iGEM 2013 — Team WLC-Milwaukee

[![](/wiki/skins/common/images/wiki.jpg)"](/Main_Page "Main Page")

* [Page](/Team%3AWLC-Milwaukee/Notebook)
* [Discussion](/wiki/index.php?title=Talk:Team:WLC-Milwaukee/Notebook&action=edit&redlink=1)
* [View source](/wiki/index.php?title=Team:WLC-Milwaukee/Notebook&action=edit)
* [History](/wiki/index.php?title=Team:WLC-Milwaukee/Notebook&action=history)
* teams

* [Log in](/wiki/index.php?title=Special:UserLogin&returnto=Team:WLC-Milwaukee/Notebook "You are encouraged to log in; however, it is not mandatory [o]")

# Team:WLC-Milwaukee/Notebook

### From 2013.igem.org

* [![1](https://static.igem.org/mediawiki/2013/d/d3/WLC-Header.png)](https://igem.org/Main_Page)

![](https://static.igem.org/mediawiki/2013/4/4e/Wisc_Luth_Coll.png)

* [Home](https://2013.igem.org/Team%3AWLC-Milwaukee)
* Project

  |  |
  | --- |
  | [Overview](https://2013.igem.org/Team%3AWLC-Milwaukee/Overview) |
  | [Modeling](https://2013.igem.org/Team%3AWLC-Milwaukee/Modeling) |
  | [Parts Submitted](https://2013.igem.org/Team%3AWLC-Milwaukee/Parts) |
  | [Future Direction](https://2013.igem.org/Team%3AWLC-Milwaukee/Future) |
  | [Notebook](https://2013.igem.org/Team%3AWLC-Milwaukee/Notebook) |
* People

  |  |  |  |  |
  | --- | --- | --- | --- |
  | |  | | --- | | [Team](https://2013.igem.org/Team%3AWLC-Milwaukee/Team) | | [Mentors](https://2013.igem.org/Team%3AWLC-Milwaukee/Mentors) | | [Our Sponsors](https://2013.igem.org/Team%3AWLC-Milwaukee/Sponsors) | |
* [Safety](https://2013.igem.org/Team%3AWLC-Milwaukee/Safety)
* Human Outreach

  |  |
  | --- |
  | [Summer Camp](https://2013.igem.org/Team%3AWLC-Milwaukee/Camp) |
  | [Science Survey](https://2013.igem.org/Team%3AWLC-Milwaukee/Survey) |
* Human Pratices

  |  |
  | --- |
  | [Food Economics](https://2013.igem.org/Team%3AWLC-Milwaukee/Food) |
  | [Misinformation](https://2013.igem.org/Team%3AWLC-Milwaukee/MisInfo) |
  | [GMO Politics](https://2013.igem.org/Team%3AWLC-Milwaukee/GMOPolitics) |
  | [Culture Blowback](https://2013.igem.org/Team%3AWLC-Milwaukee/Culture) |
  | [The Expert](https://2013.igem.org/Team%3AWLC-Milwaukee/Experts) |

# 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.

### [List of Past iGEM Protocols](https://static.igem.org/mediawiki/2013/1/1f/Past_iGem_Protocols_Compilation.pdf)

Retrieved from "[http://2013.igem.org/Team:WLC-Milwaukee/Notebook](http://2013.igem.org/Team%3AWLC-Milwaukee/Notebook)"

[![Powered by MediaWiki](/wiki/skins/common/images/poweredby_mediawiki_88x31.png)](https://www.mediawiki.org/)

[![Attribution 3.0 Unported](https://i.creativecommons.org/l/by/3.0/88x31.png)](https://creativecommons.org/licenses/by/3.0/)

* [Recent changes](/Special%3ARecentChanges "Recent changes")
* [What links here](/Special%3AWhatLinksHere/Team%3AWLC-Milwaukee/Notebook "List of all wiki pages that link here [j]")
* [Related changes](/Special%3ARecentChangesLinked/Team%3AWLC-Milwaukee/Notebook "Recent changes in pages linked from this page [k]")
* [Special pages](/Special%3ASpecialPages "List of all special pages [q]")
* [My preferences](/Special%3APreferences)

* [Printable version](/wiki/index.php?title=Team:WLC-Milwaukee/Notebook&printable=yes "Printable version of this page [p]")
* [Permanent link](/wiki/index.php?title=Team:WLC-Milwaukee/Notebook&oldid=241750 "Permanent link to this revision of the page")
* [Privacy policy](/2013.igem.org%3APrivacy_policy "2013.igem.org:Privacy policy")
* [Disclaimers](/2013.igem.org%3AGeneral_disclaimer "2013.igem.org:General disclaimer")