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

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

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

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