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Team:XMU-China/Parts1
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| - | + | <li><a href="https://2013.igem.org/Team:XMU-China/Background">Background</a></li> | |
| - | <li><a href="https://2013.igem.org/Team:XMU-China/ | + | <li><a href="https://2013.igem.org/Team:XMU-China/Mechanism">Three Plasmids</a></li> |
| - | <li><a href="https://2013.igem.org/Team:XMU-China/ | + | <li><a href="https://2013.igem.org/Team:XMU-China/Microfluidic">Microfluidic</a></li> |
| - | <li><a href="https://2013.igem.org/Team:XMU-China/ | + | <li><a href="https://2013.igem.org/Team:XMU-China/Exploration">Exploration</a></li> |
| - | <li><a href="https://2013.igem.org/Team:XMU-China/ | + | <li><a href="https://2013.igem.org/Team:XMU-China/Modeling1">Modeling</a></li> |
| - | <li><a href="https://2013.igem.org/Team:XMU-China/ | + | <li><a href="https://2013.igem.org/Team:XMU-China/Applications">Applications</a></li> |
| - | <li><a href="https://2013.igem.org/Team:XMU-China/ | + | <li><a href="https://2013.igem.org/Team:XMU-China/Parts1">Parts & Future</a></li> |
</ul> | </ul> | ||
</div> | </div> | ||
Latest revision as of 14:19, 27 October 2013
Parts
Table 1. The Bio-bricks used in the study
|
Part |
Backbone |
Type |
Location |
Size
(bp) |
Description |
|
|
part |
backbone |
|||||
|
BBa_K546000 |
pSB1C3 |
Signaling |
2013-P1-12D |
1964 |
2070 |
Lux pL
controlled LuxR with lux pR autoinducing LuxI (lva tag)- AHL. |
|
BBa_I763020 |
pSB1C3 |
Intermediate RBS-GFP-TT |
2013-P3-11H |
914 |
2070 |
RBS-GFP
(+LVA Tag)-Terminators |
|
BBa_F2621 |
pSB1A2 |
Signaling |
2013-P2-21F |
1158 |
2079 |
3OC6HSL
Receiver Device |
|
BBa_K546001 |
pSB1C3 |
Device |
2013-P1-12F |
2135 |
2070 |
AIIA AHL Reporter
and Quencher |
|
BBa_J04450 |
pSB4K5 |
Reporter |
2013-P5-5G |
1069 |
3419 |
RFP Coding
Device |
|
BBa_J04450 |
pSB3T5 |
Reporter |
2013-P5-7C |
1069 |
3241 |
RFP Coding Device |
|
BBa_J23022 |
BBa_J23006 |
Composite |
2013-P5-3H |
234 |
2356 |
[key3d][TT] |
| W/N | Name | Type | Description | Designer | Length |
|---|---|---|---|---|---|
| W | BBa_K1036000 | Composite | lux pL controlled luxR with lux pR controlled ndh (LVA-tag) coding for NADH dehydrogenase II | Shengquan Zeng Zhaopeng Cheng | 2687 |
| W | BBa_K1036001 | Coding | the ndh gene coding for respiratory NADH dehydrogenase II | Shengquan Zeng Zhaopeng Cheng | 1366 |
| W | BBa_K1036003 | Composite | lux pL controlled luxR with lux pR controlled gfp (LVA-tag) | Xin GuoXi Xi | 4052 |
| W | BBa_K1036006 | Composite | lux pL controlled luxR with lux pR controlled sfgfp (with LVA-tag) | Zhaopeng ChengXiyu Wu | 4046 |
| W/N | Name | Type | Description | Designer | Length |
|---|---|---|---|---|---|
| W | BBa_K1036002 | Composite | gfp (with LVA-tag) under plux R control | Xin Guo Xi Xi | 2866 |
| W | BBa_K1036004 | Reporter | sfgfp with LVA-tag | Zhaopeng ChengXiyu Wu | 753 |
| W | BBa_K1036005 | Composite | sfgfp (with LVA-tag) under plux R control | Zhaopeng ChengXiyu Wu | 2880 |
Agarose Gel Electrophoresis Confirmation for all Plasmids
pSB1C3-gfp-luxI
