Difference between revisions of "Divide and Conquer Biological Challenges"
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| − | <center>[[Image:iGEM2008_design.jpg]]</center> | + | <center>[[Image:iGEM2008_design.jpg]]</center><br> |
| + | [http://partsregistry.org/AHL '''List of auto-inducers in the Registry, with names and Sigma part numbers.'''] | ||
| + | <br> | ||
| + | === * We are interested in building the [http://partsregistry.org/Part:BBa_K091100 pLac_lux promoter]. === | ||
| + | [[Image:lac_lux.jpg]][[Image:PLac Lux logic.jpg]] <br>[[Image:K091100.jpg]]<br> | ||
| + | '''If LuxR and LacI<sup>Q</sup> are always on.....when you have IPTG AND NOT AHL'''<br> | ||
| + | [[Image:pLac_lux_promoter.jpg]]<br> | ||
| + | [[Image:lac_lux_pcr.jpg]]<br> | ||
| + | Temperatures and cycles: <br> | ||
| + | 95 degrees for 5 min. <br> | ||
| + | 95 degrees for 30 sec. <br> | ||
| + | 29 degrees for 30 sec <br> | ||
| + | 72 degrees for 5 sec. <br> | ||
| + | Go back to step 2 29x <br> | ||
| + | <br> | ||
| + | 100 ng of DNA was in the PCR (Is this supposed to say 10 ug? -TE) | ||
=== * We need to get the Lux/AHL system working. === | === * We need to get the Lux/AHL system working. === | ||
| Line 5: | Line 20: | ||
* [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0062 LuxR]<br> | * [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0062 LuxR]<br> | ||
* [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0062 lux pR] <br> | * [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0062 lux pR] <br> | ||
| + | * [http://partsregistry.org/Part:BBa_I751502 hybrid plac-lux promoter] <br> | ||
| + | [[Image:LuxR_pLac.jpg]] | ||
| − | === * We need to get the Lsr/AI-2 system working. === | + | === * We might need to get the Lsr/AI-2 system working. === |
'''Our summary of the Lsr system:''' [[Davidson/Missouri_Western_iGEM2008#Lsr_.28AI-2.29_cell_signaling_system]]<br> | '''Our summary of the Lsr system:''' [[Davidson/Missouri_Western_iGEM2008#Lsr_.28AI-2.29_cell_signaling_system]]<br> | ||
* There are '''NO''' Lsr parts in the registry: <br> | * There are '''NO''' Lsr parts in the registry: <br> | ||
| + | *We will need to find information on the Lsr system:<br> | ||
| + | #We need to find where the LsrA and LsrR promoters are located. We know that they must be located in the region between the LsrR and LsrA genes (1599514..1601049 E. coli K-12 Gene) but we cannot find any papers that describe specific sequences for these promoters. The sequences are unavailable through the MG1655 sequencing center because the site does not keep track of promotor sequences. The cooridantes for the promoters are also unavailable for this strain. | ||
| + | #We know that CAP binds to the LsrA promoter. | ||
*We will need to make BB parts for:<br> | *We will need to make BB parts for:<br> | ||
# LsrR and LsrK which are adjacent to each other in the E. coli genome. [http://www.ncbi.nlm.nih.gov/projects/sviewer/?id=NC_010473.1&v=1686000..1689200 We could amplify them together with a total size of over 2600bp.] | # LsrR and LsrK which are adjacent to each other in the E. coli genome. [http://www.ncbi.nlm.nih.gov/projects/sviewer/?id=NC_010473.1&v=1686000..1689200 We could amplify them together with a total size of over 2600bp.] | ||
| − | # Lsr promoter | + | # Lsr promoter |
# LuxS that produces DPD that somehow is converted to ''R''-THMF. | # LuxS that produces DPD that somehow is converted to ''R''-THMF. | ||
# How do we get cells to make AI-2? | # How do we get cells to make AI-2? | ||
| Line 19: | Line 39: | ||
=== * We need to have an XOR logic gate produced. === | === * We need to have an XOR logic gate produced. === | ||
'''Existing Logic Gates''' | '''Existing Logic Gates''' | ||
| − | * | + | *'''AND''': <br> |
| − | + | [http://www.bio.davidson.edu/Courses/Synthetic/papers/ANDgate.pdf '''Existing But Complex AND'''] | |
| − | + | [[Image:AND_Gate.png]]<br> | |
| − | [[ | + | <br> |
| − | *NAND:<br> | + | '''[http://partsregistry.org/Part:BBa_K091101 Simple But Hypothetical AND]''' |
| + | [[Image:tet_lac.jpg]] | ||
| + | [[Image:AND_Table.jpg]]<br> | ||
| + | We would need to have: <br> | ||
| + | # [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0040 Starts with pTetR] | ||
| + | # over production of [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 LacI<sup>Q</sup> repressor] but note this link is plain LacI | ||
| + | # over production of [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0040 TetR repressor]<br> | ||
| + | # '''Therefore, we will have to build 1) modified promoter and 2) LacI<sup>Q</sup>''' | ||
| + | <br> | ||
| + | *'''NAND''':<br> | ||
# [http://partsregistry.org/Part:BBa_I732914 I732914] | # [http://partsregistry.org/Part:BBa_I732914 I732914] | ||
| − | *NOR:<br> | + | <br> |
| + | *'''OR''':<br> | ||
| + | '''Simple but Hypothetical OR''' | ||
| + | [[Image:Simple_OR_gate.jpg]][[Image:OR_Logic.jpg]] | ||
