Team:NRP-UEA-Norwich/Project Mo-Flipper


NRP UEA Norwich iGEM 2014

Golden Gate Modular Flipper - 'Mo Flipper'

The little plasmid that could!

Current iGEM standard

As part of the registry standard at iGEM all parts need to be in the same format, much like a computer file. If the files are incompatible (ie, different parts) then they will not work with the rest of the registry. The iGEM registry standard is the BioBrick cloning method; the method utilises 4 type II restriction endonucleases (RENs): EcoRI and XbaI are used in the prefix (the sequence of DNA before the region of interest) and SpaI and PstI are used in the suffix (the sequence of DNA after the region of interest. Systematic use of the 4 type IIs RENs allows new sections of DNA to be inserted into others.

A possible new standard

For our project (Green Canary) we utilised the GoldenGate method of cloning, for more information on the GoldenGate method please see our GoldenGate page. As previously described the parts submitted to the registry need to be in the same format (BioBrick), but, as all our wet lab work was carried out in GoldenGate, we had to convert our parts to BioBrick for submission. We developed the MoFlipper (Modular Cloning Flipper) which provides users with a oneway swap between GoldenGate and BioBrick. The standard pSB1C3 plasmid was improved and altered to include the GoldenGate REN recognition sites allowing for (near)scarless transfer from GoldenGate to BioBrick. The modified pSB1C3 contains two divergently orientated BsaI sites between the 4 BioBrick sites which will allow the GoldenGate fragments to be dropped into the (now) BioBrick vector in a one-pot, one-step, digestion-ligation GoldenGate cloning reaction. The improved pSB1C3 part now contains the GoldenGate recognition site as well as a RFP coding device that will allow for red white selection of the transformants.

As discussed in our GoldenGate page the modules have specific 5’ and 3’ overhangs which correspond to their final position within the level 1 and then 2 constructs. We have developed 4 MoFlippers that will accept constructs designated promoters (GGAG —> AATG), coding sequences (AATG —> GCTT), terminators (GCTT —> CGCT) and a whole construct (GGAG —> CGCT) using the 4 base pair overhangs shown.

The image shown below gives an example of how the Flipper is designed to work in this way. The additional sequences, inserted between the RFP and the BioBrick prefix and suffix, are inverted BsaI sites as shown in orange. This enables the flipper to accept MoClo Pro + 5U parts, which begin GGAG and end AATG. The other 3 flippers work in much the same way but are designed with overhangs to accept coding sequence parts, terminator parts and whole transcriptional units as detailed above.

Figure 1: Promoter Mo flipper showing the inverted BsaI sites that allow the module to accept Pro + 5U parts, converting GoldenGate promoter parts into Biobrick form.

As GoldenGate uses type IIs restriction enzymes (that cleave outside of their recognition sequence), the sequence or part can be inserted into the MoFlipper, and not leave the recognition sites of BsaI, and then is fully functional as a BioBrick.

The NRP-UEA iGEM team are submitting this as an improved part of the pSB1C3 backbone used as the registry standard to allow more teams to work in the GoldenGate method of cloning whilst still being able to submit parts to the registry in the standard. The improved parts Bba_K1467100 (Promoter module), Bba_K1467200 (Coding sequence module), Bba_K1467300 (Terminator module), Bba_K1467400 (Transcription unit module) are detailed on our Parts Page. Below is a picture of one of our plates showing a part inserted into our MoFlipper via a GoldenGate reaction. As you can see the reaction is highly efficient with only two colonies on the plate being untransformed.

A big thank you to our sponsors