From 2009.igem.org
Targeted Biobrick Construction
Synthesis of Targeted Biobrick. A.pAdEasy-1 plasmid extraction; B. First round PCR to modify fiber gene; C.Second round PCR to modify fiber gene.
In this section, we were contructing two contructs in paralell. First, to contruct a standardized Targeted Biobrick for parts registry, where Targeted Biobrick can be served for further use by other teams or researchers. Second, to incorporate Targeted Biobrick into an expression vector pET-28a to test its expression. Targeted Biobrick was contructed via two rounds of PCR.
pET-28a+Targeted Biobrick
Identification of the Targeted Biobrick positive clones. Desired bands are at 1260 bp
The expression vector was used for Targeted Biobrick characterization, while the standardized Targeted Biobrick has been submitted to iGEM headquarters for parts registry. We have successfully gained the desired Targeted Biobrick protein of 44184.58 Daltons.
SDS-PAGE of the purified Targeted Biobrick. The desired Targeted Biobrick protein is 44.2 KD. The left most lane is the marker, where there are bands of 14.4 KD, 20 KD, 30 KD, 43KD, 66 KD and 97 KD respectively
In conclusion, we have successfully contructed Targeted Biobrick for both part registry and expression (most of the results have not been showed here to make our wiki concise). Of course, the next step should be the characterization of its targeted function.
C-Targeted Biobrick Fusion
Targeted Biobrick Characterization by Fusing with GFP
Tranfection of pEGFP+Targeted Biobrick into HeLa Cells. A.tranfected HeLa cells by pEGFP+Targeted Biobrick at bright field; B.tranfected HeLa cells by pEGFP+Targeted Biobrick under fluorescent microscopy at the same field; C.control HeLa cells at bright field; D.control HeLa cells under fluorescent microscopy at the same field. 10X10 magnification
Tranfection of pEGFP+Targeted Biobrick or pEGFP empty vector into HeLa Cells. A.tranfected HeLa cells by pEGFP+Targeted Biobrick under fluorescent microscopy; B.tranfected HeLa cells by pEGFP empty vector under fluorescent microscopy. 10X10 magnification
Selected Cells for Specificity Testing before co-incubation. A.MRC-5 human diploid fibroblast cells. B.Hela cells. 10X10 magnification
Specificity testing after co-incubation with purified Targeted Biobrick (MRC-5). A.MRC-5, pEGFP+Targeted Biobrick, at bright field; B.MRC-5, pEGFP+Targeted Biobrick, under fluorescent microscopy; C.MRC-5, pEGFP empty vector control, at bright field; D. MRC-5, pEGFP empty vector control, under fluorescent microscopy
Specificity testing after co-incubation with purified Targeted Biobrick (HeLa). A.HeLa, pEGFP+Targeted Biobrick, at bright field; B.HeLa, pEGFP+Targeted Biobrick, under fluorescent microscopy; C.HeLa, pEGFP empty vector control, at bright field; D.HeLa, pEGFP empty vector control, under fluorescent microscopy. 10X10 magnification
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