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First day in the lab. We were given all of the necessary safety training and debriefing. We toured our new lab area and cleaned out our lab space. At the end of the day we prepared a bacteria culture containing constitutive GFP (K584001) to grow overnight (E. coli in LB with chloramphenicol)
We learned the protocols of preparing plates by making and pouring the media. Because our overnights failed to grow the previous night, we performed a transformation in order to replicate our DNA with the constitutive GFP. We plated half of them on the plates we made that morning (containing chloramphenicol) and used the other half for another overnight batch.
We performed a mini-prep on our overnights with the constitutive GFP in order to isolate plasmids and verify that they were present through spectrophotometry. We also saw that a good amount of colonies had grown on the plates from the day before. We then started two separate 3A assemblies in order to piece together the pBAD promoter with the miraculin gene and the constitutive GFP gene to the SRNBC gene (homolog to Hok/Sok).
We performed a transformation in order to clone our new plasmids, one containing the pBAD promoter and miraculin gene and the other containing SRNBC and the constitutive GFP. We plated these to grow overnight. We also transformed β-cyclase, AppY, and CRTBEY genes in bacteria to grow on plates overnight.
We found that the RE cloning for the pBAD and miraculin gene construct produced colonies on our plates (the growth was not optimal but it was a good amount). Unfortunately, nothing grew on the SRNBC and constitutive GFP cloning plates. We then started a new RE cloning using 3A assembly for the SRNBC and constitutive GFP construct along with pBAD, constitutive colicinFy, and lysostaphin. Our final products were SRNBC + constitutive GFP, SRNBC + constitutive colicinFy, and pBAD + lysostaphin. At the same time that we prepared these digests, we also ran these 5 different DNAs through a gel in order to cut out the specific bands and later purify them in case the 3A assembly does not work. We were able to cut out all of the DNA except for the constitutive colicinFy gene and the pBAD promoter. We also prepared overnights for the pBAD + miraculin construct that grew on the plates and the β-cyclase, AppY, and CRTBEY genes. Day 6- 6/2/15
We performed minipreps on the overnights we had prepared the day before (for the β-cyclase, AppY, and CRTBEY genes) and shipped them off to be sequenced. We also performed transformations on the SRNBC + constitutive GFP, SRNBC + constitutive colicinFy, and pBAD + lysostaphin digests and let them grow overnight.
SRNBC + constitutive colicinFy plate grew, other two didn’t
RE digest on β-cyclase, AppY, CREB, and CRTBEY genes along with the PBS1C3 backbone and the pBAD + miraculin out of the PBS1A3 backbone so that we could move the genes to that backbone
Ran gel with all parts and cut out the bands (accidentally froze samples overnight)
miniprep on SRNBC + constitutive colicinFy and shipped off to be sequenced
performed gel purification on gels from day before but failed as didn’t show up in the spectrophotometer
still tried ligation on pBAD with PSB1C3 and PSB1A3
both transformations grew
order G blocks
epsilon cyclase, pyosin-->find cDNA→ optimize
Hi guys this is where we'll log what we did each day. It'll be especially helpful for people who miss days.
Make LB broth
make ampicillin (50mL of 2.5 g) 500X
We visited the UMD Bioprocess Scale-Up Facility which is “dedicated to the development and scale-up of fermentation biotechnology products and processes, specifically bacteria and yeast.” Although our project is more focused on purifying protein from E. coli it was good to tour the facility and learn about the possibilities of commercializing experiments, especially with the resource being right on campus. http://www.brep.umd.edu/bsf.html
We performed minipreps on pBAD+Miraculin in the PSB1C3 backbone as well as the const. GFP+SRNBC which we then performed RE digests on-- EcoRI and PstI on pBAD+mir and XBa1 and Pst1 on SRNBC+const. GFP. We ran culture of pBAD+miraculin was induced with 0.1% arabinose at OD of 1 arabinose acts like lac operon
arabinose binds to AraC repressor
want to activate promoter in log phase bc have greatest amount of growth, highest metabolic energy, generally in the middle OD=> A_600nm
tomorrow:
lysing:
chemical: P2 buffer→ chemical lyses using sodium hydroxide because base destroys the ester bonds in membrane
SDS→ has hydrophilic end and hydrophobic end
mechanical:
sonication
French press
under very high pressure, pushing bacteria through tiny hole smaller than them=> liquid mixture
doesn’t effect proteins→ still soluble
centrifuge
put into FPLC (Fast Protein Liquid Chromatography)--beads with cobalt which his tags will bind to so that miraculin is stuck on cobalt
elute proteins
SDS PAGE-- (sodium dodecyl sulfate polyacrylamide gel electrophoresis)=> SDS makes protein linear and gives uniform charge
enzymatic
thermal
electrical
Performed a gel extraction of SRNBC, const. GFP, and pBAD which we then ligated them together to make the SRNBC construct. We also attempted to purify the miraculin out of the induction culture by first using a French Press to lyse the cells and then running it through cobalt bead column by FPLC (Fast Protein Liquid Chromatography). We then ran the resulting 62 elutions through an SDS-PAGE.
Results from day before:
There were no bands in the gel, concluding that the purification process failed.
We performed transformations of the constitutive GFP+SRNBC construct. We also performed PCR on the PSB1C3 backbone in order to amplify it in preparation for a Gibson Assembly with the Hox/Sox gene.
Update: transformations of SRNBC/const. GFP failed
Made overnights of pBAD+Miraculin culture to prepare for test inductions tomorrow.
Discussed future of lutein project with Dr. Cunningham who was able to make E. coli that produce lutein.
We inoculated 3 test cultures of pBAD+Miraculin in LB broth with Ampicillin. We then induced the tubes with 0.05%, 0.1%, and 0.2% arabinose in order to initiate production of miraculin at an OD of 0.4.
We prepared the induction samples for SDS-PAGE by separating out the media, supernatant after lysing with 200 uL SDS and then the supernatant after lysing with SDS again (pellet supernatant). We ran samples of 0%, 0.05%, 0.1%, and 0.2% arabinose through the gel. Unfortunately, no bands showed up for any of the samples.
In order to move the Hok/Sok gene in the PSB1C3 backbone, we performed an RE digest on the PSB1C3 backbone with XbaI and SpeI and then ran it through a gel to separate it out. We extracted it from the gel and then ran it through a gel to determine the concentration of the sample.
Week 4 summary:
RE cloning with pBAD+Miraculin and SRNBC