Team:CLS CLSG UK/Methods
Credit - Michael Longmire@unsplash
Methods
Introduction
We planned out all of our lab work in the hope that we would be able to get into the labs during summer. We were unable to do this, however, due to the pandemic. Given this, with our school reopened and all our protocols planned we were still eager to try and run some experiments. And so, we ran a set of experiments to test one of our genetic constructs, the synthetic adhesin. Included below is our protocol details for this experiment as well as the details for our planned experiments.
Experiments
Test of adherence of Synthetic Adhesive expressing E coli to antigen bound ELISA plates: 100µL of Purified GFP (Sigma) at a concentration of 10µg/mL was adsorbed into ELISA plates (Fisher Scientific), with PBS (Sigma) being used as a negative control, overnight at 4°C. The antigen covered plates were washed with 200µL of PBS one time before being blocked for 1 hr at room temperature with PBS containing 3% (w/v) microbiology skimmed milk (Sigma). Next 100µL of bacterial suspension at a transmission of 9 ± 4% at 590 nm on the colorimeter (WPA colour wave C07500) was added. The suspension was made up of LB broth (see supplementary materials) that was inoculated with either the S.A. E. Coli (Spanish department of microbiology biotechnology) or K12 E. Coli (Breckland Scientific). The plates were then incubated for 1 hr at 37°C before being washed. One plate was washed with PBS and the other was washed with our synthetic sewer water (see supplementary materials), each column was washed a different number of times (columns 1 and 8 were washed once, columns 2,3 and 9,10 were washed three times and columns 4,5 and 11,12 were washed five times. This, as well as the layout of our plates in a visual way can be seen in the supplementary materials). After this we stained each well with 1% crystal violet solution before washing with distilled water.
N.B. any materials detailed above or in the supplementary information in without the supplier included was from school stores.
Plate Layout
We ran two 12x8 Enzyme Linked Immunosorbent Assay (ELISA) plates that were laid out as so:
Both plates were laid out in the same way, the only difference was the solution used for the final washing of the plate: plate 1 was washed with PBS while plate 2 was washed with the synthetic sewer solution.
By laying out the plates as so we were able to test the Synthetic Adhesin expressing E. coli against various conditions, and those conditions against a K12 E. coli strain, allowing us to conclude that it was in fact the very nature of the antigenic surface combined with the Synthetic Adhesin present on the membrane that was the cause of the greater ability to adhere. As well as this, by washing different rows a different number of times we are also able to test the effect that washing has on the ability of both K12 and the Synthetic Adhesin expressing E. Coli to adhere to surfaces.
LB plate composition
This protocol makes 500mL LB agar for 20-25 LB agar plates. It can be scaled up and down according to needs.
Please click here to see full protocol.
N.B. this protocol was taken directly from the High School guide made by the Calgary 2014 iGEM team, it can be found here.
LB Broth Composition
This protocol makes 500mL LB agar for 20-25 LB agar plates. It can be scaled up and down according to needs.
Please click here to see full protocol.
N.B. this protocol was taken directly from the High School guide made by the Calgary 2014 iGEM team, it can be found here.
Synthetic Sewer Media
Our lab work was focused on testing the effectiveness of synthetic adhesins incorporated into the chromosome of E.coli in comparison to normal strains of K12 E.coli. To ensure accurate testing of our adhesion assays, we used a synthetic sewer media to wash our plates with (instead of PBS), in order to replicate the biologically significant conditions present in the sedimentation tank. We adapted the river media designed for the Athabasca River by the 2012 Cornell iGEM team to apply to our analysis of the water quality and chemical composition of the River Thames.
Please click here to see full protocol.
Lab notebook
18/9/20
Making up of plates of the K12 and SA strain from a slope and stab respectively.
23/9/20
Made up the first set of suspensions of both strains also practised micropipetting into epindorphs and wells. Also learnt about aseptic technique and ran through full method. Made up suspensions from the plates.
30/9/20
Tested the first set of suspensions with the colourimeter and made up a new batch of suspensions for further testing.
1/10/20
Further safety briefing focussing on waste disposal and general practise in the lab in order to start proper lab work. Also new plates made up of both strains, from the old plates.
5/10/20
Tested most recent sets of suspensions, but the growing rates didn’t fit with the previous rates, it was thought that refrigerating the broths before incubation as well as having the lids on tighter may have caused the difference in growing rates.So, inoculated more broths and incubated them, the incubator was also slightly butchered in order to be fitted with a shaker to help them grow.
6/10/20
Tested the new broths and they seemed to be growing at an expected and similar rate to each other.
8/10/20
Made up a batch of new broths that were left incubating with the shaker and the same conditions as the previous set. Also prepared a slide of the e coli in order to confirm that simply staining with crystal violet was a legitimate method for visualising our bacteria, and while the stain was rather thick it seemed to work, maybe further experimentation with safranin or other stains would be useful.
12/10/20
We measured the transmittance of the most recent broths, they now all follow the same trend and we have a very large amount of data on the growing rates of e coli strains now. We made up the final set of suspensions for our proper lab work.
13/10/20
With the pandemic I never thought we would get here, but we have! Lab work started today and we made up a solution of GFP in PBS and micropipetted it into our ELISA plates, as well as PBS into some wells as a control. The ELISA plates were then left in the fridge. We also used one of the leftover plates to make up slides of the E. coli and stained them with both safranin and crystal violet in order to compare and contrast the effectiveness of the different strains. We think we have settled on crystal violet now.
