor detection chip. Meanwhile, they are used to catch cutinases for plastic degradation. We even make them into a fusion to test if the enhancement could be better. And we use its amphipathicity to achieve protein separation, where they act as a special purification tag, and the system could be as simple as polymer, detergent and water.

Biology

Janus could be produced by filamentous fungi, such as Ascomycetes and Basidiomycetes, and their scientific name is hydrophobin. Many different aspects of fungal development have been attributed to Janus. For example, they are thought to play a role in the formation of aerial hyphae and fruiting bodies. One of the most important features of Janus is that they are able to assemble spontaneously into amphipathic monolayers at hydrophobic–hydrophilic interfaces.

There are two classes of Janus, which are divided by the stability of their self-assembly. inJanus from Grifola frondosa belongs to Class I. They could generate very insoluble assemblies, which can only be dissolved in strong acids such as trifluoroacetic acid or formic acid.




Protein Expression

This year, Nankai helped us to express inJanus in pichia pastoris.
To express inJanus in pichia pastoris, Nankai chose fed-batch fementation carried out in bioreactor with 1L initial volume. When the cell fresh weight reached to 200g/L, Nankai started inducement for 96h. What’s more, there is a higher production when dissolved oxygen was maintained at 15%-25% by turning up the methanol speed.
Follow is the figure shows the SDS-PAGE and western blot analysis of inJanus. And the inJanus molecule weight was 8KDa. In addition, inJanus in the broth was mainly monomer and dimer. The latter binds better with the primary antibody of Janus.

Tianjin_co4.png
Figure 1. (a)silver staining along with SDS-PAGE and (b)western blot analysis of inJanus fermentation broth(lane 2,3), lane 1 was pure inJanus as control

To measure the yield of inJanus, Nankai measured the concentration of inJanus by UPLC. And Nankai used standard sample of sJanus of different concentration (0.1, 0.05, 0.025 mg/mL) to get a standard curve of concentration with adsorption area of UPLC.

Tianjin_co3.png
Figure 2.(a) silver staining along with SDS-PAGE of Janus fermentation broth of different induction time(lane 1-4 24,48,72,96 h).(b)inJanus production and cell fresh weight in different time.

Tianjin_co5.png
Figure 3.HPLC of pure inJanus HPLC


Super Protein Chip

Results

1. The optimal concentration of inJanus is 200μg/ml.
2. The optimal concentration of antigens (CEA, AFP and CA15-3) is 2.5μg/ml.
3. The optimal concentration of antibodies is 5μg/ml.

Tianjin_chip1.png
Figure 4.Hydrophobins sJanus show good effects of antibodies fixing. Here we use antibodies with green fluorescence tag as example. The spots with strong fluorescence represent successful binding between capture antibody (probe) and antigen (tumor marker).

Tianjin_chip3.png
Figure 5.Hydrophobins inJanus tend to show weak fluorescence signal. After compared experiments, we discovered that increasing the incubation time helps to improving detecting sensitivity.

Project Achievements
1.We introduced a new method of protein chip substrate modification by using Janus, and verified its feasibility.
2.We confirmed the optimal concentration of antigens and antibodies in the detection of tumor markers, which helps to save materials to the most extent and keep the reliability of the results at the same time.
3.The experiments were conducted with different kinds of antigens and antibodies, ensuring the universality of the tumor detection chip under a certain range.

Optimization

1.In the beginning, due to our inexperience, the concentration of antigens and antibodies was too high, which leads to excessive strong fluorescence signal intensity.
At first we thought it was caused by the improperly operation of microscope, but after literature reviewing and analyzing, we tried lowering the concentration about four times and achieved good results.

Tianjin_chip5.png
Figure 6.Here is the excessive strong fluorescence signal intensity caused by high concentration.

Tianjin_chip7.png
Figure 7.Good results were achieved after lowering the concentration.

2. In mid-stage, many crystallized structures appeared in the protein spot, and hence influenced the image quality and observation results.

To solve this problem, we consulted large quantities of data and information, and discovered a similar case arising when solutions of antibodies and antigens were stored for too long, leading to degeneration. So we reformulated all the solutions we need, and crystallization disappeared. From this we learned that reagents should be used right after they were ready. In this case, another possible cause was that we used TBST in the last washing step, which may also lead to crystallization. In the upper course of experiments we used water instead of TBST.

Tianjin_chip9.png
Figure 8.Crystallized structures appeared.

Tianjin_chip11.png
Figure 9.Crystallized structures disappeared after reformulating all the solutions we need.

Stimulated Plastic Ezymolysis

Pre-experiment

Overview

We have found out appropriate concentration of the cutinase we made and suitable pH for our enzyme Thc-Cut1, the right time to detect the product of hydrolysis of PET.

Result

We use absorbance in 600 nm to measure the turbidity of the liquid in tube. We tried different concentration of enzyme with one piece of plastic, decided to use 2mg/ml to conduct our experiment afterwards.

Tianjin_result39.png
Figure 8. This curve describe the OD600nm after 3h’s reaction changes through the concentration of Thc_Cut1. We can see clearly at 2mg/ml, the curve reaches a peak, at which concentration we will compare the hydrolysis effect.

We also conducted our system in various pH, found that the activity of Thc_Cut1 doesn’t change much in different pH, so we chose pH 7.0 as one of our experimental condition.

Tianjin_result41.png
Figure 9. The curve shows the absorbance at 600nm changes along with pH. The activity of enzyme doesn’t change much in various pH.

In order to find right time to detect, we detect OD600nm in several time, here’s the time line.

Tianjin_result43.png
Figure 10. The timeline clearly indicates the hydrolysis rate rises along with time. Considering we need an appropriate detect time to measure the hydrolysis effect, we set our detect time at 3h.

Main experiment

Overview

We discovered the right temperature to conduct various way to mix up our sJanus (or inJanus) and Thc_Cut1.And we have found out the best way to increase the hydrolysis rate.

Result

We conduct our trail in different temperature ,realizing 50℃ is the proper temp for the enzyme and our Janus to combine.

Tianjin_result45.png
Figure 11 Different color represent different ways of mix. Thc_Cut1+Janus means enzyme had incubated with Janus for 16h before the plastic was added into the system. And Plastic+Janus means plastic had incubated with Janus before adding enzyme. And we also conduct these incubation separately in 4℃,37℃.50℃, values at 50 degree are higher than that under the other temperature.

Based on the experiment above, we compared these two different ways with simple mix, here’s the result.

Tianjin_result47.png
Figure 12. The tag with “*” means simple mixture. We can conclude that pre-incubation does increase the hydrolysis rate, some ways(Plastic+sJanus) even boost 200%+ react rate comparing with Thc_cutL1* in our conditions!! Another interesting phenomenon is sJanus usually works better than inJanus, and the reason behind it need our further exploration. On the whole, Our Janus works a lot!

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