From 2007.igem.org

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Please read the background for this system. It will aid in making sense of the following.

Contents 1 Aug 2007: Order ComP and ComA from GENEART 2 Aug 2007: Primer design for PCR stitching 3 Sep 2007: PCR stitching and Ligation of construct into biobrick plasmid 3.1 First round PCR 3.2 Second round PCR 3.3 Digestion of product and ligation into Death Plasmid 4 Sep 2007: Construction of ComP/ComA reporter system 5 Oct 2007: Creation of sfrA biobrick 6 Primers used

Aug 2007: Order ComP and ComA from GENEART

We were counting on [http://partsregistry.org/Part:BBa_J51000 ComP] and [http://partsregistry.org/Part:BBa_J51001 ComA] being available in the 2007 iGEM distribution. As this was not the case, and we had DNA synthesis bases to burn, we ended up ordering them from GENEART. The original sequences, as described in the above links, were optimized for expression in e.coli and all cis-acting sites and assembly restriction sites were removed. We also included biobrick prefix/suffix and vf2 and vr primer binding sites. The two sequences, now labeled ComA' and ComP' took around 3 weeks after confirmation of order to be dispatched. The sequences have been uploaded below:

ComA' sequence (As revised by geneart)

ComP' sequence (As revised by geneart)

Upon receiving the DNA, we digested and ligated into the death plasmid, as detailed here These parts are now biobricks!

Aug 2007: Primer design for PCR stitching

In order to stitch NpSopII-NpHtrII to ComP at the sites detailed here, these primers were designed. We aimed to have at least 18bp on each half of the stitch, while adhering to the standard primer design guidelines.

Sep 2007: PCR stitching and Ligation of construct into biobrick plasmid

First round PCR

Two separate PCR reactions for each construct. One to amplify the NpSopII-NpHtrII half and the other to amplify the kinase domain of ComP. Detailed protocol. Gel purification of ~1.2kb band from both reactions. Some non-specific bands observed in some reactions.

Second round PCR

Gel purified products from first round used as templates/primers in the second round PCR.PCR mix and program. Products were quite smeared, but we excised a band of ~2.3kb and gel purified. We now have a set of seven constructs, referred to as {BL_A1, BL_B2,.....,BL_G7}

Digestion of product and ligation into Death Plasmid

Products were digested with X/P, and ligated into [http://partsregistry.org/Part:BBa_P1010 BBa_P1010](AmpR)( X/P digested and gel purified), and [http://partsregistry.org/Part:BBa_J61035 BBa_J61035](S/P digested and gel purified). Ligated at RT for 5 minutes using Promega's Rapid Ligase kit. Then transformed into competent DH5-alpha. Plated over night, cultured and miniprepped the next day. Details.

We also ordered BL_seq_v1 sequencing primer spanning the region of joining, so that we can make sure that the construct has been correctly stitched at the desired point.

Sep 2007: Construction of ComP/ComA reporter system

We want to know that the ComP/ComA reporter system works, so as a test harness, we're creating a reporter line using the ComA and the ComP received from Geneart. This involves attaching RBS, terminator and constitutive promoter sequences. (PostScript: We got up to the RBS and terminator, but no further than that.) Details week 13 onwards from general lab book.

Oct 2007: Creation of sfrA biobrick

For this all to work, we do need a promoter sequence for the phosphorylated ComA to act on. We received the pJS34, containing the PsrfA promoter (sequence) from Prof. Grossman's lab at MIT. Luckily for us, there are no restriction sites that need to be removed. In order to turn this into a biobrick, we designed PsrfA pre/sux primers and PCR'd it up. Unfortunately, we were unable to complete this stage of the project before we decided to pull the plug on labwork.

Primers used