Regulatory

Part:BBa_K4325010

Designed by: Minchun He   Group: iGEM22_SZPT-CHINA   (2022-09-16)
Revision as of 15:04, 8 October 2022 by Bozaigao (Talk | contribs)

pDawn without LVA tag

Description

Our reduce the leakage level of pDawn system by removing the degradation tag LVA in the original repressor cI .

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2171
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 63
    Illegal NgoMIV site found at 195
    Illegal NgoMIV site found at 289
    Illegal NgoMIV site found at 582
    Illegal NgoMIV site found at 1076
    Illegal NgoMIV site found at 1094
    Illegal NgoMIV site found at 1184
    Illegal AgeI site found at 414
    Illegal AgeI site found at 1542
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1643
    Illegal BsaI.rc site found at 525

2022 SZPT-China

Biology

Our goal is to reduce the leakage level of pDawn system by removing the degradation tag LVA in the original repressor cI as increasing the concentration of cI could enhance the repression of PR promoter.

Figure 1. Sequence alignment of pDawn(cI-LVA) before and after removing LVA tag

Usage

The plasmid containing the blue light induced kill switch was transformed into E. coli TOP10. Sixteen colonies were picked and grown on two new LB plates at 37 °C for 16 h. One plate was placed in the dark and the other was placed under blue light. The number of the candidates that survived the dark but failed to grow under blue light but survived in the dark was counted. For the E. Coli strain containing pDawn(cI-LVA)-RBS070-LKD-pSEVA331, the number is 5. For pDawn(cI)-RBS070-LKD-pSEVA331, the number is 10. The leakage expression of the lysis cassette LKD could be associated with this discrepancy in functionality. The new pDawn in which the LVA tag of cI is deleted improves the effectiveness of the blue light responsive lysis system.

Figure 2. (a1) Colony growth of E. coli TOP10 containing the plasmid of pDawn(cI-LVA)-RBS070-LKD-pSEVA331 in the dark

(a2) Colony growth of E. coli TOP10 containing the plasmid of pDawn(cI-LVA)-RBS070-LKD-pSEVA331 under blue light

(b1) Colony growth of E. coli TOP10 containing the plasmid of pDawn(cI)-RBS070-LKD-pSEVA331 in the dark

(b2) Colony growth of E. coli TOP10 containing the plasmid of pDawn(cI)-RBS070-LKD-pSEVA331 under blue light

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