Part:BBa_K3814030:Design
GmR gene
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 502
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
USYD's 2021 team aimed to generate a naturally transformable (NT) E. coli. We planned to insert the 23 genes responsible for NT from A. baylyi into a strain of E. coli. We designed eight clusters of genes to do this, and used a novel recombineering strategy called Babushka Blocks.
One key element of the Babushka block design is that each gene cluster needed a unique selectable marker. Selectable markers need to be used in each gene cluster to be able to select for colonies that have successfully incorporated them into the E. coli chromosome. There are many selectable markers available, but we needed to also consider that the clusters need to mostly stay under 5kb as well, due the limits set by the company we are ordering from (Twist).
We found that the following configuration was possible:
Table 1. Assigned selectable markers to gene clusters.
| Cluster | Selectable Marker Used | Total Cluster Length (bp) |
| 1 | TpR | 4174 |
| 2 | AmpR | 4274 |
| 3 | fosC2 | 4876 |
| 4 | CmR | 5015 |
| 5 | GmR | 4948 |
| 6 | TcR | 3370 |
| 7 | malS | 2958 |
| 8 | qacE | 5128 |
This selectable marker would be used in Cluster 5.
Where possible, restriction enzymes were removed to minimise off-target effects. Substitute bases were chosen to most closely match the natural codon frequency in bacteria.