Difference between revisions of "Part:BBa K1152015:Design"
(→‎Design Notes)
 
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[[file:ccdB_TOP10.png|200px|''Figure 1'']]
 
[[file:ccdB_TOP10.png|200px|''Figure 1'']]
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{|class="wikitable"
 
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|+ Table 1: Primer for removal of internal RFC[10] cutting sites in indC using a CPEC approach
 
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!Name!!Primer Sequence 5' - 3'
 
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|1_K1152013_fw||AAGTGGATTGAACAGACAGACTCTAAAAC
 
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|2_K1152013_rv||AGTATCTGTATGTAATGGCACCAATAGACGC
 
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|3_ccdB_fw||TGCCATTACATACAGATACT ACTGGCTGTGTATAAGGGAGCCTGAC
 
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|4_ccdB_rv||AGAGTCTGTCTGTTCAATCCACTT CGCGTGGATCCGGCTTAC
 
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In a 6 ul PCR Master Mix (NEB Phusion High-Fidelity in High Fidelity buffer), all fragments were put together in an equimolar ratio. CPEC assembly was performed using an optimized protocol (Table 2).
 
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{|class="wikitable"
 
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|+Table 2: CPEC assembly Cycler Parameters
 
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!Cycles!!Temperature [°C]!!Time [s]
 
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|1||98||30
 
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|rowspan="3"|5||98||5
 
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|53||15
 
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|72||60
 
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|1||72||180
 
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Transformation was performed with 5 ul of the CPEC reaction product.
 
  
  
 
Using the same strategy as for introducing the ccdB gene, we replaced the ccdB gene with the novel T-domain. Using this strategy, every colony on the plate with transformed cells contains the new T-domain.
 
Using the same strategy as for introducing the ccdB gene, we replaced the ccdB gene with the novel T-domain. Using this strategy, every colony on the plate with transformed cells contains the new T-domain.

Latest revision as of 02:05, 6 October 2013

IndC Indigoidine Synthetase device with T-domain of plu2642


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 4087
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1467
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 2730


Design Notes

Figure 1: ccdB CPEC assembly strategy for exchanging T-domains in indC

We amplified the plasmid K1152008 without the T-domain with primers 1/2 and the ccdB cassette from pDONR with primers 3/4. In a second CPEC assembly, the indC-ccdB part was created and transformed into OneShot ccdB survival cells.

Table 1: Primer for removal of internal RFC[10] cutting sites in indC using a CPEC approach
Name Primer Sequence 5' - 3'
1_K1152013_fw AAGTGGATTGAACAGACAGACTCTAAAAC
2_K1152013_rv AGTATCTGTATGTAATGGCACCAATAGACGC
3_ccdB_fw TGCCATTACATACAGATACT ACTGGCTGTGTATAAGGGAGCCTGAC
4_ccdB_rv AGAGTCTGTCTGTTCAATCCACTT CGCGTGGATCCGGCTTAC

In a 6 ul PCR Master Mix (NEB Phusion High-Fidelity in High Fidelity buffer), all fragments were put together in an equimolar ratio. CPEC assembly was performed using an optimized protocol (Table 2).

Table 2: CPEC assembly Cycler Parameters
Cycles Temperature [°C] Time [s]
1 98 30
5 98 5
53 15
72 60
1 72 180

Transformation was performed with 5 ul of the CPEC reaction product.

The prepped plasmid was then transformed into both E. coli TOP10 and OneShot cells, outlining that there are no background colonies (Figure 1).

Figure 1


Using the same strategy as for introducing the ccdB gene, we replaced the ccdB gene with the novel T-domain. Using this strategy, every colony on the plate with transformed cells contains the new T-domain.