Difference between revisions of "Team:Marburg/Collaborations"

In our Google form we asked our participants to tell us all about their difficulties with the experiments and Vibrio natriegens and to answer some general questions. When asked if they could imagine working with Vibrio natriegens in the future, 7 out of 8 teams answered yes (figure 3).
We could identify four main critique points based on their feedback:

1. The shipping
   We received a lot of feedback on our shipping. Some of the plates broke during transport because we didn’t pack them as well as we should have. For the future we recommend using bubble wrap around the plates or enough crumpled paper to really cushion the plates. Relying only on parafilm and paper towels wasn’t enough to send plates in an envelope instead of a box. Boxes provide more protection against mechanical impact and are therefore better suited for this.


2. Transformation
   Some teams had problems transforming the plasmids into Vibrio natriegens and needed two attempts until they were able to insert the plasmid into V. natriegens. Two of the teams couldn’t perform the transformation, so we sent them the strains. In our lab we observed that the more experience you have, the higher the success rate of transformations. We believe that with guidance and a few more tries the teams would have been able to complete this step, but it would have taken precious time away from their projects.


3. Fast growth
   Some teams were so used to good ol’ E. coli they weren’t prepared for Vibrio natriegens. The fast growth of V. natriegens surprised them and threw off their lab routine. In some cases they ended up overgrown plates. We think that we can solve this problem in the future by simply spreading the word on Vibrio natriegens. If people are confident that the organism will deliver on its promises and grow fast, they can plan their workflow accordingly.


4. Not storable in the fridge
   In the beginning of our project we realized that Vibrio natriegens had problems surviving in the fridge. If left outside in room temperature the plates became overgrown. We are working on a strain that is able to survive cold temperatures to solve this problem. We want to insert a KatG catalase into our VibriClone strain, but we haven’t completed this experiment as of now.

 

 

 

 

 

 

 

 

We calculated the mean emission of each device for each team and the strongest fluorescence was emitted by device 5. Device 1 and 4 were very similar and came in at about half the fluorescence signal as 5. Devices 2 and 6 had a quite low emission and device 3 almost no signal.

Growth Curves of V. natriegens

We compared our curve to the growth curves from other teams.
The data points from our measurement aren’t connected because we didn’t measure inbetween. After their first growth curve didn’t show the results we expected NTNU was kind enough to repeat the experiment, that is why they have two curves. In two hours NTNU reached an OD₆₀₀ of about 3,5 both times, Thessaloniki reached an OD₆₀₀ 5 and we an OD₆₀₀ eached 12.

 

 

 

 

 

 

Science

Vibrigens InterLab

It can be observed that Vibrio natriegens produces a much weaker fluorescence signal at the same OD when compared to E.coli using the same plasmids. In some cases, the fluorescence with a plasmid meant to express GFP is weaker than the blank. This happens because the cells block light more than they emit fluorescence at high densities. According to our measurements the strongest promoter was Test Device 5, followed by 4, 2, 1, 3 and 6. The other teams also found device 5 to be the strongest, but followed by 1, 4, 2, 6 and 3. Device 5, Part BBa_J364008 with the promoter J23104 is unequivocally the strongest. The experiments showed similar results, except that Device 1 was much weaker in our measurements than for the other teams. Device 6 and three had such weak GFP expression that small variations can change their place in the order of strength. We had some difficulty comparing resulting because the units of fluorescence are not standardized. We could only compare results and not analyze them on their own. Some teams had a much higher values then the other. This doesn’t mean that they measured a higher fluorescence, just that the values were given in another frame. The only quality control we could conduct on the data given to us were common sense checks, for example, the negative control had to show a lower fluorescence than the positive control. These checks excluded one team from our experiment.

