Difference between revisions of "Part:BBa K1582003"
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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. We did some mutations to it, and we call it sJanus-m.
 
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. We did some mutations to it, and we call it sJanus-m.
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===Protein Expression===
 +
Pre-expression:<br>
 +
The bacteria were cultured in 5mL LB liquid medium with kanamycin in 37℃ overnight. After taking samples, we transfer them into 1L LB medium with kanamycin.<br>
 +
 +
Cultured in bottles:<br>
 +
After 4 hours culturing in 37℃ in bottles, we used 500μM IPTG induced in 16℃ for 8-12h. We used McAc 0 and 20 to wash off the bulk proteins, and used McAc 200 to wash off aimed proteins. <br>
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<p style="text-align: center;">
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https://static.igem.org/mediawiki/2015/4/45/Tianjin_result03.png<br>
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'''Figure 1.'''The expression of sJanus-m
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</p>
 +
We finally can use McAc 0 and 20 to wash off the bulk proteins and use McAc 200 to wash off aimed proteins.<br>
 +
The concentration of IPTG, temperature and time we used in sJanus-m’s expression are same to those of inJanus-m, except that we used TB liquid culture medium rather than LB to raise bacteria to get more proteins. <br>

Revision as of 16:52, 23 September 2015

sJanus-m


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. We did some mutations to it, and we call it sJanus-m.

Protein Expression

Pre-expression:
The bacteria were cultured in 5mL LB liquid medium with kanamycin in 37℃ overnight. After taking samples, we transfer them into 1L LB medium with kanamycin.

Cultured in bottles:
After 4 hours culturing in 37℃ in bottles, we used 500μM IPTG induced in 16℃ for 8-12h. We used McAc 0 and 20 to wash off the bulk proteins, and used McAc 200 to wash off aimed proteins.

Tianjin_result03.png
Figure 1.The expression of sJanus-m

We finally can use McAc 0 and 20 to wash off the bulk proteins and use McAc 200 to wash off aimed proteins.
The concentration of IPTG, temperature and time we used in sJanus-m’s expression are same to those of inJanus-m, except that we used TB liquid culture medium rather than LB to raise bacteria to get more proteins.