Team:SUSTC-Shenzhen/Parts

From 2014.igem.org

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=='''Our RFC'''==
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=='''Our New Standard'''==
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We made a new standard and we built three parts based on that this year.[http://parts.igem.org/Part:BBa_K1431201 BBa_K1431201],[http://parts.igem.org/Part:BBa_K1431301 BBa_K1431301],[http://parts.igem.org/Part:BBa_K1431302 BBa_K1431302]
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We made a new standard and we built three parts based on that this year. ([http://parts.igem.org/Part:BBa_K1431201 BBa_K1431201],[http://parts.igem.org/Part:BBa_K1431301 BBa_K1431301],[http://parts.igem.org/Part:BBa_K1431302 BBa_K1431302])
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The BbsI restriction enzyme recognize the sequence 5’ GAAGAC3’ and cut downstream two bases to six bases. If we make two reverse directions based on BbsI site, we can cut a part and insert another one with seamless. (Mechanism shown below)
+
The BbsI restriction enzyme recognize the sequence <span style="color: red"> GAAGAC <span style="color: black"> and cut downstream two bases to six bases. If we make two reverse directions based on BbsI site, we can cut a part and insert another one with seamless. (Mechanism shown below)
<center>{{SUSTC-Image|wiki/images/a/ab/Our_RFC.png}}</center>
<center>{{SUSTC-Image|wiki/images/a/ab/Our_RFC.png}}</center>
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<center>'''Fig.1  The Machanism of Our RFC'''</center>
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<center>'''Fig.1  The Machanism of Our Standard'''</center>
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Let us make the above sequence as an example. If you want to insert a coding sequence between promoter and polyA, the prefix of forward primer you design must contain GAAGACNNtaaaNNNN and reverse must contain GAAGACNNagttNNNN. The middle two Ns would be any bases for them will be cut off and the four Ns should be begins or ends of the coding sequence. You can get parts by above primers and digest by BbsI restriction enzyme. Then you can insert the parts into promoter and polyA seamless. The PCR product must be such sequence, including the protect bases, shows below.
+
Let us make the above sequence as an example. If you want to insert a coding sequence between promoter and polyA, the prefix of forward primer you design must contain '''GAAGACNN<span style="color: red">taaa<span style="color: black">NNNN''' and reverse must contain '''GAAGACNN<span style="color: red">agtt<span style="color: black">NNNN'''. The middle two Ns would be any bases for them will be cut off and the four Ns should be begins or ends of the coding sequence. You can get parts by above primers and digest by BbsI restriction enzyme. Then you can insert the parts into promoter and polyA seamless. The PCR product must be such sequence, including the protect bases, shows below.
<center>{{SUSTC-Image|wiki/images/1/1b/PCR_product_by_new_RFC.png}}</center>
<center>{{SUSTC-Image|wiki/images/1/1b/PCR_product_by_new_RFC.png}}</center>
<center>'''Fig.2  PCR Product Sequence'''</center>
<center>'''Fig.2  PCR Product Sequence'''</center>
 +
 +
The purpose to make such design because we do not know how many or what kind of bases interval between different promoters and polyAs will make the highest transcription efficiency. Just like the data of ''[http://en.wikipedia.org/wiki/Kozak_consensus_sequence Kozak consensus sequence]'', we can measure it by changing the base numbers or kinds and finally find it.
=='''Favorite Parts'''==
=='''Favorite Parts'''==
 +
==='''Design'''===
 +
{{SUSTC-Image|wiki/images/7/77/TRE_3G_pro%2BpA.png}}
 +
 +
TRE-3G promoter + SV40 PolyA([http://parts.igem.org/Part:BBa_K1431301 BBa_K1431301]) would be the best parts what we designed this year and also be our favorite parts. The combination of TRE-3G promoter and SV40 PolyA make people insert coding sequence directly without subsection or 3A assembly. The application of our new standard, BbsI site, make a seamless gether which coule test the highest efficiency.
 +
 +
TRE-3G promoter(P<sub>TRE3G</sub>) will be switched on after combine with the compound of Tet-On 3G([http://parts.igem.org/Part:BBa_K1431101 BBa_K1431101]) protein and doxycycline(Dox) mix.
 +
 +
<center>https://static.igem.org/mediawiki/parts/3/37/SUSTC-Shenzhen_Tet_On_3G.png</center>
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<center>Figure 1.The Tet-On 3G systems allow inducible gene expression only in the presence of doxycycline. When Dox binds, the transactivator undergoes a conformational change allowing it to bind tet operator (tetO) repeats within the <br>TREG Promoter. The transactivator activates expression through transcription activation domain repeats.</center>
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 +
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==='''Data'''===
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 +
The plasmid transfected into Hela Cell shows below.
 +
 +
<center>{{SUSTC-Image|wiki/images/9/9b/Plasmid_transfected.jpg}}</center>
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<center>'''pBX-093 PB5-HS4-SV40-puro-2A-tetON3G-pA-HS4-TRE-AzaminGreen-2A-T'''</center>
 +
 +
 +
