Difference between revisions of "Part:BBa I714891"

Latest revision as of 13:09, 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 amended 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_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).

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
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 14: / Line 14: @@
 [https://parts.igem.org/Part:BBa_K2559005# Team:SCAU-China 2018]
-<p>The BBa_K2559005 is a full-length EGFP coding part improved from BBa_l714891.
+<p>The [https://parts.igem.org/Part:BBa_K2559005# BBa_K2559005] is a amended eGFP coding part improved from BBa_l714891.
 ===Usage and Biology===
-The part BBa_K2559006 has an improvement on the basic part submitted by iGEM07_Peking (BBa_l714891) which is cording the SDY_EGFP. We had added 16 bp DNA to fill the length of EGFP cording sequence in BBa_l714891 to crease the BBa_K2559005. Because, we found out a 16 bp deficiency in the EGFP starting cording region of BBa_l714891, after we checked the sequence of BBa_l714891 in NCBI.
+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.
-In order to test the function of BBa_K2559005, we design a new E.coli expression vector with our new part, BBa_K2559003, a strong E.coli endogenous promoter (PrplJ). The full length EGFP in BBa_K2559005 was driven by PrplJ, and expressed in DH10B. Attention, we also used the BBa_K2559005 in the promoter strength analysis of our other two new parts, the BBa_K2559004 and BBa_K2559011 which are weak E.coli endogenous promoters (PdapA and PcaiF).
+To test the function of BBa_K2559005, we designed a new E.coli expression vector containing our new part termed as [https://parts.igem.org/Part:BBa_K2559003# 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 [https://parts.igem.org/Part:BBa_K2559004# BBa_K2559004] and [https://parts.igem.org/Part:BBa_K2559011# BBa_K2559011] which are relatively weaker E.coli endogenous promoters (PdapA and PcaiF) (Figure 1).
-https://static.igem.org/mediawiki/parts/c/ce/Scau-china-2018-11.png
+[[File:Scau-china-2018-11.png|800px|thumb|center]]
-https://static.igem.org/mediawiki/parts/f/f7/Scau-china-2018-12.png
-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.
+[[File:Scau-china-2018-12.png|800px|thumb|center|Figure 1: Fluorescent intensity of amended eGFP driven by by PrplJ, PdapA, PcaiF promoter.
+]]
-Because the BBa_I714891 is not available, we chose the [https://parts.igem.org/Part:BBa_J04450# BBa_J04450]stored 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 [https://parts.igem.org/Part:BBa_K2559009# 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.
+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 the[https://parts.igem.org/Part:BBa_J04450# BBa_J04450]stored 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 [https://parts.igem.org/Part:BBa_K2559009# 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).
+[[File:T-SCAU-China-Red_and_green_colonies.jpg|200px|thumb|center|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!
 <!-- Add more about the biology of this part here