Part:BBa_K3024004:Experience
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Applications of BBa_K3024004
This composite part is formed by the constitutive promoter J23119, followed by two tet-Operators, and the gene that encodes for C protein fused with FLAG-tag (DYKDDDDK epitope). This tag allows us to detect C protein with immunological techniques, since there is no antibody against C commercially available. Finally, RFP gene is expressed downstream as a real-time cell reporter. This module was designed by iGEM Stockholm 2019 as a component to regulate the P2 phage Switch plasmid.
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UNIQ869127db71dfe680-partinfo-00000000-QINU UNIQ869127db71dfe680-partinfo-00000001-QINU
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iGEM Stockholm 2019 |
This biobrick was created to characterize C-protein, a key regulator of the P2 phage switch; and the constitutive promoter, as this is required to influence the switch system. For detection, we made use of a Flag-tag, and RFP reporter gene, which allows to report expression using different methods. Our results show no RFP expression through spectrophotometric analysis. Yet with Western blot, we detected the protein of interest using the incorporated Flag-tag. We hypothesize that this indicates C-protein is expressed but the RFP is not, or is broken down too fast, due to the degradation tag that we added. Aside from this, the growth curve shows arabinose-dependent toxicity, whereas no such thing was detected in the empty vector control sample. This contradicts the expectation as arabinose should have no effect on either the promoter or the Top10 cells. However, we were unable to determine the cause of this anomaly.
Figure 1. FluOstar readout of a growth curve. Absorbance OD600 measured at a cycle interval of 5 min, 120 cycles. Blank; media. AraC3: negative control. Samples: TOP10 cells transformed with BBa_K3024004.
Figure 2. ClariOstar measurement of mRFP(ex:550-20/em:605-40). Cells were grown in a plate-reader for 10 hours, 120 cycles with 5 min intervals, 20 flashes for each cycle. Samples 0%-0.4% are replicates of BBa_ K3024004. AraC3 empty vector was used as negative control. As a positive control we used the RFP coding device, BBa_J04450 in the pB1C3 backbone.
Figure 3. Western blot of BBa_K3024004. Transfected TOP10 cells were grown in LB-media for 5 hours at 37C. Samples were collected at 1. 180 min, 2. 240 min and 3. 300 min. L= ladder -C= negative control. +C x= positive control for RFP only. Anti-GAPDH was used as a loading control.
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