Flip One Pancake
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'''Flipping One Pancake''' | '''Flipping One Pancake''' | ||
| + | |||
| + | '''Plan A''' | ||
The plan is to construct a plasmid that contains the following. Recombination would be detected as production of Tet resistant colonies. | The plan is to construct a plasmid that contains the following. Recombination would be detected as production of Tet resistant colonies. | ||
# Amp resistance gene as in pSB1A3 | # Amp resistance gene as in pSB1A3 | ||
| - | # Hin recombinase gene driven by lacP (inducible in JM109 by IPTG or lactose) | + | # Hin recombinase gene driven by lacP (inducible in JM109 by IPTG or lactose) |
| - | # RE (recombination enhancer) | + | # RE (recombination enhancer) |
# One Pancake test construct: | # One Pancake test construct: | ||
araBAD promoter, inducible with arabinose | araBAD promoter, inducible with arabinose | ||
<br> RBS | <br> RBS | ||
| - | <br> hixL recombination site | + | <br> hixL recombination site |
<br> CDS for Tet resistance in reverse direction | <br> CDS for Tet resistance in reverse direction | ||
| - | <br> hixR recombination site, synthesized by | + | <br> hixR recombination site |
| + | <br> double forward terminator, BBa_B0015 Plate DNA-1, Spot 1I | ||
| + | |||
| + | '''Plan B''' | ||
| + | |||
| + | The plan is to construct a plasmid that contains the following. Recombination would be detected as RFP expression. | ||
| + | # Amp resistance gene as in pSB1A3 | ||
| + | # Hin recombinase gene driven by lacP (inducible in JM109 by IPTG or lactose), cloned from PCR product by Davidson team | ||
| + | # RE (recombination enhancer), synthesized by Davidson team | ||
| + | # One Pancake test construct: | ||
| + | <br> hixL recombination site | ||
| + | <br> araBAD promoter, inducible with arabinose | ||
| + | <br> hixR recombination site | ||
| + | <br> RBS | ||
| + | <br> CDS for mRFP | ||
<br> double forward terminator, BBa_B0015 Plate DNA-1, Spot 1I | <br> double forward terminator, BBa_B0015 Plate DNA-1, Spot 1I | ||
| + | |||
'''Flipping Procedure (draft)''' | '''Flipping Procedure (draft)''' | ||
Revision as of 15:39, 8 June 2006
Flipping One Pancake
Plan A
The plan is to construct a plasmid that contains the following. Recombination would be detected as production of Tet resistant colonies.
- Amp resistance gene as in pSB1A3
- Hin recombinase gene driven by lacP (inducible in JM109 by IPTG or lactose)
- RE (recombination enhancer)
- One Pancake test construct:
araBAD promoter, inducible with arabinose
RBS
hixL recombination site
CDS for Tet resistance in reverse direction
hixR recombination site
double forward terminator, BBa_B0015 Plate DNA-1, Spot 1I
Plan B
The plan is to construct a plasmid that contains the following. Recombination would be detected as RFP expression.
- Amp resistance gene as in pSB1A3
- Hin recombinase gene driven by lacP (inducible in JM109 by IPTG or lactose), cloned from PCR product by Davidson team
- RE (recombination enhancer), synthesized by Davidson team
- One Pancake test construct:
hixL recombination site
araBAD promoter, inducible with arabinose
hixR recombination site
RBS
CDS for mRFP
double forward terminator, BBa_B0015 Plate DNA-1, Spot 1I
Flipping Procedure (draft)
- Grow JM109 in minimal media to exponential (A600 = 0.5?)
- Induce Hin recombinase with IPTG (how much, how long?)
- Allow recombination (how long?)
- Turn off recombination by spinning down, resuspending with no IPTG
- Induce araBAD promoter and Tet resistance with arabinose (how much, how long?)
- Spread on Amp plates first, then replica plate on Tet plates, look for resistant colonies
Flipping To Do List
- Plasmid prep pSB1A3, digest with EcoRI + PstI, Pase, gel purify
- Anneal hixL, hixR, and hixC oligos, then ligate into vector, transform
- Start overnights from colonies for double forward terminator, pBAD, mRFP
- Decide on resistance gene to be flipped, do transformations
