Part:BBa_K914007

Meganuclease I-SceI controlled by pBad

I-SceI homing endonuclease expression is controlled by pBad promoter. Expression is induced with IPTG if LacI is present in the cell. I-SceI doesn't have an LVA degradation tag. Read about this biobrick part [http://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme in the context of the project].

Characterization

We performed a set of experiments to show that I-SceI meganuclease is expressed and active in eliminating restriction-site harbouring plasmid.

Experimental setup

Firstly, we transformed two plasmids with different antibiotic resistances into NEB Turbo E.Coli strain:

1. First plasmid: Version of the low copy plasmid with encoded generator to express I-SceI meganucllease regulated by pBad.
   • Backbone: pSB3C5
   • Resistance: Chloramphenicol
   • Origin of Replication: p15a
2. Second plasmid: High copy plasmid with encoded I-SceI restriction site, K175027. This biobrick was a kind gift of the [http://2012.igem.org/Team:TU-Delft TUDelft iGEM team] which we further characterized.
   • Backbone: pSB1AK3
   • Resistance: Ampicillin and Kanamycin
   • Origin of Replication: modified pMB1 derived from pUC19

We expected to perform transformation with both plasmids, and plate with two antibiotics in order to select for double transformants. We would then induce I-SceI expression in those clones to measure its efficiency.

Selection: Cm

Selection: Amp

Selection: Cm & Amp

We suggested two hypotheses to explain the results:

1. Those two plasmids are not compatible. Plasmids could have different origins of replication. That might be the reason why double transformation is unsuccessful.
2. Our system works. Our system perfectly works, but there is some leakage in the promoter leading to the expression of I-SceI meganuclease. In such case, it very efficiently cuts I-SceI restriction site, digesting the second plasmid with ampicillin resistance.

First, we decided to check the hypothesis 1, and verify if two plasmids are compatible with each other.

Control for plasmid compatibility

As control experiment, we decided to trasform two plasmids into NEB Turbo E.Coli strain. The first plasmid in this experiment is analogous to the one from the previous experiment, but with GFP insted of I-SceI meganuclease; the second plasmid is the same as in the previous experiment:

1. First plasmid: Analogous plasmid with encoded generator to express GFP regulated by pLac (low copy version).
   • Backbone: pSB3C5
   • 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

Only the version with pLac promoter was tested, because they all have the same backbone plasmid, and consequently the same replication origin. Read more

We expected to have colonies on both type of plates: firstly, on plates with one antibiotic (Chloramphenicol & Ampicillin), secondly, on plates with both antibiotics. Our expectations became true, and our cells expressed GFP, so we conclude these two plasmids have compatible replication origins, and there should be nothing preventing the I-SceI from being expressed in the previous experiment (See results). That means that our circuits work, but there is some leaky expression of I-SceI meganuclease that leads to a very efficient digestion of the plasmid carrying the Ampicillin antibiotic.

To avoid leakage, in the next experiment we tried to recover cells after transformation and plate it in the presence of glucose that represses the pLac promoter. Results are presented on the next photos:

Selection: Cm

Selection: Amp

Selection: Cm & Amp

Even if we recover the double transformants in the presence of Glucose to tightly repress the expression of the meganuclease, we still have no colonies on the plates containing both Amp and Cm. This is a sign that our construct is leaky, and the expression and function of I-SceI is so efficient that it digests all plasmids containing the corresponding restriction site, and the cells are no longer resistant to Amp.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 125
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 65
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]