Week 1
Tuesday, May 20 - Sunday, May 25
iGEM 2014 had their first meeting with Dr. Salis and Dr. Richard. Ashlee, Emily, Clay, and Sam met each other. Emily will help Ashlee continue work on her Honors Thesis with the project "Engineering a Biodetoxification Pathway for Lignocellulosic Feedstock". Ashlee began to show Emily around the lab and instruct her during her first cloning experiences while they continued Ashlee's semester research trying to add a terminator upstream of the HMF pathway where dCas9 would be inserted. The dCas9 system has a transacting RNA sequence which could disrupt upstream genes if it was not turned off correctly.
Clay and Sam began work for Clay's Honors Thesis in Biological Engineering on the "Codon Optimization" project. Clay and Sam began preliminary research and worked on constructing a program to optimize GFPs.
Week 2
Monday, May 26 - Sunday, June 1
Ashlee and Emily saw no success with adding the terminator and must move on to a new approach. With the advice of Dr. Salis and graduate student Iman Farasat, they decided to insert the HMF pathway and the dCas9 system into the genome using homologous recombination and the Lambda Red Recombinase system.
Clay and Sam successfully design a program in Excel to optimize genes, continue working on one to be used in MATLAB. Work begins on designing a plan for obtaining useful data to show efficacy of codon optimization, as well as a plan for this cloning in more specific terms.
Week 3
Monday, June 2 - Sunday, June 8
Ashee and Emily constructed parts of Plasmid 1 via PCR Rescue and colony PCR. Genome overlaps 1, 2 and the kanamycin resistance cassette and ColE1 replication origin were constructed and assembled via Gibson CBA. Other plasmids necessary for the completion of the project were prepared.
Decisions made to use restriction site cloning to introduce synthetic GFPs into pFTV. Also, determined that best method of obtaining the genes is ordering them as gblocks from IDT. Problem of RBS library anticipated, synthetic leader sequence developed in order to work around the problem of the coding sequences being different.
Week 4
Monday, June 9 - Sunday, June 15
The 4-Part Gibson CBA was sequenced and determined to have all the correct junctions - our Plasmid 1 is complete. Attempts were made to insert the dCas9 system into the Plasmid 1 construct via ligation.
Project Plan updated extensively as mistake in design of gblocks realized (truncation) as well as the addition of a fifth gblock (slow insertion time). Programs written to optimize for insertion time as well as total the insertion time for existing genes. Restriction enzymes re-chosen and gblocks ordered. Project plan modified to include a gene first, RBS later strategy.
Week 5
Monday, June 16 - Sunday, June 22
However, the ligation from the previous week was not successful. We believe this was due to recently expired ClaI restriction enzyme or expired DNA ligase buffer. Both were replaced. We attempted to insert the dCas9 system and Lambda Red Recombinase system simultaneously (bypassing the need for ClaI) by performing a 2-part Gibson CBA. Upon PCR amplifying and running on a gel, the CBA also failed.
Gblocks amplified with rescue PCR, digested along with pFTV, ligated and transformed into cells. Additional design steps taken toward finding suitable RBS library. Began to work on website and other iGEM forms.
Week 6
Monday, June 23 - Sunday, June 29
The Gibson CBA of Lamba Red Recombinase and dCas9 system was repeated at 25 femtomole per DNA product and 50 femtomole. Emily and Ashlee worked on their presentation for the Center for Science and the Schools. We met with Dr. Richard to discuss incorporating his biomass hydrolyzer into our research and to evaluate the economic impact of our project by using the NREL AspenPlus model and economic analysis of a biorefinery. We had our weekly meeting with Dr. Salis and Dr. Richard to update them on our progress. Dr. Salis helped to tweak the website and offered suggestions for improving the design.
Colonies sent for sequencing, discovered that no insert was present. Decision reached to attempt digestion, ligation, transformation again.
Week 7
Monday, June 30 - Sunday, July 6
We prepared and digested 10 colonies of Plasmid 1 to ensure that the backbone is correct. Four of the digested plasmids were sent for sequencing. More work was done to update and design the website.
Problems realized with inverse PCR. Several attempts made to optimize this reaction. Success near end of week as strong bands observed. These will be used in the next digestion, ect. Digestion, ligation, transformation carried out again, cells plated. No colonies observed.
Week 8
Monday, July 7 - Sunday, July 13
We prepared the Plasmid 1 backbone and dCas9 system for ligation. New backbone colonies were sequenced and cryogenic storage was prepared. We met with Matt Johnson from the Center for Science and the Schools and he gave us good feedback on how to structure our presentation to the NewBio teachers.