Transfect Hela Cell  with different doxycycline(Dox) concentration by lipofectamine3000 and get the data after 30hours.
 +
 +
===='''fluorescence microscope'''====
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 +
{| class="table"
 +
!{{SUSTC-Image|wiki/images/7/7f/30h_0_BF.jpg}}
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!{{SUSTC-Image|wiki/images/5/52/30h_0_GF.jpg}}
 +
|-
 +
|<center>0 ng/mL doxycycline after 30h</center>
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|<center>0 ng/mL doxycycline after 30h</center>
 +
|-
 +
|-
 +
!{{SUSTC-Image|wiki/images/9/98/30h_100_BF.jpg}}
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!{{SUSTC-Image|wiki/images/2/2d/30h_100_gf.jpg}}
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|-
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|<center>100 ng/mL doxycycline after 30h</center>
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|<center>100 ng/mL doxycycline after 30h</center>
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|}
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===='''Flow Cytometry '''====
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{| class="table"
 +
!{{SUSTC-Image|wiki/images/5/59/Transfected_cells_induced_in_0_ngmL_doxycycline.png}}
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!{{SUSTC-Image|wiki/images/e/ec/Transfected_cells_induced_in_0.1_ngmL_doxycycline.png}}
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!{{SUSTC-Image|wiki/images/c/c8/Transfected_cells_induced_in_1.0_ngmL_doxycycline.png}}
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|-
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| <center>0 ng/mL doxycycline</center>
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| <center>0.1 ng/mL doxycycline</center>
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| <center>1.0_ng/mL_doxycycline</center>
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|-
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|-
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!{{SUSTC-Image|wiki/images/1/1b/Transfected_cells_induced_in_10_ngmL_doxycycline.png}}
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!{{SUSTC-Image|wiki/images/8/8f/Transfected_cells_induced_in_100_ngmL_doxycycline.png}}
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!{{SUSTC-Image|wiki/images/9/95/Transfected_cells_induced_in_1000_ngmL_doxycycline.png}}
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|-
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| <center>10_ng/mL_doxycycline</center>
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| <center>100_ng/mL_doxycycline</center>
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| <center>1000_ng/mL_doxycycline</center>
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|}
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We could find that without Dox, TRE-3G promoter hardly be activated because the fluorescent protein do not expressed. The higher concentration of Dox,the higher number of fluorescent protein expressed. But if the concentration reached 1000 ng/ml, the expression become low, that may be toxic to Hela cell for too high concentration.
 +
=='''Chromoprotein Collection'''==
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{|class="table"
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![http://parts.igem.org/Part:BBa_K1431812 BBa_K1431812]
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|{{SUSTC-Image|wiki/images/2/29/1431812.jpg}}
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|-
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![http://parts.igem.org/Part:BBa_K1431813 BBa_K1431813]
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|{{SUSTC-Image|wiki/images/0/01/1431813.jpg}}
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|-
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![http://parts.igem.org/Part:BBa_K1431814 BBa_K1431814]
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|{{SUSTC-Image|wiki/images/8/88/1431814.jpg}}
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|-
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![http://parts.igem.org/Part:BBa_K1431824 BBa_K1431824]
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|{{SUSTC-Image|wiki/images/6/69/1431824.jpg}}
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|-
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![http://parts.igem.org/Part:BBa_K1431832 BBa_K1431832]
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|{{SUSTC-Image|wiki/images/0/0c/1431832.jpg}}
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|-
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![http://parts.igem.org/Part:BBa_K1431834 BBa_K1431834]
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|{{SUSTC-Image|wiki/images/0/04/1431834.jpg}}
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|}
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'''J23100 in 2014 Distribution Kit is probably mutated as shown by sequencing.'''
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<groupparts>iGEM014 SUSTC-Shenzhen</groupparts>
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=='''Parts Table'''==
 +
<center><groupparts>iGEM2014 SUSTC-Shenzhen</groupparts></center>
{{SUSTC-Shenzhen/main-content-end}}
{{SUSTC-Shenzhen/main-content-end}}
{{SUSTC-Shenzhen/wiki-footer}}
{{SUSTC-Shenzhen/wiki-footer}}
{{SUSTC-Shenzhen/themeJs}}
{{SUSTC-Shenzhen/themeJs}}

