Plasmid Construction

Following our experimental strategy we built two plasmid backbones on which to insert the Lux constructs.

Neutral recombination plasmid

This plasmid backbone was constructed by standard assembly techniques. Recombination sites (BBa_K743000 and Part:BBa_K743001) were Biobricked by Gibson assembly and were consenquently ligated to neighboring parts and KanRB0015 in parallel assembly. Afterwards, we ligated both composites through a standard assembly reaction. Final construct was validated by digestion (see gel image at right) and corroborated through sequencing.

After obtaining BBa_K743006, we proceeded assembling our constructs through Gibson assemblies.

If you are wondering why the plasmid was constructed by standard assembly and not by gibson assembly from scratch, please refer to Gibson assembly for small parts

Insertion of LuxAB genes

Starting from K743006 as a plasmid backbone, we were able to build our LuxAB constructs for the bacterial luciferase using two different versions available at the registry (from Photorhabdus luminiscent, BBa_K743014 and Vibrio fisherii, BBa_K743015) under an endogenous Synechocystis's promoter (transaldolase Reference???). Resulting constructs were verified by digestion (see gel images below) and corroborated by sequencing.

Lane 1 = Ladder 1Kb, Lane 6 = K743014. Band at 4000: RS1-Pta-LuxABvf-Kan-RS2partial, band at 2000: pSB1C3, band at 500: RS2partial.

Lane 1 = Ladder 1Kb, Lane 3 = K743014. Band at 4000: RS1-Pta-LuxABxl-Kan-RS2partial, band at 2000: pSB1C3, band at 500: RS2partial.

Suceptibility construct

pSB1C3_IntS was constructed in two successive Gibson assemblies. The first assembly inserted the CopS region (the entire RS5 & RS6 recombination locus) to a chopped-down pSB1C3 lacking the whole VF2 to VR region. The second assembly inserted the Spectynomycin resistance selection marker and the whole VF to VR region of plasmid pSB4K5-J04450 right in between of the RS5 & RS6 CopS region. This construct yields red colonies if correctly assembled (see image at left). Red colonies were colony PCRed, verificated by digestion (see image down right) and corroborated by sequencing.

A couple of picked red colonies are shown in this plate.

Insertion of Lux substrate biosynthesis genes under twilight promoters

We have amplified all the parts of the substrate production pathway (LuxCD and LuxEG) for the final construct under the Pcaa3 promoter (which has also been Biobricked with code [K743002]. Also, we are amplifying one remaining part (LuxCD with 5' homology to PsigE) to do Gibson assembly of the LuxCDEG construct under PsigE. Gibson reaction will be done along with the substrate production construct under Pcaa3 in pSB1C3_IntS for both constructs.

Transforming Synechocystis PCC 6803

Once constructs were corroborated by sequencing, we proceeded to transform synechocystis. Prior to transforming, we verified that our Synechocystis cultures were in exponential growth by comparison to our growth curve characterization.

Transformation in Synechocystis takes about two weeks to reveal transformant colonies, which in turn have to be restreaked again before PCR verification. This makes working in Synechocystis pressing as mistakes can take long to unravel.

This image shows various time points during a Synechocystis PCC. 6803 transformations. Be advised that it is only to be used as a reference of what to expect during the first week of transformation.

LuxABxl under transaldolase promoter transformation

Transformation of Synechocystis PCC 6803 with K743014 on the 14th day of transformation. The left plate is the control transformation plate (no DNA) on Kanamycin 25ug/mL and on the right, successfully transformed Synechocystis colonies growing throughout the Millipore membrane on the same antibiotic.

LuxABvf under transaldolase promoter transformation

Transformation of Synechocystis PCC 6803 with K743015 on the 14th day of transformation. The left plate is the control transformation plate (no DNA) on Kanamycin 25ug/mL and on the right, successfully transformed Synechocystis colonies growing throughout the Millipore membrane on the same antibiotic.

It is important to note that to our knowledge we are the first iGEM team to do a succesful direct transformation of Synechocystis PCC. 6803 with naked DNA.

sfGFP with LVA tag for describing circadian behaviour

To describe the circadian behaviour of the promoter we built a fast-degrading reporter consisting of sfGPF I746916 with a LVA degradation tag in the C-terminal end of the protein. This construct will serve as a real-time reporter of promoter activity, you may find more information about the half-life of proteins with the LVA tag | here. The construct has been verified by digestion and corroborated by sequencing.