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 (K1212016), pBAD+RS 2+GFP (K1212009), and pBAD+RS 1+GFP (K1212010) 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 (J04450) or pBAD+RS 2+GFP (K1212009). 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 pBAD+RS 1+GFP (K1212010) and pBAD+RS 2+GFP (K1212009) in pSB3K3 backbones. We also may have successfully assembled pTet+TBS 2+GFP (K1212016), 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 pBAD+RS 1+GFP (K1212010), pBAD+RS 2+GFP (K1212009), pBAD+RS 2+TAL 1 (K1212011), and pBAD+RS 1+TAL 1 (K1212014) correctly assembled. However, these constructs are in a pSB3K3 backbone. We did a cotransformation involving pTet+TBS 2+GFP (K1212016) and pBAD+RS 2+TAL 8 (K1212012) 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 pBAD+RS 2+TAL 8 (K1212012), pBAD+RS 1+TAL 1 (K1212014) and pTet+TBS 2+GFP (K1212016) 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 pBAD+RS 1+TAL 8 and pBAD+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, pBAD+RS 1+GFP, pBAD+RS 2+GFP, pBAD+RS 1+TAL 8, and pTet+TBS 2+GFP. We did several ligations involving pBAD+RS 1+TAL 1 or pBAD+RS 2+TAL 8 with pTet+TBS 2+GFP. We made glycerol stocks of pBAD+RS 1+GFP and pBAD+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
We measured the turbidity of our starter cultures containing pBAD+RS 1+GFP and pBAD+RS 2+GFP. We diluted them down to .01 for growth. We then put the appropriate amounts of the new culture into a 96 well plate. The cells were treated with arabinose and theophylline at concentrations varying from 0-1% and 0-2 mM, respectively. We also did a gel extraction and ligation of pBAD+RS 1+GFP and pBAD+RS 2+GFP into a pSB1C3 backbone. The ligated product of these constructs were used for several transformations. In an effort to finish any assembly reaction that did not work through Golden Gate we tried PCR SOEing several of our parts of interest. In total we tried 13 different reactions with single components of our total constructs. Tomorrow we will run these PCR reactions on a gel.

9/7/13
With the exception of pSB3K3, all of the PCR fragments for SOE were properly amplified. We did a PCR purification and then did the assembly step of the protocol with up to three components in one reaction. After assembly, we did transformations with the assembled reactions and will screen for colonies tomorrow. We did colony PCR on the parts pTet+TBS 1+GFP and pBAD+RS 2+TAL 8. The bands looked to be the correct sizes, so we grew up the cultures for minprep tomorrow. A cotransformation of pBAD+RS 2+TAL 8 and the fixed pTet+TBS 2+GFP was performed. The fixed pTet+TBS2+GFP was also transformed by itself. Minipreps were carried out for the colonies that showed up for R1T1 + GFP, R2T8 + GFP, R0040+TBS2+GFP, constructed through Standard Assembly. Will be sequenced on Monday. More R1T8 was grown up from glycerol stock for miniprepping.