The following is writing for my 10th grade colicin engineering project. I was 15 at the time. I was kicked out of the science fair for doing this work. I personally believe their reasons were good in theory, but bullshit in practice. I did not do anything dangerous, and I knew that I did not do anything dangerous. Teens might make poor decisions sometimes, but they are not stupid, and I was not stupid. At the bottom is their full email.
If you're interested in pictures, please check out these PDF files! They have lots of pictures of my process.
I took a lot of pictures because I wanted to prove that I could do a project at home, and do it well, better than those kids who get into their parent's labs and do whatever shit a graduate student has been doing for years. I had the opportunity to lie, to just say I did the project at LA Biohackers or somewhere else, but I specifically decided against it and held steadfast to my principles. Didn't get me that far in the science fair, at least.
Antibiotics are slowly losing their effectiveness as new resistant strains of bacteria emerge, and are dangerous if used accidently used against communal gut bacteria. There, however, is a natural alternative. These are the bacteriocins.
Bacteriocins that are produced by bacteria specifically target their own species, with a resistance protein encoded on a plasmid. One in particular, colicin V (also named microcin V, and will be referred to as ColV) is an bacteriocin-producing operon has been heavily studied in E coli cells.
Microcins, a specific type of bacteriocin, are small peptides exported from the cell using dedicated transport proteins. They are mostly found on mobile plasmids, which use the antibiotic properties to gain an advantage in the population.
The ColV system can be used in the lab to kill rival populations of E coli cells. I have verified this, and also present the idea of the “critical concentration” using new simulated real time data. In addition, I have disproven theories on using ColV as a selection mechanism for transforming cells, supporting the idea presented above.
I have also cloned plasmids to test if different types of bacteriocins can be fused to the ColV toxin, allowing a cell to produce various amounts of different toxin proteins, increasing its antibiotic properties. In the future, this can be used to make probiotics that make humans immune to several different gut based infections.
All materials used in the experiment have been acquired by the student prior to beginning. Because of the shear variety of different materials that are used in synthetic biology, it is best to refer to materials needed protocol by protocol.
Specifics on each protocol are included in the back of the experimental notebook
PCRs were done according to the recommended NEB protocol:
https://www.neb.com/protocols/2013/12/13/pcr-using-q5-high-fidelity-dna-polymerase-m0491
Gibson assemblies were done with Hifi Mastermix by NEB:
https://www.neb.com/products/e2621-nebuilder-hifi-dna-assembly-master-mix#tabselect0
Electrophoresis gels were done according to the addgene protocol:
https://www.addgene.org/plasmid-protocols/gel-electrophoresis/
Gel and DNA cleanup were done according to the mobio protocol:
http://www.mobio.com/images/custom/file/protocol/12100-300.pdf
DNA transformations were done according to the lucigen protocol:
http://lucigen.com/docs/manuals/MA010-Ecloni-10G-Chem-Comp.pdf
Minipreps were done according to the mobio protocol:
http://www.mobio.com/images/custom/file/protocol/12300.pdf
All protocols used to construct this plasmid are listed below. Sequenced by genewiz.
Unless otherwise noted, protocol here-https://www.neb.com/protocols/2013/12/13/pcr-using-q5-high-fidelity-dna-polymerase-m0491 was followed. Tms were calculated using http://tmcalculator.neb.com/ .
(1) ColV_full
Primers
ColV_Full_for (3-13) 55c
ColV_Full_rev (3-14) 56c
Conditions
Tm: 58c
L: 2:30s (4.4kb)
T: ColV gDNA
(2) pUC19-ColV
Primers
pUC19-ColV_for (3-19) 55c
pUC19-ColV_rev (3-20) 55c
Conditions
Tm: 58c
L: 2:30 (2.6kbp)
T: pUC19
Unless otherwise noted, gels were cleaned up using this kit with it’s attached protocol- http://www.mobio.com/images/custom/file/protocol/12100-300.pdf
Unless otherwise noted, used PCR reactions and HiFi protocol from neb- https://www.neb.com/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol
(1) pUC19_ColV-gib
7µl ColV_full
3µl pUC19-ColV
10µl 2x HiFi Mastermix
Unless otherwise noted, used lucigen’s 10G cells with attached protocol- http://lucigen.com/docs/manuals/MA010-Ecloni-10G-Chem-Comp.pdf
Unless otherwise noted, mobio’s 6 minute miniprep is used http://www.mobio.com/plasmid-minipreps/ultraclean-6-minute-mini-plasmid-prep-kit.html
The Critical Concentration is the idea that there must be a certain concentration of colV producing bacteria to effectively overcome the metabolic burden of the system and take over the population.
The charts above are from data collected during the log growth experiment. In essence, the log growth experiment tested pColV in simulated “real time”, observing the population dynamics of two rival E coli strains, one with pUC19 and one with pColV.