14/10/20
We continued with our lab work today, we first washed the GFP from our plates, before blocking them with a microbiological milk solution. And leaving them covered for an hour at room temperature. We then washed the plates three times with PBS, before micropipetting in 100microlitres of bacterial suspension in. This was incubated at 37 degrees for an hour before the final washing with either PBS or our synthetic sewer water, we washed different rows a different number of times, see the full practical details for which rows and how many. We then stained them with crystal violet solution and washed it off with distilled water. We only had a chance to have a quick look under the microscope before needing to leave for cleaning, so the plates were placed in the fridge for observation and recording of data the following day.
15/10/20
We had not really anticipated the fact that a high powered microscope lens would be unable to focus on our bacteria due to the thickness of the bottom of the wells, due to this we don’t think we will be able to get any objective quantitative data. However we have collected strong qualitative data that supports the results of the original paper and these will be published on our wiki. In order to attempt to obtain some quantitative data we have created a scale by which to compare each well to and so rank it, this will also be published on the wiki.
Safety
All members of the team taking part in wet lab work were briefed on the safety rules prior to carrying out any experiments. This included safety protocols on how to ensure that bacteria remained uncontaminated, correct measures of disposal to prevent bacteria or harmful substances from leaking into the wider environment and protocols to carry out if any of the previously mentioned events occurred. Although all lab workers have had experience carrying out experiments in the lab, we were reminded of our school laboratory safety regulations, attached here [INSERT LINK]
As a High school in the United Kingdom we are required to comply with very strict government regulations, which we followed word for word. All work carried out in the lab was supervised by a teacher of the school Biology department who has experience in microbiology and aided by a laboratory technician to enforce the lab safety rules and answer any questions regarding safety during the experiment.
As well as this, all members of our team had a briefing from the school biosafety officer, a previous iGEM judge and PI. Due to this he was able to brief us in depth both on general lab safety but also more specifically on lab safety with respect to iGEM and genetically modified organisms.
Further to this, any individuals partaking in lab work attended the safety seminar put on by the iGEM ambassadors as part of the preparation for the lab work and to help us begin thinking about safety when planning our experiments and when filling out the safety form. We also filled out the safety form in good time before our lab work so that any feedback we received from it could be taken into consideration, as such the feedback was all positive and we received confirmation that all was in order regarding the safety form before we began lab work. Having the utmost safety in mind we also checked in one of the organisms we would be using even though this was not required as we felt that this was the best way to ensure the safety of those performing the work and the safety of the wider environment.
Whilst the labs were not in use, they were locked and only accessible with an access card belonging to a member of staff; no students are permitted to enter labs unless supervised by a staff member. Any biological material that we were not using or was being left out for the purpose of the experiment was kept securely locked in the lab technicians store room.
Handling Bacteria
The two E. Coli strains our team used were SA (an e Coli strain producing Synthetic Adhesins, enabling to stick to certain antigenic surfaces) and K12 (used in our experiment to give a comparison) which are both biosafety level 1 and therefore presented very minimal hazard to lab workers and environment. Nevertheless, workers made sure to clean the work area with 70% concentrated ethanol and use sterile equipment prior to handling the bacteria. A bunsen was also kept on yellow flame, whilst bacteria were exposed to the air and any equipment which came into contact with the bacteria was immediately put into a beaker containing Virkon.
Micropipetting
Workers pipetted purified GFP, skimmed milk (used for blocking) and PBS (used for washing) into allocated wells on ELISA plates. Workers avoided directly breathing over any wells as it could cause contamination of bacteria. Furthermore, when bacteria was being pipetted into wells, a new pipette tip would be used after each well was filled and the previous tip would be put into Virkon for disinfection.
Waste disposal
Excess PBS or skimmed milk after washing was not poured down the drain but instead emptied onto paper towels which were put into autoclave bags then taken to be autoclaved. We also did this with used pipette tips, gloves and plates.
Chemical Safety
None of the substances used were corrosive or toxic. Nevertheless, all workers wore microbiology laboratory coats and safety spectacles for the entirety of lab work. In the case of any spillages, we were briefed on what to do depending on the substance. We were also told that any breakages of glass should be immediately reported to the teacher supervising and made aware of the location of the glass disposal bin.
Spillages
Spillages were covered in 70% ethanol or virkon and left for 10 minutes before being wiped up and the tissue then disposed of in an autoclave bag. Before the surface could be used again it was wiped down.
Accidents
All accidents, including trips and slips, as well as near accidents were recorded and documented in the official and correct channels by those individuals supervising us, be it technicians or teachers. Luckily we had no need for this as there were no accidents are close calls besides the occasional spillage.
Planned Labwork
Unfortunately we were unable to carry out all the lab work we wanted to, due to the pandemic. But despite this we had still planned out everything in full, so included below is the protocol list we would’ve followed as well as the complete inserts and plasmids.
This is a reduced version of our full spreadsheet (so that it could fit on the wiki):
If you would like to access the full spreadsheet it can be downloaded here (link).
As we intended to use the mutant form of the enzyme, we initially constructed this insert:
However when we came to order this from IDT there were various issues regarding complexity. The first was that the mutant form, even after codon optimization was too complex, and so our lab work would have to be performed with the wildtype enzyme. As well as this, the kill switch was also too complicated to synthesise as a whole. After running codon optimization IDT would have been able to synthesise it in two parts, requiring another set of digestion and ligations. And we would aim to create this as our final insert:
We would’ve inserted this into a standard backbone and giving us this:
We have also included some of our other preparatory work about different genetic vectors and how to deal with the problems some of them pose, it can be found here.