IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-6-28
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Newer entries at the top.
Phone conversation with Professor Golden
- Hybrid plasmids
- Hybrid plasmids exist, there are a few of them out there.
- Professor Golden made some of the first ones, which were improved upon by other labs.
- Professor Golden's lab primarily worked with transcription, so they don't use endogenous plasmids anymore.
- They'd probably be fine for what we want to do but Professor Golden doesn't use them because they caused minor changes in promoter expression when putting them in e. coli.
- Using non-replicating vectors to insert our genes into the cyanobacteria chromosome is more stable and well behaved.
- Golden says it probably won't make a difference for our purposes whether we use endogenous or exogenous plasmids.
- Our promoters are part of our insert and so don't need to be a part of the plasmid.
- Endogenous plasmid is 8Kb; fairly large.
- If placing 3kb into the endogenous plasmid, the total size will be 11 kb.
- Problem: miniprepping.
- If placing 3kb into the endogenous plasmid, the total size will be 11 kb.
- The neutral sites can accommodate all 3 kai genes.
- Professor Golden stated that she may be able to find a version of her hybrid plasmid(s) for us.
- Light sources
- Professor Golden uses cool white fluorescent lights and says that we can throw in grow lights for more red.
- Be aware that conversions between lux and µE aren't perfect. Grow lights have more energy in the 400-700 nm range.
- Light intensity drops with the square of the distance, so the lights should be a foot, foot and a half above the flasks.
- Grow lights provide more red light, which is happily absorbed by cyanobacteria. However, they are not essential-- fluorescent lights should suffice.
- Nevertheless, the more light the better, and we're not in danger of giving cyanobacteria too much light.
- Air
- Enough gas exchange within plates (so the plates need not be left uncovered).
- Use flasks that give dead air space, shake them a little to exchange gas.
- Professor Golden is worried that commercial caps may release toxins into the flasks over a long period of time. Her lab uses cotton wrapped in cheesecloth to plug the flasks, over which they fit a loose covering of aluminum foil. Everything is autoclaved together.
- Plate storage
- Plates can be stored in a drawer at room temperature.
- Reporters
- GFP is unsuitable for cyanobacteria.
- GFP is too stable (doesn't decay in the troughs of expression).
- GFP does not glow in cyanobacteria.
- YFP and CFP work (in van Oodenaarden's lab), but they haven't done anything circadian with it.
- Additional note: GFP can be destablized but this may take 2 weeks.
- Measuring fluorescence requires its own special equipment
- Arguments for Luciferase.
- Luciferase doesn't require you to illuminate the cells with high energy (fluorescent) light (which damages them).
- The damage is referred to as photobleaching.
- You can passively measure bioluminescence in real time since you don't need to shine a light on the cells
- Luciferase is non-invasive according to Professor Golden.
- Don't need a great CCD if you're not trying to measure single colonies.
- Splotches on a plate and a fairly good camera are needed for imaging.
- Luciferase doesn't require you to illuminate the cells with high energy (fluorescent) light (which damages them).
- GFP is unsuitable for cyanobacteria.
- Isolating and measuring KaiC
- Tagging KaiC has caused problems for other labs (oscillation ceases)
- Kondo lab has a new paper in press where they succesfully tagged some of the Kai proteins.
- Professor Golden offered to do Western blots for us if we are able to create the cells.
- There's skill to running the SD-PAGE.
- Reporter gene
- RNA polymerase is very similar between cyano and E. coli; but the other factors may cause problems (Professor Golden's area of research).
- Professor Golden states that the problem is not the RNA polymerase, but what connects to the RNA polymerase.
- The RNA polymerase and the sigma factors are similar, but there are a lot of other unknown facters between KaiC and gene regulation.
- Building back the gene expression pathway is the hard part.
- What we can reasonably do
- Make a BioBrick, try to get the oscillation going in E. coli. Might as well try a reporter gene, but the easiest thing is to directly measure the KaiC phosphorylation.
Questions for Professor Golden
- Are plant lights viable for growing cyanobacteria?
- Can we use GFP as a reporter in cyanobacteria instead of luciferase? (We don't have the right equipment for automatically measuring bioluminescence).
- Do we need to aerate our cultures with CO2? If so, how do we prevent contamination and dry out?
- How do we store plates for a long time?
- For isolating and measuring the phosphorylation of KaiC, what are the pros and cons of using KaiC antibody assay versus purifying with HisTag?
- Why not use endogenous autoreplicating plasmids for transformation, instead of non-replicating vectors?
Incubator status
Incubator lighting
Per Susan Golden's email, we will grow our cyanobacteria in constant light conditions, only cycling to dark when we need to synchronize them.
Agar longevity
We put clean agar plates on the shaking floor of the incubator to test how quickly they dry out. One plate is partially open while the other plate is closed but not sealed.
Green growth
Some of our PCC 6803 flasks showed growth since yesterday (single 2006-6-26 and streak 2006-6-26). Nick vortexed the streak 2006-6-22 to disperse the fungus poof.
Email from Professor Golden
We received an email from Professor Golden with lots of information.
- Professor Golden's expertise lies with PCC 7942. She warned that growth conditions and the circadian oscillator may be very different in other strains.
- We shouldn't be worried about giving our cyanobacteria too much light. The more light the better. We can even keep them in constant light conditions instead of the 16h light / 8h dark cycle we've been using.
- Professor Golden is willing to send us plasmids from her lab.
- Getting KaiC phosphorylation to oscillate in E. coli would be a major achievement by itself. We can still try to transplate RNAP and sigma factors from cyanobacteria into E. coli, but we'd be heading into unknown territory. We should make sure that KaiC will phosophorylate on a circadian rhythm in E. coli.
