Part:BBa_M36666:Experience

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Stanford Location

plasmid name: pJ607

DNA 2.0 gene number: 151014

organism: E. coli

selection marker: ampicillin resistance

device type: HeLa beta-galactosidase actuator with mammalian secretion tag

barcode numbers of glycerol stocks: 0133010228, 0133010061, 0133009660

box label: BIOE44 S14

User Reviews

UNIQaa56636a5abf3ba5-partinfo-00000000-QINU UNIQaa56636a5abf3ba5-partinfo-00000001-QINU Upon transfection into HeLa cells, we did not detect any significant β-galactosidase activity in assays on both media and cell lysate samples. We did, however, run an RT-PCR on samples transfected with our plasmid, and confirmed via gel electrophoresis that our device was undergoing transcription. This means that our device likely produced misfolded or nonfunctional protein. This could be due to a number of causes. Because chaperone proteins are different in HeLa cells than they are in E. coli, the chaperones in the transfected cells may not have been equipped to help fold the β-galactosidase protein in the way that bacterial chaperones do. It is also possible that the N-terminus secretion tag we added may have interfered with proper protein folding.

Most likely, however, is that our sequence was incorrect and produced a nonfunctional protein. In the designed sequence, we placed the lacZ alpha fragment next to the lacZ omega fragment. However, after doing further research on this topic, we discovered that the lacZ alpha gene and lacZ omega gene need to be simultaneously expressed, but not physically connected to one another. In E. coli cells, the lacZ omega fragment is contained on the bacterial chromosome, and the gene can be expressed if a plasmid containing the lacZ alpha fragment is transformed into the cells. The omega fragment codes for most of the protein, but the protein does not become functional until it combines with the subunit coded for by the alpha fragment.

Since we planned to transfect our plasmid into HeLa cells after amplifying it in E. coli, we designed our plasmid to contain both the lacZ alpha and lacZ omega fragments, so that both would be expressed together in HeLa cells. In the gene we designed, we did not place a stop codon after the lacZ alpha sequence before adding the lacZ omega sequence, which likely caused the entire gene to be transcribed and translated as one large nonfunctional protein, instead of producing two separate alpha and omega fragments. Additionally, we should have included a linker sequence between the stop codon of the alpha fragment and the start codon of the omega fragment in order to ensure that the two fragments did not sterically interfere with one another during translation.

It is possible that the misfolded or nonfunctional protein we likely produced was secreted but that the assays we ran on the media samples came back negative because they relied on β-galactosidase activity.

We hypothesize that if we had designed our gene so that the lacZ alpha fragment was separated from the lacZ omega fragment by a stop codon and a linker sequence, the functional β-galactosidase protein would have been produced. However, without further testing, we cannot speak to whether or not the protein would have been secreted with this new gene sequence.