 

Growth curve

In the five hours in which the experiment was performed by the other team, we reached an OD600 of about 12, and they reached one of 3,5 to 5. There are also some small factors that can combine to truly impact growth. Vibrio natriegens needs a lot of oxygen, so it’s important to take big baffled flasks and keep them shaking at all times. It is also very sensitive to cold, so taking the flasks out of the incubator to take samples can have an impact on the growth speed. These small adjustments come with experience, and because the other teams are much more used to working with E.coli they treated the organism similarly, which worked well, but not as well as it did in our lab (after we gained experience). We think these small tricks created the difference between our growth curves. With experience it would be possible for them to achieve the same growth rate we started with growth curves that only reached an OD600 of about 1, and now we are able to produce ones with an OD600 of 14.

 

GFP/LUX

From all the data that we gathered from this experiment we formed an opinion on whether GFP measurements are adequate to realize the InterLab studies full potential. We think that by replacing GFP by Lux and measuring luminescence instead of fluorescence we could make a more universal experiment, easier to reproduce not only from lab to lab, but also from organism to organism. One of the Lux operon advantages is the almost complete absence of background signal without reporter expression. This is the reason why the Lux operon is suitable to analyze extremely low levels of expression caused by weak promoters or terminator readthrough. We also noticed we have a lower detection threshold when using Lux in our lab, which is why we did all measurements using Lux. The variability in the interLab results, represented by the large error bars and the order of strength changing, is in part a result of the low accuracy of GFP measurements in general. With fluorescence there is an excitation beam that produces light scattering and can falsify your emission measurement, resulting less accurate data. Lux has a lower detection threshold because there is no light being emitted to excite the Chromophore, instead a chemical reaction causes luciferin to emit light. There is no background! One big drawback of Lux is that it would be much more difficult to calibrate. For GFP, you can simply use a known concentration of fluorescein.

 

General

Organization

First of all, we were surprised by how complicated it was organizing and running an InterLab study. Writing universally understandable protocols for students on different levels of knowledge proved difficult. We only had to manage 11 teams, but this small number should not be underestimated. Each country has its own postage rules, which we had to learn about before we could send anything. 11 packages, each with about 40 aliquots needed to be prepared, labeled and wrapped individually took a lot of manpower and patience to pack. We can’t imagine what a logistical nightmare working with 340 teams would be. Preparing the InterLab earlier would have been better. Tasks you never thought about will have to be completed and something will inevitably go wrong, so it is very important to leave margin for error while planing. Also sending the kits out when there was more time until the wiki freeze might have given more teams, not just NTNU (thank you!), time to repeat experiments. Having undergraduate students test out your protocols, not team members who know the organism, would have been beneficial to find where small tips that we see as general knowledge at this point should be inserted into the protocol. We thought the language barrier might present a problem, but were pleasantly surprised by how easy understanding each other was. It was still difficult to communicate with the other teams at times. Email is somewhat formal and because we are young and inexperienced it still takes effort to compose emails (especially in a foreign language), which leads to much slower communication. It would have been better to have instant messaging where we could reach the teams fast and casually instead of email. To help with the experiments a group chat for all teams to exchange tips would have made getting information out faster, easier and possibly yielded better results. Another problem we encountered was that teams didn’t fill in our excel sheets instead making their own with the data we asked for. Maybe if we had explained how we would interpret the data the participants would have known to fill in the correct excel sheets.

 

Shipping

From this experience we learned that there is much room for improvement. Not only should we have packed the plates in a more secure manner, sending duplicates would have made the experiment less susceptible to human error. Unfortunately our lack of experience combined with our lack of resources made it so that we didn’t have boxes big enough to accommodate extra bubble wrap and duplicates. We didn’t realize we needed those until it was too late, at which point we had already bought the shipping containers.

 

Outlook

If we were to conduct an InterLab type experiment again, we would take all that we learned and mentioned above, and implement it. If the team next year works with Vibrio natriegens aswell, they could examine if teams that have worked with V. natriegens before had better results because of their experience with the organism. Probably by then we would have the VibriClone strain that can live in colder temperatures, eliminating the fridge problem.

Our vision is to establish our Vibrigens Interlab study over the next years by improving everything we learned this year, and subsequent years. Vibrio natriegens made an appearance in many labs in europe, but next year we want to expand to other continent, to share with more people how this organism can truly change the day to day workflow in labs everywhere.