Favourite Constructs

M.ScaI Methyltransferase [http://partsregistry.org/wiki/index.php?title=Part:BBa_K874000 BBa_K874000]

This part codes for the [http://rebase.neb.com/rebase/enz/M.ScaI.html M.ScaI] methyltransferase protein. M.ScaI is a type II methyltransferase (subtype beta) that recognizes site on the DNA of the following sequence 5..AGTACT..3. It methylates this site at the 5th (Cytosine) nucleotide leaving an N4-methylcytosine (m4). This methylation type (m4) is not found in native E. coli nor is the recognition site methylated by any of E. coli's native methylation systems (Dam, Dcm). Also this specific methylation inhibits restriction by M.ScaI's prototype restriction enzyme ([http://rebase.neb.com/rebase/enz/ScaI.html ScaI]).

Data-mining of the [http://rebase.neb.com/rebase/rebms.html REBASE (m4) methyltransferase database] revealed that M.ScaI was the best candidate based on the following parameters:

• Methylation (m4) done by this M.ScaI inhibits its prototype (ScaI) restriction enzyme ability to restrict the site [1]
• E. coli's native methylation systems do not methylate the recognition site and thus can not interfere with systems using M.ScaI
• E. coli's native restriction systems do not restrict the recognition site (in either methylated and unmethylated form) and thus can not interfere with systems using M.ScaI
• Recognition site has high specificity (1 in 4048 random sequences)
• Its prototype restriction enzyme is commercially available

It can be assumed that M.ScaI is expressed and folds properly in E. coli (Dh5alpha & LacIq) because its function has been verified by the iGEM Amsterdam 2012 team. More detail about this can be found on the BBa_K874100 & BBa_K874101 parts registry pages.
However it might be worth noting that the protein is natively found in [http://rebase.neb.com/rebase/enz/M.ScaI.html Streptomyces caespitosus] which is a bacteria that has an optimal growth temperature of 26C and thus might not be expressed optimally in E. coli.
For more information or design considerations refer to the iGEM Amsterdam 2012 Wiki or the Part Design page of this part.

IPTG inducible expression of M.ScaI methyltransferase
(IPTG -> M.ScaI) [http://partsregistry.org/wiki/index.php?title=Part:BBa_K874100 BBa_K874100]

This BioBrick contains the first proof of concept and contains the both the Reader and the Sensor described in our molecular design. Therefore this is also the most extensively studied BioBrick in our project, for an in dept view of the experiments we performed using this BioBrick you can look at the Experimental Setup section. We managed to insert this BioBrick in both the pSB1AT3 and pSB1C3 backbones but all testing was done in pSB1AT3.

Arabinose inducible expression of M.ScaI methyltransferase
(ARA -> M.ScaI) [http://partsregistry.org/wiki/index.php?title=Part:BBa_K874101 BBa_K874101]

After the assessment of the first BioBrick we deemed it nessesary to change te promotor to the pBAD (Arabinose) promoter. This therefore is still the first proof of concept but contains a different Reader as described in our molecular design. This BioBrick was also extensively studied so for an in dept view of the experiments we performed using this BioBrick you can look at the Experimental Setup section. We managed to insert this BioBrick in both the pSB1AT3 and pSB1C3 backbones but all testing was done in pSB1AT3.