Difference between revisions of "Part:BBa K175041"

Revision as of 16:41, 27 September 2012

p(LacI) controlled I-SceI homing endonuclease generator

p(LacI) controlled I-SceI homing endonuclease generator, inducible with IPTG if LacI is present in the cell. I-SceI has an LVA degradation tag.

Measuring the I-SceI efficiency by [http://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design iGEM 2012 Paris Bettencourt team]

In 2009, [http://2009.igem.org/Team:TUDelft/SDP_Overview TUDelft iGEM Team] has already tried to design a Self Destructive Plasmid based on I-SceI meganuclease. For their experiments, they designed a generator to produce I-SceI meganuclease and used the same pLac promoter, but they had two essential differences with our system (BBa_K914005, BBa_K914007, BBa_K914008):

• They used a strong RBS.
• I-SceI meganuclease they used had an LVA tag.

Moreover, they didn't submit the meganuclease alone as a biobrick, so it couldn't be used for constructing new composite biobricks controlled by other promoters, which would be very useful for the modularity of our system. That is why we started working on our own constructions of meganuclease first. However, we asked TUDelft to send us two plasmids that they designed, so that we can test and characterize them:

1. First plasmid: High copy plasmid with encoded generator to express I-SceI meganucllease, BBa_K175041:
   • Backbone: pSB1C3
   • Resistance: Chloramphenicol
   • Origin of Replication: modified pMB1 derived from pUC19

2. Second plasmid: High copy plasmid with encoded I-SceI restriction site, K175027:
   • Backbone: pSB1AK3
   • Resistance: Ampicillin and Kanamycin
   • Origin of Replication: modified pMB1 derived from pUC19

Step 1

To mesure the efficiency of I-SceI from TUDelft parts, we proceeded in the same way as for the characterization of our own designs: we transformed two plasmids with different antibiotic resistances into NEB Turbo E.Coli strain.

The transformation was successful.

Step 2

We followed the following protocol:

• Pick a colony and start a liquid culture with both antibiotic resistances (Amp and Cm).
• Incubate at 37°C until optical density (OD) reaches 0.5
• Pellet and wash cells to remove Amp and Cm.
• Re-dilute them in the same volume of LB.
• From each of those two tubes, start two liquid cultures:
  1. The first culture with Cm and w/o IPTG.
  2. The second culture with Cm and IPTG.
• Incubate overnight at 37°C

Step 3

Plate colonies from each tube on two different plates:

1. Selection with Cm and Amp.
2. Selection with Cm.

Results

To analyze data, we counted and compared the number of colonies on four plates corresponding to 4 conditions.
First, we compared the number of CFU formed by non-induced cells, plated with a single antibiotic and with both antibiotics. We expected that the plate with only Cm selection would have the biggest number of colonies. A smaller number would be on the plate with two antibiotics (Amp and Cm). It could be explained by the loss of the plasmid carrying Amp resistance.
Next, we compared the number of CFUs formed by the cells where we induced the I-SceI expression, plated with a single antibiotic and with both antibiotics. We expect that there would be much less colonies on the double antibiotic plate, because the Ampicillin carrying plasmid would be lost not only due to the absence of selection during growth (as for the first two cells), but also due to the digestion by the restriction enzyme.

The combination of plasmids:

• First plasmid: pLac & RBS & I-SceI [Cm]
• Second plasmid: I-SceI restriction site [Amp]

Selection: Cm Plated colonies: w/o IPTG	Selection: Cm Plated colonies: IPTG induced
Selection: Cm & Amp Plated colonies: w/o IPTG	Selection: Cm & Amp Plated colonies: IPTG induced

CFU number.

From photos and bar graph above we can see that our expectation came true.

Sequence and Features