Cre-recombinase functionality assay
We assayed doubly-transformed E. coli. BL21(DE3) cells with the both the CRY2-CreN/CIB1-CreC construct and the reporter construct with loxP-flanked RFP (EforRED) followed by a kanamycin-resistance gene (NPTII) (below).
We expected that in the dark, the CRY2-CreN and CIB1-CreC fusion proteins would be disassociated in solution. In the reporter construct, RFP would be expressed with transcription terminating at the STOP codon. When activated by blue-light, we expected CRY2 and CIB1 to associate, bringing together the fused Cre-recombinase fragments. The reconstituted Cre-recombinase would recombine the loxP sites of the reporter construct, excising RFP and allowing expression of the kanamycin resistance gene.
After growing these cells up in LB-amp/chlor to an A600 of 0.3, we aliquoted 40uL of these cells to the middle of each test plate as a control with no induction by IPTG (to activate expression of both constructs) or blue-light (to activate association of the split Cre-recombinase).
We then induced expression of both constructs with 0.2mM IPTG at room temperature for 15 minutes under blue-light from a gel imaging system for different intervals as indicated. After the each time interval, 50uL of the culture was diluted in 1mL LB and then 50uL aliquots spotted on the antibiotic-supplemented plates (ampicillin, chloramphenicol and 1x, 2x or 5x kanamycin). BL21(DE3) cells not induced with IPTG (plates 1,3 and 5) and cells transformed only with the CRY2-CreN/CIB1-CreC plasmid (plates 7 and 8) growing on LB-chlor were used as controls.
RFP was observed on all plates after a few days growth in the fridge (not shown), indicating leaky expression of the reporter construct. Growth of cells in the absence of IPTG or blue-light induction comparable to induced cells indicated leaky read-through of the reporter construct, despite the STOP codon at the end of the RFP reporter gene. To try to decrease this read-through, we incorporated a second terminator sequence after the EforRED gene. However, we observed similar results upon assaying this new construct.
This is in contrast to the study by Kennedy et al. (2010), where they observed low levels of recombination of the reporter construct alone and equivalent levels with CRY2-CreN and CIB1-CreC in the dark (attributed to background levels of the reporter spontaneously recombining and not light-independent CRY2-CIB1 interaction). Blue-light exposed cells showed a 158-fold increase in reporter. We used different light pulse and incubation times to induce our cells with light, though the activity of the reporter construct in the absence of the CRY2/CIB1-Cre-recombinase construct and light induction indicates that that device may also be faulty.
We hypothesised that a layer of kanamycin-resistant cells growing at the surface of the plate created a barrier to the antibiotic, allowing growth of non-resistant (and non-recombined) cells on top of this layer. If this is the case, then background levels of recombination might not be as high as suggested by these plates. In applied systems such as our NAD biosynthesis system, low levels of read-through might not be highly detrimental to the system, although death of a proportion of cells may decrease the efficiency of the system.
Runsheet
0-10 minutes: Students are supplied with nametags and the session begins with a quick ice breaker game. We decided on the Link game due to similarities with protein folding. In this game, in groups, students close their eyes and link up their hands with other members in their group. The goal is to, without letting go of each other’s hands, unravel the knot completely.
10-15 minutes A quick introduction of our team members is followed by an explanation of the practical. We decided to demonstrate the role of enzymes within pineapples in breaking up gelatine at different temperatures. Due to the length of time needed for jelly to set, the practical was set up ahead of time. One sample of pineapple juice will be heated and the other set at room temperature so that the heated sample would contain only denatured proteins thus jelly would be able to form. This will be used to motivate how radiation might change protein function a la optogenetics.
15-30 minutes We give a short presentation on proteins including a basic definition, their function, and various examples and pictures. The key here is not scientific understanding but more an intuition of how proteins work linking back to the pineapple experiment as well as the link game. We specify the importance of the shape of the protein. We explain that the function of proteins can be affected by various factors such as heat or light and give necessary examples. This then sets us up to introduce DNA very vaguely as the blueprint which “constructs” proteins. Now that students are familiar with how proteins influence biological function, they can now acquaint themselves with the idea that changing DNA, changes proteins, which change biological function.
45-55 minutes: We introduce the idea of genetic engineering on the above basis. Students are asked to brainstorm examples of genetic engineering they are familiar with. We use reflective questions to guide students into thinking about the social “good” derivable from certain genetically modified organisms and how this links with their ethical intuitions. We then conclude the session by checking on the jelly to make sure it has set. If not, we will prepare spares. We will then handout some prepared jelly for students to take with them into recess.