Protein_Domain

Part:BBa_K2559009

Designed by: Weiteng Xu   Group: iGEM18_SCAU-China   (2018-10-12)


LacI-eGFP

Expression vector of eFGP, it is a composite part modified from BBa_J04450 based on our another improved part BBa_K2559005

Usage and Biology

The part BBa_K2559005 has a sequence improvement on the basic part submitted by iGEM07_Peking (BBa_I714891) which encodes the SDY_eGFP. However, we found out a 16 bp nucleotides redundancy in the eGFP starting coding region in BBa_I714891, after checking the sequence of BBa_I714891 from NCBI. Therefore, we decided to delete the redundant 16 bp nucleotides in BBa_I714891 to amend the length of eGFP coding sequence. The amended eGFP coding biobrick is the BBa_K2559005. To test the function of BBa_K2559005, we designed a new E.coli expression vector containing our new part termed as BBa_K2559003under a strong E.coli endogenous promoter (PrplJ). Therefore, the amended eGFP in BBa_K2559005 was driven by PrplJ promoter, and expressed in DH10B. In addition, we also applied the BBa_K2559005 in the promoter intensity analysis of our other two new parts, the BBa_K2559004 and BBa_K2559011 which are relatively weaker E.coli endogenous promoters (PdapA and PcaiF) (Figure 1).

Scau-china-2018-11.png


Figure 1: Fluorescent intensity of amended eGFP driven by by PrplJ, PdapA, PcaiF promoter.


We summarized that our improvedpart, the amended eGFP coding biobrick BBa_K2559005 worked well in DH10B. We also hoped that our improvement on the BBa_I714891 can help their future applications by other groups in the future. However, it is difficult for us to perform additional experiments with BBa_K2559005 and BBa_I714891 due to the unavailable BBa_I714891.

To expand the application of BBa_K2559005, we searched theBBa_J04450stored in registry and do another improvement in the BBa_J04450. The BBa_J04450 is a strong RFP expression vector in E.coli. As the main page of BBa_J04450 mentioned, the E.coli colonies with BBa_J04450 were in red color under normal light after about 18 hour culture on LB plate (Figure 2). We used the BBa_K2559005 to replace the RFP region in BBa_J04450, the modified part is BBa_K2559009. We transferred the BBa_K2559009 to DH5α by heat-shock, and found that the fluorescence signal can be observed under the UV (Figure 2).

Figure 2 : Figure 2 Colonies with BBa_J04450 (red colony) and BBa_K2559009 (green colony) were visualized under UV lightbox

So, we confirm that our improved part BBa_K2559005 can work in different E.coli expression system. We are also looking forward to more application of the BBa_K2559009!

Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 781
    Illegal AgeI site found at 893
  • 1000
    COMPATIBLE WITH RFC[1000]


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