Difference between revisions of "Part:BBa K3814018:Design"
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===Design Notes=== | ===Design Notes=== | ||
| − | |||
| + | 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. The Babushka doll method requires all the clusters to have a unique antibiotic resistance marker. There are many selectable markers available, but we needed to also consider that the clusters need to mostly stay under 5kb as well. | ||
| + | We found that the following configuration was possible: | ||
| + | Table 1. Assigned selectable markers to gene clusters. | ||
| + | |||
| + | {| | ||
| + | | Name || Selectable Marker Used || Total Cluster Length (bp) | ||
| + | |- | ||
| + | | Cluster 1 || TpR || 4174 | ||
| + | |- | ||
| + | | Cluster 2 || AmpR || 4274 | ||
| + | |- | ||
| + | | Cluster 3 || fosC2 || 4876 | ||
| + | |- | ||
| + | | Cluster 4 || CmR || 5015 | ||
| + | |- | ||
| + | | Cluster 5 || GmR || 4948 | ||
| + | |- | ||
| + | | Cluster 6 || TcR || 3370 | ||
| + | |- | ||
| + | | Cluster 7 || malS || 2958 | ||
| + | |- | ||
| + | | Cluster 8 || qacE || 5128 | ||
| + | |} | ||
| + | |||
| + | |||
| + | Name | ||
| + | Selectable Marker Used | ||
| + | Total Cluster Length (bp) | ||
| + | Cluster 1 | ||
| + | TpR | ||
| + | 4174 | ||
| + | Cluster 2 | ||
| + | AmpR | ||
| + | 4274 | ||
| + | Cluster 3 | ||
| + | fosC2 | ||
| + | 4876 | ||
| + | Cluster 4 | ||
| + | CmR | ||
| + | 5015 | ||
| + | Cluster 5 | ||
| + | GmR | ||
| + | 4948 | ||
| + | Cluster 6 | ||
| + | TcR | ||
| + | 3370 | ||
| + | Cluster 7* | ||
| + | malS | ||
| + | 2958 | ||
| + | Cluster 8 | ||
| + | qacE | ||
| + | 5128 | ||
| + | |||
| + | |||
| + | |||
| + | |||
| + | 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. | ||
===Source=== | ===Source=== | ||
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Design Notes
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. The Babushka doll method requires all the clusters to have a unique antibiotic resistance marker. There are many selectable markers available, but we needed to also consider that the clusters need to mostly stay under 5kb as well.
We found that the following configuration was possible:
Table 1. Assigned selectable markers to gene clusters.
| Name | Selectable Marker Used | Total Cluster Length (bp) |
| Cluster 1 | TpR | 4174 |
| Cluster 2 | AmpR | 4274 |
| Cluster 3 | fosC2 | 4876 |
| Cluster 4 | CmR | 5015 |
| Cluster 5 | GmR | 4948 |
| Cluster 6 | TcR | 3370 |
| Cluster 7 | malS | 2958 |
| Cluster 8 | qacE | 5128 |
Name
Selectable Marker Used
Total Cluster Length (bp)
Cluster 1
TpR
4174
Cluster 2
AmpR
4274
Cluster 3
fosC2
4876
Cluster 4
CmR
5015
Cluster 5
GmR
4948
Cluster 6
TcR
3370
Cluster 7*
malS
2958
Cluster 8
qacE
5128
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.
Source
n/a