Flip One Pancake
From 2006.igem.org
Flipping One Pancake
Plan A: Flipping the Coding Region
Option 1 - Flipping TetR
The plan is to construct a plasmid that contains the following. Recombination would be detected as production of Tet resistant colonies.
Advantages of this plan are that flipping of the CDS for Tet resistance could be selected for and that the DNA to be flipped is similar in size to the region flipped in Salmonella (1 kb, according to Nanassy and Hughes, 1998).
Disadvantages of this plan is that it involves a larger number of assembly steps than plan B (this could be address if certain assembly intermediates could be found) and that reversing the CDS of Tet resistance using Not1 will switch biobrick ends. It may be difficult or impossible to determine the percentage of cells in which flipping has occurred since the use of Tet resistance focuses solely on the survivors and does not lead to a method to count the dead.
- 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, BBa_I13453 Plate DNA-2, Spot 13D
RBS - BBa_B0030 Plate DNA-1, Spot 3G
hixL recombination site
CDS for Chl resistance in reverse direction (see below)
hixR recombination site
double forward terminator, BBa_B0015 Plate DNA-1, Spot 1I
CDS for Chl resistance in forward direction is BBa_P1000, Plate DNA-2, 23H, pSB1AC3
Option 2 - Flipping mRFP
Similar to Option 1, except flipping mRFP instead of Tet resistance.
Advantages of this plan are that it is around 1 kb (as opposed to flipping a small promoter as in Plan B below) and that visual inspection might be possible to determine the percentage of cells in which flipping has occurred.
A disadvantage of this plan is that we would need to construct (probably no in the registry) a backwards mRFP.
Plan B: Flipping the Promoter
Option 1 - Flipping promoter in front of mRFP
Mission Statement: The plan is to construct a plasmid that contains the following. Recombination would be detected as RFP expression.
Advantages of this plan would be that you could visually detect recombination by the color of the colonies, and that an assembly intermediate reduces the number of assembly steps. Following plasmid prep, we can usual visual identification to identify percentages of plasmids with flipped regions at various time scales allowing for a possible understanding of the flipping mechanism through mathematical modelling.
Disadvantage is that you cannot select for flipping.
- 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 BBa_I13453 Plate DNA-2, Spot 13D
hixR recombination site
RBS, CDS for mRFP, double forward terminator, BBa_I13507 Plate DNA-1, Spot 16N
Option 2 - Flipping promoter in front of Tet resistance
Similar to Option 1 except use Tet instead of mRFP. Could select, but can not get percentages.
Option 3 - Flipping promoter in front of Tet resistance and mRFP
Since we are not flipping the CDS here, but rather only the promoter region, we could include both coding regions in our construction and therefore run both visual analysis for mRFP and survivability analysis using the Tet resistance.
Is this a good thing? Comments invited.
Building A House of Pancakes, in the Fewest Steps Possible
We are going to start with Plan B: Flipping a Promoter This has the most parts ready to go.
- We need to know how to put the Hix sites around the promoter and coding regions so we can build things one time only.
- What would it take to create coding regions or promoters that are in both orientations? If we use the NotI method, can we continue to add onto them with the reversed and modified BB ends?
- Using Ara promoter is a good idea so we can separate transcription from flipping.
- We need to use PCR to amplify the Tet, Chloramphenicol, Kanamycin resistance genes.
- We will need these +/- Hix sites. Do we need them with terminator included? Probably to keep things simple. Alternatively, we could leave TT at the end and never flip it.
- Do we need to compare with low copy number plasmid?
Control Experiments For One Pancake Flippin'
- We need to test Lac promoter in front of Hin (with RFP downstream perhaps for easy measuring?)
- We need to test Ara Promoter +/- AraC.
- We need to test Ara Promoter with Hix sites +/- AraC.
- We need to flip a promoter with no stuffer DNA.
- We need to flip a promter with stuffer DNA so it is about 1 kb total length.
- We need to flip coding region of about 1 kb in length downstream of a non-flippable promoter.
- We need to see if flipping stops on its own.
- We need to see if chloroquine can regulate flipping.
- We need to see if we can measure kinetics of flipping (i.e., how fast does it start once we induce Hin?).
Flipping Procedure (draft)
- Grow JM109 in minimal media to exponential growth phase (A600 = 0.5?)
- Induce Hin recombinase with IPTG or lactose (how much, how long?)
- Allow recombination to occur, taking out aliquots every 2 minutes, up to 20 minutes
- Spin down cells in refrigerated centrifuge, lyse cells, do plasmid preps
- Transform fresh JM109 competent cells, plate on minimal with arabinose (how much?), no lactose
- If looking for expression of tet resistance, spread on Amp plates first, then replica plate on Tet plates, look for resistant colonies. If looking for mRFP expression, count white and red 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
- Design PCR primers for 3 drug resistance genes (Not AmpR). Amplify and clone into BioBrick plasmid.
