Lab Notebook Summary

July 7- July 13

This was the first time this summer that the team had a chance to get together.  We spent this week designing primers for our genes of interest BglS, XynA, and YesZ along with conformation primers for Psb1A3, Psb1C3, Bsb1K3, and Psb1T3.  This week we also ordered the parts BBa_K215002 and BBa_K215000 from the registry. 

July 14- July 20

After we received BBa_K215002 and BBa_K215000 from the Parts Registry we streaked the stabs, selected for single colonies, and streaked again.  From the restreaks of the agar stabs, liquid cultures were taken for plasmid extraction and glycerol stock creation.  After plasmid extraction the plasmids were digested with XbaI and SpeI for visualization in a 1% agarose gel. 

July 21- July 27

We just received the Bacillus Subtilis Subtilis 168 in the mail from the Bacillus Genetic Stock Center and the primers for our genes of interest!  Since the primers were created using the B. Subt. Subt. 168 genome we had to wait for the spores to come in before starting amplification of the cellulolytic, pectinolytic, and xylanolytic genes.    Under the guidance of Dr. James Henkel we isolated and glycerol stocked the Bacillus Subtilis Subtilis 168 from the recieved spores so that future research at WLC can fully trust and rely on the integrity of the stocks.

It was during this week that  we rehydrated and chemically transformed BBa_K206000, BBa_I13453, BBa_B0015, BBa_K87004, BBa_B0034,  and BBa_K206001.

July 28- August 3

This was the week of miniprep reckoning where we performed culture and miniprep isolation of all of the genes that had been transformed and glycerol stocked up to this point.  Because our larger bucket centrifuge, normally used for 15mL conical tubes,  we were forced to use the table top centrifuge that can only do 4 tubes at a time.  To say the least we spent more time spinning down the original cultures than anything. However, we isolated a large amount of the plasmids that contained the inserts that we needed for our project. 

Also after the PCR of the Bacillus Subtilis Subtilis 168 for BglS, XynA, and YesZ we characterized the band length of the amplified genes.

BBa_B0013, BBa_K314200, and BBa_J04450 were transformed into chemically competent E. Coli cells. 

A Qubit analysis was performed on all of the isolated plasmids to determine concentration for proper digestion.   This occurred just before we took our first shot at digesting the plasmids for subsequent ligation.

After selection for successful transformants, we got to reap the benefits of transformation by restreaking the successful transformants for liquid culture and plasmid isolation. 

August 4- August 11

All of the successful transformants were isolated, cultured, and minipreped for plasmid isolation.  The isolated plasmids were then characterized on a 1a% garose gel.

 After size characterization of the isolated plasmids we had all we needed to begin assembly of the one ring.  However, this could not be done all at once.  Thus, we started by attaching promoter sequences and ribosomal binding sites to the parts that required them. 

It was a learning experience for all of the members on the team as we learned together how to create chemically cells for more efficient transformations through chemical transformation. Nick and Ben were exceedingly giddy at the prospect of expanding their knowledge base.  

August 12- August 16

The students have arrived for the first annual WLC/ MSOE synthetic biology summer camp (or SBSC for short)!  Nick and Ben are eager to teach what they know to the kids along with the professors of WLC!  Sierra, Anna, Matt, and Steve had a great time as camp counselors seeing the camp both as the campers did and as senior members of the WLC iGEM team. 

All the while the research continued.  BBa_K215104 came in the mail and so culture, isolation, and size characterization of this part happened.  This week was also spent doing more trials on the earlier ligations and restriction enzyme digestions, after some troubleshooting of the protocols happened.

August 17- August 24

This is the week that we received the E. Coli NIssle 1917 (both with and without a genome integrated CymR gene) from Team Triste!  Isolation and glycerol stocking was done for pure and reliable cultures of the bacteria. 

During this week we ran PCR on the Bacillus Subtilis Subtilis 168 genome for isolation of the biobrick parts for submission.  Size characterization of all of the parts was done after restriction enzyme digestion and isolation by a 1% agarose gel. 

August 25- August 31

This was the first week of school for everyone on the team, but research continued despite the fact.  This week we experimented with the use of electroporation for our transformations.  We all had a chance to learn how to make electrocompetent cells for use in our electroporation transformations.  Despite the decreased availability of the team members due to classes we made sure that the workload was evenly distributed, along with the coffee.  

It was during this week that we did a large amount of trouble shooting with our enzymes both for restriction enzyme digests and ligation. 

September 1- September 7

During this week we continued the trouble shooting of our ligations and the electroporation protocols.  For the electroporation protocols we focused on the bacteria that we were using, experimenting with DH5α, Nissle 1917, and other K-12 E.Coli strains. 

September 8- September 17

During this week In a final attempt to put together the One Ring we began by creating liquid cultures of all of the parts necessary to create it.  From this we performed plasmid extraction, digestion, ligation, and electroporation every day, step by step, synthesizing the part one day and size characterizing the part the next day.  At the end of this period the parts were sent into iGEM.

September 18- September 21

Our time was spent researching and mixing the differential medias used for the characterization of our parts.  The Congo Red dyes and Ruthidium Red dyes were ordered for the staining of the differential medias to see if the enzymes were successfully excreted into the media, digesting the plant polymers, creating a zone of monomers, shown as a clearing zone. 

September 22- September 27

Everyone came together to compile all of the data and protocols we used throughout the project to stock our wiki for your viewing pleasure.