Difference between revisions of "Part:BBa I714891"

Revision as of 10:41, 16 October 2018

SDY_EGFP

a strong reporter

• 

  measured by fluorescence microscope

[http://2018.igem.org/Team:AFCM-Egypt# Team:AFCM-Egypt 2018] provided specific characterization for this part by using eGFP for experimental characterization of lentiviral transfer plasmid transfection efficacy and apoptotic effect on colorectal cancer cell line RKO.

Team:SCAU-China 2018

The BBa_K2559005 is a full-length EGFP coding part improved from BBa_l714891.

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_K2559003 under 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).

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

We conclude that our improved new part, full length EGFP cording biobrick , BBa_K2559005 can work well in DH10B, and we also hoped that our improvement on the BBa_l714891 can help the future application of this part.

Because the BBa_I714891 is not available, we chose the BBa_J04450stored in Registry and tried to expand the application of BBa_K2559005.The BBa_J04450 part is a strong RFP expression vector for E.coli because of the strong RBS. As the main page BBa_J04450 mentioned, the E.coli colonies with BBa_J04450 were red in color under natural light after about 18 hour culture in LB plate. We used the BBa_K2559005 to replace the RFP region in BBa_J04450, the restructured vector from those two parts is BBa_K2559009. We transferred the BBa_K2559009 to DH5α by heat-shock transformation, and found that the fluorescence signal can be observe clearly under the UV.

Sequence and Features