Standard enzymatic digestion, ligation, attempted one more time, with sufficient optimization from the last two attempts. Used Chiam's electrocompetent cells, as it is suspected that ours are bad. Colonies observed, DNA harvested, digested to see if expected bands observed, sent for sequencing. Sequencing results show presence of an insert, but also include significant regions of ambiguity. Attemps made to send for sequencing on campus. Numerous improvements made to plasmid harvest protocol. Improvements made to journal and updates made to website. Skills gained in html, CSS coding. Problem with inverse PCR is discovered. The reverse primer tail was incorrectly designed and was fixed. Unfortunately, this means that virtually everything earlier than this was likely failing because of this error, which cost us a lot of time. Primer re-ordered.
Week 9
Monday, July 14 - Sunday, July 20
Ashlee and Emily digested 12 dCas9-Plasmid 1 ligation colonies and sent them for sequencing. The size of the band on the gel were not what we expected, so we hope the sequencing results will shed some light on this. We continued to work on the presentation for the Center for Science and the Schools. We made preparations to attempt our first homologous recombination to insert the dCas9 system into the genome.
The great miniprep adventure of summer 2014! All five GFPs re-cloned, and all of the five variant GFPs had colonies on their respective plates. The decision was made to pick six (6) colonies from each plate and prepare them for plasmid harvest. These thirty (30) samples were picked, minipreped, and streaked so that they could be saved for future analysis if needed. All of the samples had acceptable concentrations after plasmid harvest, so each sample was digested with Xma1 and Sac1 to see if the correct bands would appear when run on a gel. The samples that showed the bands in the correct part of the ladder were selected to be sent for sequencing in the upcoming week.
Week 10
Monday, July 21 - Sunday, July 27
Before Ashlee and Emily left on vacations, they plasmid prepped eight plasmids containing dCas9. These and three CBA colonies were sent for sequencing. The team had a meeting with Dr. Richard to discuss some upcoming dates and to get feedback on the progress of our projects.
More results of cloning sent for sequencing. Unfortunately it appears that the R2 sequencing primer is bad (probably too much secondary structure, as it was erroneously designed to anneal within the terminator) and if so, this is the source of a lot of our headaches. Developed a presentation for SCIENCE U, an interactive presentation to give to high schoolers at a science camp at PSU.
Week 11
Monday, July 28 - Sunday, August 3
After returning from vacation, Ashlee and Emily checked the sequencing results. Only one colony from the ligation (termed "L6") had dCas9 inserted in the plasmid. No other colonies were found to have the proper sequencing. L6 is now the most important plasmid we have! More digestions and transformations were done to keep the flow of the project moving well.
Science U presentation goes extremely well. Instructors are happy, kids were very engaged and seemed to learn a lot. Cloning and sequencing continue, with the goal being the confirmation of all five variants so that progress can be made to inserting the RBS library and characterizing.
Week 12
Monday, August 4 - Sunday, August 10
Sequencing confirms two of the GFPs correct, two more likely correct, and one (GFP4) still lagging behind in cloning. Progress made by inserting dRBS and transforming cells. Unfortunately, numerous tries are needed until this is done successfully. Oligos containing the dRBS are re-annealed more than six times, each time with minor improvements to the protocol. Sequencing continues, and G4 is re-cloned.
Week 13
Monday, August 11 - Sunday, August 17
Construction of Plasmid 2 and 3 started.
Finally, lots of colonies found on plates with G2 and G3 that contain the dRBS. Colonies glow, which is encouraging, cryo stocks prepared, then TECAN measurements began for G3. Old plates cleaned out, cryo stocks prepared for anything that seems like it might be useful, and rest is throw out. Clay leaves for Spain.
Week 14
Monday, August 18 - Sunday, August 24
Week 15
Monday, August 25 - Sunday, August 31
Week 16
Monday, September 1 - Sunday, September 7
Week 17
Monday, September 8 - Sunday, September 14
Lab move! Our lab needed to be moved to a new building as the old one was beginning renovations. This week consisted of organizing and packing up all of our materials and DNA samples. This was a lengthy process and took up the time we needed to make progress on the projects.
Week 18
Monday, September 15 - Sunday, September 21
Lab move! This week the lab needed to obtain specific access to our new building for weekends and nights. We set up and organized the new lab area and got ourselves adjusted to everything. Research will begin next week!
Week 19
Monday, September 22 - Sunday, September 28
Things are finally up and running in our new lab space. More efforts have been put into the Codon Optimization project due to the time constraints before the Jamboree and Wiki Freeze.
Week 20
Monday, September 29 - Sunday, October 5
Emily spent time in lab over the weekend plasmid prepping and transforming as well as adding to the Wiki and making things look nicer.
Week 21
Monday, October 6 - Sunday, October 12
Things with codon optimization started to pick up again. Ashlee and Emily spent time coordinating with Sam to make sure that adequate process was being made. They helped to fill in the blanks when it was hard for Sam to come in to lab based on classes and work schedule. They were excellent team players and helped to make sure that we had a part available to send to the registry!
Week 22
Monday, October 13 - Sunday, October 19
Improvements made to codon project page. Text streamlined and visual elements added.
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