Open Access Research

I happen to have a certain research paper for government due tomorrow morning, and I am not quite sure if I can use personal experiences in it. However, I have an official website, so I can post them here, and reference myself. 

 

Years ago, when I was in 7th and 8th grade, I consumed any biology tutorials and any cloning methods I could. A good half of what I researched I could never access myself. Never has there been the capability to educate so many people. There are those out there doing valuable research for the masses that are actively discriminated against with paywalls. It is frustrating to see scientists, such as Jeffrey Beall, who have never in their life been deprived of research due to a paywall. 

http://triplec.at/index.php/tripleC/article/view/525/514

From the above. “The OA movement is an anti-corporatist movement that wants to deny the freedom of the press to companies it disagrees with”. This is a wholesomely incorrect statement. I am an open access advocate because it affects me. I taught myself not from expensive textbooks and state-of-the-art libraries, I taught myself from the resources available to me as a middle schooler learning biology. I, personally, would only publish my work in open journals on a moral basis. I want the world to be better for people like me, not just put it behind a paywall. Saying open access “sacrifice[s] the academic futures of young scholars” is simply not true. I am a young scholar, and open access is the only thing that allowed me to get to where I am. 

While I respect Jeffrey Beall’s list of potentially predatory publishers, I believe there ought to also be a voice for many of the DIY biologists who can’t afford research. Though we all know where to get the articles… well.. we should not have to break the law in order to do good research for the benefit of all humanity.

Mesoplasma florum growth media

Lately I have tested out Mesoplasma growth media. It appears that it cannot grow in SOB + Tween80 but I suspect this may have something to do with sugar levels. When I analyzed the genome on KEGG, it was very simple, and therefore may not have all the genes needed to derive sugar from some other source than just plain sugar I add to the media. Interestingly enough, it has proteins required for the uptake and use of extracellular sucrose, unlike E coli. Really shows its background of being from a lemon tree rather from an animal. I wonder if other Mycoplasmas have this trait.

 

I’ve been slowly working on Vibrio natriegens. Going to try a basic electroporation according to the new published paper. Unfortunately, this does not help my Mesoplasma research because I require high transformation efficiency and conjugation machinery, something it does not have.

Vibrio natriegens transformations

So far I have gotten vibrio natriegens transformants using electroporation with pRL1342, which is addgene Plasmid #70691. They grow into large colonies overnight at room temperature.

Chloramphenicol stocks have to be diluted approx. 1/25 from E coli usage because V. nat is very sensitive to that condition. I had originally tried room temperature creation of electrocompetent cells according to “Vibrio natriegens, a new genomic powerhouse”, and not gotten transformants due to this. I will be attempting this protocol again at room temperature.

I got decent transformation efficiency, but nowhere near what I need for actual cloning. Will be investigating possible knockouts to increase efficiency. (50 colonies with 50ng plasmid approx)

Annealing oligos with CutSmart

To optimize annealing conditions, I tested the annealing of the following primers-

OligoSim_I-SceI-1_for AGAggtctcaccctCGCTAGGGATAACAGGGTAATGGCtacatgagaccGGC
OligoSim_I-SceI-1_rev GCCggtctcatgtaGCCATTACCCTGTTATCCCTAGCGagggtgagaccTCT
OligoSim_I-SceI-2_for ccctCGCTAGGGATAACAGGGTAATGGC
OligoSim_I-SceI-2_rev tgtaGCCATTACCCTGTTATCCCTAGCG

OligoSim-iSceI-1

 

In different NEB buffers. The following are the plate transformations (plated on chemically competent TG1 cells made with zymo mix and go)

Plate_19) I-SceI_1 primers + NEB 1

Plate_20) I-SceI_1 primers + NEB 1

Plate_21) I-SceI_1 primers+ NEB 1

Plate_22) I-SceI_1 primers +NEB 1

Plate_23) I-SceI_1 primers + Cutsmart

Plate_24) I-SceI_2 primers + NEB 1

Plate_25) I-SceI_2 primers + NEB 2

Plate_26) I-SceI_2 primers + NEB 3

Plate_27) I-SceI_2 primers + NEB 4

Plate_28) I-SceI_2 primers + Cutsmart

All were prepared using a CAS1200 robot with 200ul conductive tips. On attached plates, GFP colonies appear see through, and I also circled them. On some, TG1 was at the side of the tube, and a full 10ul wasn’t plated, and those plates were labeled with an L. It appears Cutsmart and NEB 1 were the most efficient and that I-SceI_2 was more efficient. This is a small test, and nothing was fully verified.

Overall, using bare oligos in cloning reactions looks viable.

Plate_28 Plate_27 Plate_26 Plate_25 Plate_24 Plate_23 Plate_22 Plate_21 Plate_20 Plate_19

UPDATE: sequenced OligoSIM and got 100% correct.

Largest phage

Bacillus megaterium phage G has a massive genome, sized 497,531bp

 

This phage is nearly the size of minimal bacterial cell. Would be cool to modify, but not enough is known.

http://ictvonline.org/proposals/2009.010a,bB.v1.Myoviridae_phageG.pdf

OligoSim

Currently I am testing whether or not I can directly use oligos to clone with in GoldenGate. Because of methods like PaperClip and BASIC, and also use of direct cloning to make sgRNAs, I assume this answer is yes. Planned experiments-

Best duplex buffer? IDT duplex or NEB RS buffers more efficient?

BsaI sites or raw overhangs? Is including BsaI sites at the end of oligos increase the efficiency because there is no requirement for T4 PNK, or less efficient, because it sequesters BsaI?

Complex parts? Can I anneal and clone terminators with high secondary structure?

Efficiency vs normal parts? (self explanatory)

OligoSim Map

Also shout out to protoza noah, your comments have been inspirational

pKGs1 cloning

I am cloning  F1 origin into pKGs2, which will then be used as a template for my other reactions on this ‘base’ vector. Should have it done very soon. Will update upon completion.

 

In other news, I bought a MacConnell MP4800 for automated minipreps. I am going to do a full write up on the machine later. Also bought a gene pulser electroporator. I am going to 3d print the safety part for the shock chamber, will post file when that is complete.