Week 11

9/1/13
We ran the gel involving our colony PCR. We also did a miniprep of our first successful Golden Gate constructs involving an arabinose promoter, a riboswitch, and GFP. We hope to send it out for sequencing on Tuesday. We grew up cultures that could potentially be our intended Golden Gate constructs.

9/2/13
We did a miniprep of our cultures containing pTet+TBS 2+GFP, pAra+RS 2+GFP, and pAra+RS 1+GFP in an effort to determine whether they were false positives. We transformed them into the strain BW22826 that doesn’t express LacI. This will help us determine whether the colony PCR was showing a band for Plac+mRFP or Para+RS 2+GFP. The cells wills be red to demonstrate that they are in fact false positives.

9/3/13
Some of the transformants from yesterdays did not turn red, which means we hopefully have a successfully assembled construct of Para+RS 1+GFP and Para+RS 2+GFP in pSB3K3 backbones. We also may have successfully assembled Ptet+TBS 2+GFP, but that still awaits sequencing along with a few of the riboswitch+TAL repressor constructs. We set up reactions for lots of Sanger Sequencing and hope to get the results by tomorrow. In the meantime, we are still doing more Golden Gate Assembly reactions in order to assemble the rest of our constructs as well as have back ups in case our current constructs turn out to be false positives.

9/4/13
After checking the Sanger sequencing it appears that we only have some of the correct constructs. The parts involving our tet promoter, the TAL binding site, and GFP seem to be correct except that the promoter is missing one of its operators. So far, we have Para+RS 1+GFP, Para+RS 2+GFP, Para+RS 2+TAL 1, and Para+RS 1+TAL 1 correctly assembled. However, these constructs are in a pSB3K3 backbone. We did a cotransformation involving Ptet+TBS 2+GFP and Para+RS 2+TAL 8 in hopes of investigating the functionality of the truncated tet promoter. Later we will do an additional step of Standard Biobrick Assembly where we will reinsert the properly functioning tet promoter. We decided to rehydrate R0040 and transform it into MG1655Z1. We also ran a restriction digests of Para+RS 2+TAL 8, Para+RS 1+TAL 1 and Ptet+TBS 2+GFP to obtain one of our constructs and a negative control. Tomorrow we will ligate these constructs together and transform them.

9/5/13
We grew up cultures involving the successful cotransformations. We also sent out our Golden Gate assemblies involving Para+RS 1+TAL 8 and Para+RS 2+TAL 8 for sequencing. Hopefully, we can finish our Golden Gate Assemblies by the end of the week. We did minipreps of R0040, Para+RS 1+GFP, Para+RS 2+GFP, Para+RS 1+TAL 8, and Ptet+TBS 2+GFP. We did several ligations involving Para+RS 1+TAL 1 or Para+RS 2+TAL 8 with Ptet+TBS 2+GFP. We made glycerol stocks of Para+RS 1+GFP and Para+RS 2 +GFP for later use and grew them up for use tomorrow in our Tecan run to characterize them in our newly made M9 minimal media with glucose added. We are also continuing to do more Golden Gate Assembly in order to get the constructs that we are still having problems with or are unsure about. Another idea we had to fix the missing operator in our Ptet+TBS2+GFP construct was to use standard assembly to put another Tet promoter in front of our existing construct and see if it can possibly work with three operators. Additionally, we are using standard assembly to move our current constructs in pSB3K3 into pSB1C3 for testing and eventually part submission.

9/6/13


9/7/13