Reading/ Thoughts
Is there a way to quantitatively measure rodZ expression. Direct method, not based on GFP.
Alyahya et al. used 10h growth in arabinose, 2h IPTG
Microscopy
Light microscopy Cells were spotted on a 1% agarose-padded slide containing appropriate antibiotics or inducers.
Fluorescence microscopy Grown in LB or M9 supplemented with 0.2% glucose, 1ug/ml thiamine and 0.05% casamino acids
Today
Digestion Clean-up of
1) rodz-gfp, XbaI, PstI
2) rfp, XbaI, PstI
3) rfp, XbaI
Ligations
1) Plac in pSB1A2 with SpeI & PstI + gfp with XbaI & PstI
2) Plac in pSB1A2 with SpeI & PstI + rodZ-gfp with XbaI & PstI
Transformation
Today: RodZ-GFP PCR products
FINALLY, we got the promega 5 minute ligation kit to work. The trick (for me) was to clean up the digestion products before ligation. I suppose the ligase is really sensitive to the 5x ligase buffer.
Gel purification awaits.
Ligations failed
yapC-gfp, rodz-gfp did not yield PCR products of correct size.
Possible next steps
1) Clean-up of digestion instead of heat treating.
2) Check ligation on gel after heat treating, apparently the ligase is very sticky
Today
Gel analysis of ligation reactions
The ligation reactions for 1) yapC-gfp 2) rodz-gfp and 3) plac-gfp in pSB1A2 plasmid, did not show any bands. The DNA ladder was visible and I adjusted the settings to increase exposure but the lanes for the ligations were as clean as a sheet. I suspect that this is due to low amounts of DNA as unligated fragments were also not visible.
I think we should just go ahead with the PCR reactions using yapC forward + gfp reverse, rodZ forward + gfp reverse. If we get PCR products, it will indicate that the ligations worked. Also, positive results will allow us to be ‘confident’ that plac-gfp ligation also worked before we proceed with transformation.
Agenda for next week
Monday
rodZ-gfp construct
Digest yapC and rodz PCR products with SpeI
Digest gfp with XbaI
Inactivate at 70degreesC for 30minutes.
Ligate overnight.
Plac-gfp control
Digest Plac miniprep with SpeI and PstI
Digest gfp with XbaI and PstI
Ligate overnight
Tuesday
Transformation with Plac-gfp ligation
Fusion PCR of yap-gfp and rodz-gfp
Wednesday
Digest Plac miniprep with SpeI and PstI
Digest rodz-gfp with XbaI and PstI
Ligate overnight.
Thursday
Transform with Plac-rodz-gfp overnight.
Today
PCR of rodz and gfp was successful. Gel analysis showed products of correct size. Both were purified.
Agenda
PCR
1. rodz
a) gDNA prep 1
b) gDNA prep 2
c) cell suspension boiled at 100degreesC for 5 minutes
2. GFP (BBa_E00240)
Today
1. Miniprep of Plac transformations. Double digest with EcoRI and PstI. Good size bands, need sequencing.
2. PCR of rodz unsuccessful. Will try with different annealing temperature.
Demonstrating synthetic morphology using prokaryotic systems
Introduction
One popular definition of synthetic biology is ‘the re-design of existing, natural biological systems for useful purposes’ (Synthetic Biology, 2009). Coupled with our understanding of developmental biology, synthetic biology has the potential to provide a foundation for ‘synthetic morphology’. If successful, we can genetically program cells to have the ability to organize themselves into designed arrangements, structures and tissues. In this view, the project’s undertakings will consider ‘synthetic morphology’ by employing an engineering approach rather than a hypothesis-driven approach.
The lofty goals of synthetic morphology include medical applications such as constructing structures that are not within the normal developmental program of the body and creating artificial interfaces between body tissues and artificial limbs (Davies, 2009). Another conceivable application is the treatment of type I diabetes. Cell clusters can be engineered to secrete insulin, when embedded into endogenous tissues, they substitute for damaged pancreatic islets (Davies, 2009).
However, before any of these aspirations can be fulfilled, we have to lay the foundations of ‘synthetic morphology’. As such, we turn to proof-in-principle demonstrations of synthetic morphological systems. The objective of building a rudimentary system is to demonstrate that synthetic genetic networks can control morphogenetic behaviour of cells.
Project
In 2008, Shiomi and associates demonstrated that the cytoskeletal membrane protein, RodZ, is crucial for maintaining bacterial rod shape. They observed that RodZ controls cell size predominantly by regulating the length of the long axis of cells with RodZ deficient cells being round or oval. Building upon their observation, this project aims to use a genetic system comprising of rodZ (the effector) to investigate inducible morphogenic behaviour in E. coli.