This plasmid consists of two parts of target genes: gfp and luxI, and both of them are regulated by quorum sensing promoter. High copy number. (Fig.7-1)
 |
| Fig.7-1 AGE confirmation of plasmid pSB1C3-gfp-luxI construction |
pSB1C3-sfgfp-luxI
This plasmid consists of two parts of target genes: sfgfp and luxI, and both of them are regulated by quorum sensing promoter. sfGFP gene is included into this plasmid as a parallel experiment group of pSB1C3-gfp-luxI. High copy number. (Fig.7-2)
 |
| Fig.7-2 AGE confirmation of plasmid pSB1C3-sfgfp-luxI construction |
p3H-GFP-luxI
This plasmid consists of two parts of target genes: gfp and luxI, and both of them are regulated by quorum sensing promoter. Middle copy number. (Fig.7-3)
 |
| Fig.7-3 AGE of p3H-gfp construction |
p3H-sfGFP-luxI
This plasmid consists of two parts of target genes: sfgfp and luxI, and both of them are regulated by quorum sensing promoter. sfGFP gene is included into this plasmid as a parallel experiment group of pSB1C3-gfp-luxI. Middle copy number. (Fig.7-4)
 |
| Fig.7-4 AGE of p3H-sfgfp construction |
pSB3T5-aiiA
This plasmid has one target gene aiiA under the regulation of quorum sensing promoter. Middle copy number. (Fig. 7-5)
 |
| Fig.7-5 AGE confirmation of plasmid pSB3T5-aiiA construction |
pSB4K5-ndh
This plasmid has one target gene aiiA under the regulation of quorum sensing promoter. Low copy number. (Fig. 7-6)
 |
| Fig.7-6 AGE confirmation of plasmid pSB4K5-ndh construction |
SDS-PAGE Confirmation for Plasmids
Please refer to Improvement (For a Better Glee) in Strains comparisons.
Future Work (Keep Practicing)
Oh, no, time is up for iGEM Competition, our glee has no more time to play, but pack their luggage and head for MIT. But let's see what they have made so far and what they still have to do if they really want to be a success.
Construction Have done:
| No. | Plasmid | ReplicationOrigin | CopyNumber | Resistance | Size(bp) | |
|---|---|---|---|---|---|---|
| Insert | Backbone | |||||
| A1 | pSB1C3-gfp-luxI | pSB1C3 | high(100~300) | Chloromycetin(Cm) | 4052 | 2070) |
| A2 | pSB1C3-sfgfp-luxI | pSB1C3 | high(100~300) | Chloromycetin(Cm) | 4046 | 2070) |
| A3 | p3H-GFP-luxI | p3H | Middle(18~22) | Ampicillin (Amp) | 4052 | / |
| A4 | p3H-sfGFP-luxI | p3H | Middle(18~22) | Ampicillin (Amp) | 4046 | / |
| B | pSB3T5-aiiA | p15A | Middle(10~12) | Tetracycline (Tet) | 2135 | 2837 |
| C | pSB4K5-ndh | pSC101 | Low(5) | Kanamycin (Kan) | 2658 | 3004 |
Need to do:
Microfluidic
Have done:Finished building two different arrays, find basic testing parameters.
To do:Confirm more suitable microfluidic parameters for oscillation.
Strain
Have done:Confirmed DH5a's incompetent in express oscillation;
To do:Make a comparison between BL21 (WT) and BL21 (DE3) in SDS-PAGE and so on.
Copy number
Have done:Have done: Compared the arrangements of high-middle-low copy number and middle-middle-low copy number's effect on oscillation.
To do:Try other arrangements..
Fast Degrading Tag
Have done:Found LVA-tag is not fast enough for our oscillation circuit.
To do:Try LAA or other fast degrading tag for comparison.