| + | *'''XOR''':<br> | ||
| + | [http://parts.mit.edu/wiki/index.php/ETH2006_xor '''See ETH's Designed but not build XOR gate.'''] [http://ieeexplore.ieee.org/iel5/4290605/4290606/04290607.pdf?tp=&isnumber=4290606&arnumber=4290607 '''They also published this here''']<br> | ||
| + | [http://www.pnas.org/cgi/reprint/101/8/2299 '''Also, there is a tunable quarum sensor here.''']<br> | ||
| + | '''Simple but Hypothetical XOR''' | ||
| + | [[Image:xor.jpg]][[Image:XOR_Logic.jpg]] | ||
| + | *'''NOR''':<br> | ||
# [http://partsregistry.org/Part:BBa_I732916 I732916]<br> | # [http://partsregistry.org/Part:BBa_I732916 I732916]<br> | ||
| − | # [http://partsregistry.org/Part:BBa_I732917 I732917] | + | # [http://partsregistry.org/Part:BBa_I732917 I732917]<br> |
| − | *Inverters:<br> | + | <br> |
| + | *'''NOT (Inverters)''':<br> | ||
| + | # [http://partsregistry.org/Part:BBa_I732205 I732205 (NOT/ Dual-repressed NOR)] | ||
''Self-Contained Inverters'' | ''Self-Contained Inverters'' | ||
# [http://partsregistry.org/Part:BBa_J23040 J23040 (AHL-dependent inverter)]<br> | # [http://partsregistry.org/Part:BBa_J23040 J23040 (AHL-dependent inverter)]<br> | ||
| Line 36: | Line 76: | ||
=== * We need to figure out how to get cells to communicate in a sequence and not stop growing too soon. === | === * We need to figure out how to get cells to communicate in a sequence and not stop growing too soon. === | ||
| − | * What if Amp<sup>R</sup> is secreted and cells are not Amp<sup>R</sup>? This would prevent cells down the chain from responding too soon. | + | * What if [http://partsregistry.org/Part:BBa_P1002 Amp<sup>R</sup>] is secreted and cells are not Amp<sup>R</sup>?<br> |
| + | This would prevent cells down the chain from responding too soon. Erin and Pallavi have conducted experiments with this for both distance and timing. | ||
Latest revision as of 14:11, 18 June 2008
List of auto-inducers in the Registry, with names and Sigma part numbers.
Contents
- 1 * We are interested in building the pLac_lux promoter.
- 2 * We need to get the Lux/AHL system working.
- 3 * We might need to get the Lsr/AI-2 system working.
- 4 * We do NOT need to use additional chemical input signals (e.g., aTc and IPTG). We will use only AHL and AI-2 added exogenously.
- 5 * We need to have an XOR logic gate produced.
- 6 * We need to figure out how to get cells to communicate in a sequence and not stop growing too soon.
* We are interested in building the pLac_lux promoter.
If LuxR and LacIQ are always on.....when you have IPTG AND NOT AHL
Temperatures and cycles:
95 degrees for 5 min.
95 degrees for 30 sec.
29 degrees for 30 sec
72 degrees for 5 sec.
Go back to step 2 29x
100 ng of DNA was in the PCR (Is this supposed to say 10 ug? -TE)
* We need to get the Lux/AHL system working.
* We might need to get the Lsr/AI-2 system working.
Our summary of the Lsr system: Davidson/Missouri_Western_iGEM2008#Lsr_.28AI-2.29_cell_signaling_system
- There are NO Lsr parts in the registry:
- We will need to find information on the Lsr system:
- We need to find where the LsrA and LsrR promoters are located. We know that they must be located in the region between the LsrR and LsrA genes (1599514..1601049 E. coli K-12 Gene) but we cannot find any papers that describe specific sequences for these promoters. The sequences are unavailable through the MG1655 sequencing center because the site does not keep track of promotor sequences. The cooridantes for the promoters are also unavailable for this strain.
- We know that CAP binds to the LsrA promoter.
- We will need to make BB parts for:
- LsrR and LsrK which are adjacent to each other in the E. coli genome. We could amplify them together with a total size of over 2600bp.
- Lsr promoter
- LuxS that produces DPD that somehow is converted to R-THMF.
- How do we get cells to make AI-2?
* We do NOT need to use additional chemical input signals (e.g., aTc and IPTG). We will use only AHL and AI-2 added exogenously.
* We need to have an XOR logic gate produced.
Existing Logic Gates
- AND:
Existing But Complex AND
Simple But Hypothetical AND
We would need to have:
- Starts with pTetR
- over production of LacIQ repressor but note this link is plain LacI
- over production of TetR repressor
- Therefore, we will have to build 1) modified promoter and 2) LacIQ
- NAND:
- OR:
Simple but Hypothetical OR
- XOR:
See ETH's Designed but not build XOR gate. They also published this here
Also, there is a tunable quarum sensor here.
Simple but Hypothetical XOR
- NOR:
- NOT (Inverters):
Self-Contained Inverters
PoPS Output Inverters
* We need to figure out how to get cells to communicate in a sequence and not stop growing too soon.
- What if AmpR is secreted and cells are not AmpR?
This would prevent cells down the chain from responding too soon. Erin and Pallavi have conducted experiments with this for both distance and timing.