Latest revision as of 03:53, 18 October 2014

Team SUSTC-Shenzhen

Parts

Our devotion to the community, in reality.

Contents


Our New Standard

We made a new standard and we built three parts based on that this year. ([http://parts.igem.org/Part:BBa_K1431201 BBa_K1431201],[http://parts.igem.org/Part:BBa_K1431301 BBa_K1431301],[http://parts.igem.org/Part:BBa_K1431302 BBa_K1431302])

The BbsI restriction enzyme recognize the sequence GAAGAC and cut downstream two bases to six bases. If we make two reverse directions based on BbsI site, we can cut a part and insert another one with seamless. (Mechanism shown below)

{{{2}}}
Fig.1 The Machanism of Our Standard


Let us make the above sequence as an example. If you want to insert a coding sequence between promoter and polyA, the prefix of forward primer you design must contain GAAGACNNtaaaNNNN and reverse must contain GAAGACNNagttNNNN. The middle two Ns would be any bases for them will be cut off and the four Ns should be begins or ends of the coding sequence. You can get parts by above primers and digest by BbsI restriction enzyme. Then you can insert the parts into promoter and polyA seamless. The PCR product must be such sequence, including the protect bases, shows below.

{{{2}}}
Fig.2 PCR Product Sequence

The purpose to make such design because we do not know how many or what kind of bases interval between different promoters and polyAs will make the highest transcription efficiency. Just like the data of [http://en.wikipedia.org/wiki/Kozak_consensus_sequence Kozak consensus sequence], we can measure it by changing the base numbers or kinds and finally find it.

Favorite Parts

Design

{{{2}}}

TRE-3G promoter + SV40 PolyA([http://parts.igem.org/Part:BBa_K1431301 BBa_K1431301]) would be the best parts what we designed this year and also be our favorite parts. The combination of TRE-3G promoter and SV40 PolyA make people insert coding sequence directly without subsection or 3A assembly. The application of our new standard, BbsI site, make a seamless gether which coule test the highest efficiency.

TRE-3G promoter(PTRE3G) will be switched on after combine with the compound of Tet-On 3G([http://parts.igem.org/Part:BBa_K1431101 BBa_K1431101]) protein and doxycycline(Dox) mix.

SUSTC-Shenzhen_Tet_On_3G.png
Figure 1.The Tet-On 3G systems allow inducible gene expression only in the presence of doxycycline. When Dox binds, the transactivator undergoes a conformational change allowing it to bind tet operator (tetO) repeats within the
TREG Promoter. The transactivator activates expression through transcription activation domain repeats.


Data

The plasmid transfected into Hela Cell shows below.

{{{2}}}
pBX-093 PB5-HS4-SV40-puro-2A-tetON3G-pA-HS4-TRE-AzaminGreen-2A-T


Transfect Hela Cell with different doxycycline(Dox) concentration by lipofectamine3000 and get the data after 30hours.

fluorescence microscope

{{{2}}} {{{2}}}
0 ng/mL doxycycline after 30h
0 ng/mL doxycycline after 30h
{{{2}}} {{{2}}}
100 ng/mL doxycycline after 30h
100 ng/mL doxycycline after 30h


Flow Cytometry

{{{2}}} {{{2}}} {{{2}}}
0 ng/mL doxycycline
0.1 ng/mL doxycycline
1.0_ng/mL_doxycycline
{{{2}}} {{{2}}} {{{2}}}
10_ng/mL_doxycycline
100_ng/mL_doxycycline
1000_ng/mL_doxycycline

We could find that without Dox, TRE-3G promoter hardly be activated because the fluorescent protein do not expressed. The higher concentration of Dox,the higher number of fluorescent protein expressed. But if the concentration reached 1000 ng/ml, the expression become low, that may be toxic to Hela cell for too high concentration.

Chromoprotein Collection

[http://parts.igem.org/Part:BBa_K1431812 BBa_K1431812] {{{2}}}
[http://parts.igem.org/Part:BBa_K1431813 BBa_K1431813] {{{2}}}
[http://parts.igem.org/Part:BBa_K1431814 BBa_K1431814] {{{2}}}
[http://parts.igem.org/Part:BBa_K1431824 BBa_K1431824] {{{2}}}
[http://parts.igem.org/Part:BBa_K1431832 BBa_K1431832] {{{2}}}
[http://parts.igem.org/Part:BBa_K1431834 BBa_K1431834] {{{2}}}

J23100 in 2014 Distribution Kit is probably mutated as shown by sequencing.

Parts Table

<groupparts>iGEM2014 SUSTC-Shenzhen</groupparts>

Maintained by the iGEM team SUSTC-Shenzhen.

Licensed under CC BY 4.0.

  • ©2014 SUSTC-Shenzhen

Retrieved from "http://2014.igem.org/Team:SUSTC-Shenzhen/Parts"