BioWire Daily Summaries

From FreeBio

Super Tuesday, 6/21

Hey all,

So a recap of what we did today. Sasha came in early and miniprepped R0079. Kang-Xing re-inoculated 2 cultures that failed to have good minipreps (I14018 and C0078). Our main focus was the digestions that had run overnight. We ran gels of these digestions (18 of them) and the bands agreed with the sizes we expected. Then we gel-extracted, ligated, and transformed. Yin pointed out that we hadn't taken into account whether to do suffix or prefix insertion and this is important as we want to put big inserts into a vector with a small brick. This luckily only messed up ligating R0065 with the RBS's (B0030-B0034).

Tomorrow,

Patrick will show us how to PCR purify the digestions that were messed up and we will run minipreps of I14018 and C0078. Then we will run analytic gels of these along with R0079 to see if the minipreps worked. Hopefully our transformations will have worked and we will innoculate the colonies from them for minipreps on Thursday. In addition we should run through the digestions we have to do in the next few weeks and make sure all planning for parts construction is squared away. And Danny and I will work on CAD.

I suggest we all come in at 10 if possible. Early start=early finish?? Have good nights- Orr

Ass Wednesday, 6/22

Hey Everyone,

Well, it was only our third day in the lab. It'll only get better.

This morning, again, Sasha came in and miniprepped 4 BB's: C0078 and I14018 (whose minipreps didn't work last time), R0079 (culture didn't grow last time), and R0065 (to make extra). Turns out that C0078 and I14018 didn't work last time because the plasmid they came in (pSB2K3) needs to be induced by IPTG in order to make enough copies. This has been added to the BioBrick to cell culture protocol. We added IPTG, after 3 hours froze the pellet and will miniprep them tomorrow.

Patrick ran a gel of the prefix insertions from yesterday (R0065 and B0030-4). We forgot to CIP the vectors, so after gel extraction we had to CIP them and then run a PCR purification. After that Sasha and Orr ligated and transformed them. They're growing in the shaker overnight and hopefully the CIP mishap will be righted.

We planned the parts for the las pathway. We took the numbers J06100-J06008. It's completely analogous to the lux pathway. We also created general parts J06400 and J06401 as subparts for the las system. Orr and Kang-Xing fixed up the lux parts (some had double ribosome binding sites or double terminators).

We caught that we were testing multiple RBS strengths on our reporters instead of on our AHL and cI. This has been fixed. Luckily it doesn't set us back any.

Orr and I met with the guy from the cleanroom who does the photolithography to talk about designing our patterns. It looks really promising; the CAD techniques should be simple.

Made another final check on BB orders.

To Do tomorrow:

1. J06001 and J06003 (sender test and sender test reporter) will be done. We can experiment with J06003 to get some practice, but we need to find an inverted microscope first. The test with the sender kinda has to wait until we've tested the receiver.

2. Always remember to check if IPTG is needed before minipreps. Miniprep the intermediate transformations that grew overnight but first make glyercol freezer stocks. Miniprep the C0078 and I14018 cultures which are pelleted and in our freezer. Not all of J06001 and J06003 should be miniprepped as they are test constructs and need no more additions (AHL sender and AHL sender YFP). Run an analytic digest and gel of the transformants. R0040's negative control had stuff growing so Orr grew up a colony of this that we can miniprep and run on a gel against the R0040 ligations.

3. Remember to check which type of digestion (prefix/suffix) is needed. Intermediate guesses are on the wiki. Digest the intermediate transformations and the BB's needed for Step Two ligations.

4. While all this is going, truly finalize parts and look at the stupid Rh1 system.

Sorry this is so long and hope you were able to read this far,

Danny and XOrr

<no-adjective> Thursday, 6/23

Hey guys,

This morning, Kang-Xing and Orr miniprepped the cultures of the step one ligation/transformations and some misc parts (R0079, C0078, I14018). Sasha set up our BioBricks database (http://d42.gotdns.com/~sasha/myphpadmin). Patrick and I went through the lux and las systems and spelled out all the digestions and ligations (except for the parts we haven't received yet, which would be hard to schedule).

We also did an analytical digest of the minipreps. Things seem to have been mislabeled to the extent that we aren't sure what's what. Patrick and Kang-Xing worked out some other enzymes to digest the transformations with in order to tell them apart. We did that digestion and will run the gels tomorrow.

We also picked colonies from the cultures of yesterday's transformations. They're growing in the shaker. These are the intermediates on the way to making J06006.x