Difference between revisions of "Part:BBa K1582001"
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There are two classes of Janus, which are divided by the stability of their self-assembly. sJanus from Trichoderma reesei belongs to Class II. The assemblies of class II Janus can be dissolved in ethanol or sodium dodecyl sulfate or through the application of pressure or lowering of the temperature.
 
There are two classes of Janus, which are divided by the stability of their self-assembly. sJanus from Trichoderma reesei belongs to Class II. The assemblies of class II Janus can be dissolved in ethanol or sodium dodecyl sulfate or through the application of pressure or lowering of the temperature.
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===Protein Expression===
 +
The bacteria ( E.coli BL21) were cultured in 5mL LB liquid in 37℃ for 6-8 hours. The antibiotic we used in inJanus-m and sJanus-m is kanamycin, and for sJanus is ampicillin. Then we used 500μM IPTG to induce in 37℃ for 4h.
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<p style="text-align: center;">
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    https://static.igem.org/mediawiki/2015/2/23/Tianjin_qing1.png<br>
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'''Figure 1.''' Pre-expression. We successfully expressed sJanus, sJanus-m and inJanus-m.
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</p>
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Expression condition:<br>
 +
 +
1. Mix the supernatant after centrifugation with the GST medium and incubate them in the shaker for 1~2h.<br>
 +
2. Add the intermixture to the column. Collect the flow-through and add it to the column again. (You can repeat the procedure for 2~3 times if necessary.)<br>
 +
3. Wash the column by 20ml 1xPBS for three times (more if necessary).<br>
 +
4. Add Prescission Protease to the column (the mass ratio of PP and your protein is 1:10). Then incubate them in the shaker overnight.<br>
 +
5. Collect the flow-through which is your sample. Purify your product by using UPLC.<br>
 +
6. Wash the column by GSH and recover the column. <br>

Revision as of 16:42, 23 September 2015

sJanus from Trichoderma reesei


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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 187
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 196


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. sJanus from Trichoderma reesei belongs to Class II. The assemblies of class II Janus can be dissolved in ethanol or sodium dodecyl sulfate or through the application of pressure or lowering of the temperature.

Protein Expression

The bacteria ( E.coli BL21) were cultured in 5mL LB liquid in 37℃ for 6-8 hours. The antibiotic we used in inJanus-m and sJanus-m is kanamycin, and for sJanus is ampicillin. Then we used 500μM IPTG to induce in 37℃ for 4h.

Tianjin_qing1.png
Figure 1. Pre-expression. We successfully expressed sJanus, sJanus-m and inJanus-m.

Expression condition:

1. Mix the supernatant after centrifugation with the GST medium and incubate them in the shaker for 1~2h.
2. Add the intermixture to the column. Collect the flow-through and add it to the column again. (You can repeat the procedure for 2~3 times if necessary.)
3. Wash the column by 20ml 1xPBS for three times (more if necessary).
4. Add Prescission Protease to the column (the mass ratio of PP and your protein is 1:10). Then incubate them in the shaker overnight.
5. Collect the flow-through which is your sample. Purify your product by using UPLC.
6. Wash the column by GSH and recover the column.