Difference between revisions of "Part:BBa K3814018:Design"

 
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===Design Notes===
 
===Design Notes===
−
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.
 
  
 +
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===

Revision as of 23:13, 21 October 2021


FosC2 gene


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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


References