Difference between revisions of "Part:BBa K3814055:Design"

 
 
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===Design Notes===
 
===Design Notes===
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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.
  
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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).
  
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===Source===
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We found that the following configuration was possible:
  
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'''Table 1.''' Assigned selectable markers to gene clusters.
  
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===References===
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{|
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|  Cluster || Selectable Marker Used || Total Cluster Length (bp)
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|-
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|  1 || TpR || 4174
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|-
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|  2 || AmpR || 4274
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|-
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|  3 || fosC2 || 4876
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|-
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|  4 || CmR || 5015
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|-
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|  5 || GmR || 4948
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|-
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|  6 || TcR || 3370
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|-
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|  7 || malS || 2958
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|-
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|  8 || qacE || 5128
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|}
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This selectable marker would be used in Cluster 1.
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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.

Latest revision as of 23:24, 21 October 2021


TpR Gene with no RBS


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

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 1.

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.