Parts

Summary of used parts

Used parts

-LacH (BBa_R0011) -pBAD (BBa_K206001) -terminators (B0014 containing B0012 and B0011) -RBS (BBa_B0032)

Synthesized parts

-Methyltransferase with myc-linker and double terminator -Memory parts 1x Znf and 2x Znf

Externally received parts

-Zinc Finger

Backbones

-pSB1AT3 -pSB1C3 -pSB3C5 -pSB4C5

Final parts in all mentioned backbones

-LacH + Mtase + MemP 1x -pBAD + Mtase + MemP 1x -LacH + Znf + Mtase + MemP 1x -LacH + Znf + Mtase + MemP 2x -pBAD + Znf + Mtase + MemP 1x -pBAD + Znf + Mtase + MemP 2x

Intermediate parts

-LacH + Mtase -pBAD + Mtase

The Lac-Hybrid

We chose this promoter because it had a lot of reference on the parts registry page and it seems like a straightforward and easy to use promoter. It has been used many times without problems and seems to be a commonly used promoter for many researchers. Suppressed by LacI and allosteric induced by IPTG it creates an on/off situation, being a strong promoter, for us to test our platform. Side note; added IPTG is not degraded by bacteria and has a long degradation time.

The Arabinose promoter

Just like the Lac-Hybrid promoter we wanted to use another reliable promoter that has been used extensively and creates another on and off situation by an inducible substrate. Induced only by arabinose it should give a clear signal with and without the presence of arabinose.

The methyltransferase

Our biggest and most essential part. The methyltransferase protein, not endogenous to E. coli, we chose to introduce in E. coli. Completely synthesized since it is a very rare protein, we actually took from N. Meningitis, and not easy to come by. It qualifies as a N-4-methyltransferase and has no direct link to E. coli, other than its ability to methylate ScaI sites. This gives us no indication that the E. coli should suffer by the presence of this protein. Using … as a substrate. We have to oblige to the iGEM rules of forbidden and avoidable sites and for that reason we had to make some adjustments to the protein. We don’t suspect this will have any influence on the protein folding or activity.

The mycosin-linker

In order to create a fusion protein of our own we need to link the two together. When checking up on our literature we found that in eukaryotes Bas van Steensel used a myc-linker for his fusion protein. After searching for some more information we found that a myc-linker is actually commonly used and seems reliable. As a bonus feature the myc part can also be used as a myc-tag for immune-blotting, providing information about protein presence and degradation. We ended up using a 39 bp linker to generate enough flexibility for the fusion protein to sustain and to be able to reach and find its specific sites.

The double terminators

We chose to put two terminators together that both occur in the part registry but not together. The both terminators did not have any bad commentary and look like reliable terminators. We chose to have these synthesized to save time and eliminate a cloning step.

The Zinc Finger

Choosing a zinc finger presented itself as a much harder task. We discovered that nature and science both give a wide selection of zinc-fingers that all have their own specialization, plus the part registry does not supply a zinc finger. To gain more information and knowledge about zinc fingers we contacted some specialists and eventually got in contact with prof. Dr. P. Hooykaas, who was interested in our project and could supply us with a zinc finger. His institute specializes in constructing and using zinc fingers fused to Nucleases. It was Dr. S. de Pater that could eventually send us two of the zinc finger nucleases she was working on to try for ourselves. Both consisting of 6 fused zinc fingers, creating a high level of specificity.

The Backbones

Using standard backbones provided by iGEM and backbones that allready contained some elements of the parts we wanted to put together we ended up using a wide array of different backbones. Using these backbones not just because they are required but also because they differ in copy-number, creating different situations inside the cells for us to compare.

Ribosomal binding site

We took this one from the part registry. It is a medium RBS which should be enough for our purpose. In later situation it might be an option to change to either a weak or strong RBS.

The memory part

Every memory system needs a memory module. We designed our own to meet our specific demands. Incorporation a ScaI site next to either one or two Zinc-finger sites. In the case of two sites the other side is anti-sense to the first since the ScaI site is blunt and the methylation can occur on either sides. Of course all lengths of DNA material between the sites is adjusted to the linker length. For the parts of ‘junk’ DNA we used a tool to generate these sequences to ensure that no genes, restriction sites or other problematic pieces of sequences could be generated.