In a ratio of 1:1 with pColV and pUC19, respectively, it is observed that pUC19 grows quicker than pColV. This is also supported by the 2 control plates. However, even if the pUC19 concentration is nearly 5 times higher than the pColV concentration, once pColV reaches a critical concentration, it takes over the entire population. In a ratio of 10:1 with pColV and pUC19, respectively, we can see clean takeover of pColV. The pUC19 population struggles to survive, but eventually fails, collapsing the entire pUC19 population. This test clearly demonstrates, along with 1:10, that pColV is capable of killing rival E coli cells, as said in literature. In a ratio of 1:10 with pColV and pUC19, respectively, the pColV population stays stable at ~500,000 cells per µl. Even with the pUC19 population vastly outnumbering the pColV, it fails to disappear from the population pool.
This data, taken together, tells a story that pColV must be at a critical concentration to become an effective killer. The minimum supported by the experiment is 5,000,000 cells per µl. Moreover, this data shows that the pColV plasmid is producing secretion machinery and actively secreting peptides. This machinery can be manipulated later to do interesting things (see future prospects)
In the past, there have been many assays into how colV functions (specifically 2-2-1-2). These functional assays almost always take place on agar dishes, giving clean data in the form of inhibition zones.
I attempted an inhibition assay without soft agar, and although it worked, the data from this was unclear. It is interesting to note that colV purified from simply supernatant has no effect on other local E coli cells (2-2-1-5). I verified these results. After .2µm filter purification, supernatant was added to bacterial lawns. Although inhibition was clearly observed with kanamycin, there was no such inhibition with colV. For this to be used as a selection marker, there has to be cells actively producing colV.
For more than a year, the DIYbiology community has had the idea of using an antibiotic resistance cassette for self-selection in transformations, getting rid of the need for antibiotics. I decided to place this idea to the test.
After transformation, I aliquoted cells directly to liquid culture without selection. After a day of growth, I plated the E coli onto agar plates, now selecting for the antibiotic. Hypothetically, if pColV was able to take over the population, there should be a plate full of ampicillin resistant cells. Unfortunately enough, this was not the result. Supported from the data from the log growth experiment, my hypothesis is that the pColV cells could not achieve the critical concentration to outcompete the faster-growing untransformed cells. It appears from this data that colV is a poor choice self-selecting transformants.
This research supports the idea that new potent probiotics can be created to take over virulent bacterial populations. These probiotics in the gut would produce a large array of different colicins and microcins. Since they would be in the gut first, the critical concentration would be reached, and the body would be immune to new pathogenic bacteria attempting to enter the system. I am currently testing the applicability of fusing new colicins to colV for a larger variety of possible toxins.
Colicin E2 is a DNase, and therefore exits the cell and enters a new cell to destroy the rival’s DNA. The implications of this are huge: a protein can be expressed in one cell, exported from that cell, imported into a new cell, and function in the new cell. Destruction of the DNase domains from E2 would give a chassis for non-quorum sensing communication. Hypothetically, this could be used to target wild populations of cells for gene specific activation, repression, or mutation.
Based on this idea, TALENs could be specifically designed not only for killing a certain species, but killing a certain plasmid inside of the species, leaving it unharmed. This ability can “disarm” any intruding virulent strains and make them docile. Off-target cutting would be minimal, as the cell not only has to have the receptor for the colicin but also the sequence for the TALEN to cut. You would be immune to pathogenic cells, and your communal bacteria would act as an immune system targeting virulent cells and specifically killing them, without the need for expensive and sometimes dangerous antibiotic treatment.
To become familiar with the colicins, I read several papers listed below. All are included in my research notebook. I included my notebook reference number, PMID #, and name for convenience.
2-2-1-1 Four Plasmid Genes Are Required for ColicinV Synthesis, Export, and Immunity
2-2-1-2 Genetic Analysis of the Colicin V Secretion Pathway
2-2-1-3 Evolution of Microcin V and Colicin Ia Plasmids in Escherichia coli
2-2-1-4 ColicinV Virulence Plasmids
2-2-1-5 The leader peptide of colicin V shares consensus sequences with leader peptides that are common among peptide bacteriocins produced by Gram-positive bacteria
2-2-1-7 Bactericidal Activity of Colicin V Is Mediated by an Inner Membrane Protein, SdaC, of Escherichia coli
2-2-1-8 Class II Microcins
2-2-1-9 Modular Structure of Microcin H47 and Colicin V
2-2-1-10 Colicin E2 is a DNA endonuclease
2-2-1-12 Structure of the Ultra-High-Affinity Colicin E2 DNase–Im2 Complex
2-2-1-13 Double-glycine-type leader peptides direct secretion of bacteriocins by ABC transporters: colicin V secretion in Lactococcus lactis
I would like to give special thanks to my parents and grandparents for being so supportive during my experimentation, and the small grants they gave me to continue my research. I would also like to thank Andreas Stuermer for sending me the ColV gDNA where I got the ColV operon from.
