Week 1
Tuesday, May 20 - Sunday, May 25

- Notebook Entries

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

- Notebook Entries

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

- Notebook Entries

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

- Notebook Entries

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

- Notebook Entries

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

- Notebook Entries

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

- Notebook Entries

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

- Notebook Entries

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.

Week 9
Monday, July 14 - Sunday, July 20

- Notebook Entries

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.

Week 10
Monday, July 21 - Sunday, July 27

- Notebook Entries

Week 11
Monday, July 28 - Sunday, August 3

- Notebook Entries

Week 12
Monday, August 4 - Sunday, August 10

- Notebook Entries

Week 13
Monday, August 11 - Sunday, August 17

- Notebook Entries