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slr0168

Part:BBa_K2559008

Designed by: Yinpin Huang   Group: iGEM18_SCAU-China   (2018-10-12)
Revision as of 12:16, 15 October 2018 by Yinpin Huang (Talk | contribs) (→‎Usage and Biology)

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Neutral site for homologous recombination

Neutral site cloned from Synechocystis sp. PCC 6803, the key component of gene expression platform

Usage and Biology

Foreign genes and even entire pathways are often ported into chassis organisms, requiring either plasmidbased expression or identification of a neutral site for genome integration. As genome integration is more stable and predictable compared to plasmid based expression,this is often the preferred method for modification,particularly for industrial microbial strains.[1] Nowadays several putative neutral integration sites have been identified in recent efforts, including the pseudogene glpK (SYNPCC7002_A2842) [2] and the genomic region between hypothetical protein genes (SYNPCC7002_A0935 and SYNPCC7002_A0936) [3], yet the neutrality of these sites remains to be verified. Liang[4] have successfully designed and constructed using synthetic biology approach two recombinant strainsof Synechococcus elongatus PCC7942 for heterologous expression of different industriallyrelevant Bacillus subtilis CotA laccase. Of the two strains the one with CotA laccase integrated in neutral site II(PCC7942-NSII-CotA) was superior in terms of growth rate and enzymatic activity towardtypical laccase substrates: ABTS [2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonate)] andsyringaldazine. That may suggest that two of the traditionally used neutral sites ofS. elongatus PCC7942 are not equally suitable for the expression of certain transgenes

Figure 1 Cultivation of transgenic strains PCC7942-NSI-CotA and PCC7942-NSII-CotA in BG 11 growth medium: (A) growth curve of PCC7942-NSII-CotA、PCC7942-NSI-CotA and PCC7942-WT ; (B) laccase activity curve in cell-free culture medium of PCC7942-NSII-CotA ，PCC7942-NSI-CotA strain, PCC7942-WT.[1]

We have cloned slr0168 gene from Synechocystis sp.PCC6803 and Microcoleus vaginatus FACHB-253.The pcr result,to some degree, proved that the slr0168 gene is highly homologous.

Figure 2The pcr results of slr0168 in Synechocystis sp.PCC6803 and Microcoleus vaginatus FACHB-253
Figure 3:Bacteria cellulose Constitutive expression cassette
Figure 4:Colony PCR of extracted extracted Synechocystis sp.PCC6803 genome

Reference： [1]Liang Y, Hou J, Liu Y, et al. Textile Dye Decolorizing Synechococcus PCC7942 Engineered With CotA Laccase:[J]. Frontiers in Bioengineering & Biotechnology, 2018, 6. [2]Qi FX, Tan XM, Lü XF. Construction and evaluation of efficient gene expression platforms in Synechocystis sp. PCC6803. Chin J Biotech, 2013, 29(9): 1332−1342. 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 1326
    Illegal BamHI site found at 1537
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 661
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 776
    Illegal BsaI.rc site found at 1886


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