Team:Groningen/Labwork/27 June 2013



Added 1 ul serva/100ml to the earlier made 0.8% agarose gel.

Today the PCR products of the signal sequences (FliZ, EstA, MotB and LytB) are purified using the Roche gel purification kit. Using nanodrop reveals the following concentrations:

signal sequence concentration (ng/µl)
FliZ 9.2
LytB 8.2
MotB 8.8
EstA 8.8

Because of the low concentrations a new PCR is done using the PCR products.

A PCR is done for some different silk constructs to start with. The following combinations are made:

  1. signal sequence - N-terminal strep tag - silk
  2. signal sequence - silk - C-terminal strep tag
  3. signal sequence - silk - no strep tag
  4. silk without tag

For every sample an annealing temperature of 50°C is used, expecting a size of 900 bp.

The samples are run over a 0.8% agarose gel. This revealed that no bands are present at all. So the gel is stained with a high concentration of Ethidium Bromide and the bands appear. A big smear is seen for all the silk products, with a higher concentrated band around 200 bp. Because of this results it is decided to do a gradient PCR on the first combination from 55-75°C.


The following bibliography summarizes the source of inspiration on which the heat-attracted bacteria sub-project is based on:
  • Thermal Regulation of Membrane Lipid Fluidity by a Two-Component System in Bacillus subtilis by L. M. Bredeston, D. Marciano, D. Albanesi, D. De Mendoza, and J. M. Delfino
  • The three adaptation systems of Bacillus subtilis chemotaxis by Christopher V. Rao, George D. Glekas and George W. Ordal
  • Chemotaxis in Bacillus Sutilis: how bacteria monitor environmental signals by Liam F. Garrity and George W. Ordal
  • Regulation of Bacillus subtilis DesK thermosensor by lipids by Mariana Martin and Diego De Mendoza