Dear Keoni,
Thank you for your SRC submission for your project "Colicins: a self-selecting alternative to antibiotics", as described below.
The three SRC reviewers had the following comments/suggestions:
reviewer 1:
Please provide a letter from the principal investigator in the lab where the project was / is completed. The letter must indicate which procedures were performed by the student and which were performed other scientists (what was the work of the student). It should also confirm that you received appropriate EHS / Biosafety training for all procedures performed in the laboratory.
Please also provide a hazardous materials certification for this project.
reviewer 2:
Comments for Keoni: Please correct your application to indicate you are performing this project at the university. Please provide a hazardous materials certification that describes the materials, procedures, safety precautions and qualified scientist. Note that if this is a continuation project submitted for ISEF, you will be judged only on work performed between January, 2014 and end of May, 2015. Please include a letter from Principal Investigator with Biosafety Committee approval for this project, confirming Committee approval, the BSL level of the work and the procedures you will be engaged in. Note that Students are prohibited from culturing certain antibiotic resistant strains (Carbapenem Resistant Enterobacteriacae) or genetically engineering bacteria with multiple antibiotic resistance.
reviewer 3:
Agree with reviewers 1 and 2 above. Student needs to provide information on university lab and supervisor. Also information on EHS/Biosafety training and which procedures were performed by student or supervisors. Hazardous materials certification form is attached with this email.
Please address all comments/suggestions from the three reviewers and resubmit your SRC forms with the needed information.
Looking forward to your SRC resubmission.
From,
SRC Committee (OCSEF), dated March 12, 2015.
I apologize for the misunderstandings.
I did not include any information about any university because I did not do this project at a university. I did this at my home DIYbiology laboratory. Because of this, I am technically the PI of this lab. I attached a hazards safety, but it may have not gone through
For the qualified scientist portion, I have worked at a university lab for almost 2 years continuously and have done synthetic biology work for over 3 years in my home lab. To prove this, I have included in this email some e-courses I had to take on safety to be able to work at UCI. I have also included pictures of my home lab.
I only worked with E coli K-12, so this is BSL 1 project. No disease related toxin proteins were ever used in this experiment. All bacteria were modified to only have the ampicillin resistance marker, no other antibiotic resistance markers.
If current training I have in synthetic biology by working in a lab and completing all necessary courses on the subject are not enough to satisfy the requirements as a qualified scientist, then I would like to remove my project from the science fair.
-Keoni
Dear Keoni,
The OSCEF SRC has determined that your current science fair project has to be withdrawn from the 2015 OCSEF/ISEF competition for the following reasons:
1. The OCSEF is required to follow ISEF regulations regarding science fair participation by pre-college students. When you submitted your science fair project to OCSEF/ISEF, the OCSEF SRC was required to check whether you complied with these regulations. For example, only some types of procedures and projects are allowed for science fair participants. In some cases, only the qualified adult supervisor (not the student) is allowed to do specific scientific procedures. The rules also specify that a home lab set-up (where you indicated your project was performed), is not allowed for all types of experiments. Summaries of the current rules/regulations are attached with this email. Universities like UCI also have their own set of regulations which are available on their university website. The university rules regarding minors in labs are not necessarily an exact duplicate of OCSEF/ISEF regulations, so each set of rules/guidelines must be read carefully , as needed.
2. The three OCSEF SRC members who reviewed your project were in agreement that your 2015 project did not follow the required rules and guidelines, and for this reason your project cannot be
included in the current 2015 OCSEF/ISEF competition finals. This does not mean that you cannot participate in future OCSEF/ISEF competitions next year and beyond. You have good potential for
science research and we hope to see you participate in future competitions with an appropriate adult supervisor who can confirm that the OCSEF/ISEF regulations were followed and adhered to.
3. It is unfortunate that you did not appear to have a scientific supervisor this year who could have caught the problems with lab set-up rules and safety guidelines early on. For the current 2015 competition it was noted on the last page of the certification of hazards control, there is a parent guardian signature ("I certify that I have reviewed the procedures described above and agree to supervise the above named student and assume primary responsibility for compliance with the existing rules and regulations pertaining to hazardous materials") and also a certification by the teacher (Marcos Cabrera): "I certify that I have reviewed the procedures described above and agree to sponsor the above named student and assume responsibility for compliance with the existing rules and regulations pertaining to hazardous materials."
Wishing you the best in your future science research endeavors. Please contact us if you need OCSEF/ISEF guidelines and rules clarified for future projects.
From,
OCSEF SRC (dated April 9, 2015).