Part:BBa_K3024011:Experience

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Applications of BBa_K3024011

Here, we put cox under the control of an inducible pBad-promoter enabling us to experimentally regulate the induction of cox and the degree of its expression by adding arabinose to the growth media. For detection, we made use of a Myc-tag and GFP reporter gene. We also included TetR as this was a necessity for our system later on, to control the P2 phage switch as part of our iGEM Stockholm 2019 project.

Arabinose-dependent toxicity was found compared to the empty vector control. Furthermore, no expression of either GFP or the Myc-tag was found.

User Reviews

UNIQ91fc227f29b22252-partinfo-00000000-QINU UNIQ91fc227f29b22252-partinfo-00000001-QINU

••••• iGEM Stockholm 2019

This is a composite, tricistronic part that co-expresses TetR, Cox and GFP under the control of pBad promoter. Cox gene is fused with cMyc tag for immunological detection (similarly to C protein, no commercial antibody for cox is available). TetR is an inhibitor acting on Tet-operator regions (tetO). Cox is a transcription factor known to activate downstream genes that cause the lytic cycle, thus this module was designed to experimentally induce the switch from lysogeny to lytic cycle. 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. Growth curve of the BBa_K3024011 in different arabinose concentrations (0%,-0.4%) over the course of 10 hours. AraC3, empty vector was used as negative control. Absorbance OD600 was measured in 5 min cycles in a FluOstar plate reader, set to orbital shaking before each cycle. Figure 2. ClariOstar measurement of GFP(ex:488-14/em:535-30). Cells were grown in a plate-reader under permissive and non-permissive conditions (0%-0.4% arabinose) for 10 hours, 120 cycles with 5 min intervals, 20 flashes for each cycle. AraC3 empty vector was used as negative control. Flourescin dye was used as positive control for GFP. Figure 3. TOP10 bacteria transformed with BBa_K3024011 were grown for 5 hours at 37C in LB-media under permissive and non-permissive conditions. Samples were collected in stationary phase at time 300 min. L= ladder -C= negative control +C=positive control for GFP only. Anti-GAPDH was used as a loading control. Visit iGEM Stockholm 2019 wiki for more details about the protocol and project design.