Imperial College/8 September 2008

=8 September 2008=

Motility

 * Assessed error with data extraction algorithm which extracts run velocity, run duration, tumbling angle and tumbling duration. Following steps were taken (and documented on OWW Wiki):
 * Used known synthetic trajectory as an input for data extraction algorithm
 * After obtaining motility data on the above 4 characteristics, posteriors were plotted. mu and sigma for run velocity, lambda for run and tumbling duration.
 * Error assessed by finding % error between parameter value for which posterior is maximised and the actual value of the parameter used to generate synthetic trajectory.
 * It was found that the errors are quite large. This is due to the low frame rate such that tumbles which are less than 1/(frame rate) do not cause the cell to be stationary. Only way to get around this problem with real data is to increase the frame rate at which our videos are captured.


 * We also tracked more cells today, tip and centre. Total number of cells tracked so far is 20, with over 5000 data points (coordinates).
 * Segmentation algorithm still does not provide us with satisfactory visual segmentation. Clustering software however, can find 2 centres, but we are not convinced whether this is of any significance.
 * This is an important issue to be resolved before we can even move onto modelling motility of bacteria.

Transformation

 * Today we confirmed that the transformation has worked! A single colony PCR confirmed that the integration of the vector pDR110 was successful. The conditions set up were as follows:
 * Single colony PCR using genomic (control) B.subtilis
 * Single colony PCR using transformed B.subtilis.
 * The following settings were used:
 * 1 cycle 95oC 30 seconds
 * 30 cycles, 95oC 30 seconds, 58/56oC 1 minute, 72oC for 2 minutes and 30 seconds.



Upper Gel

Lane 1 -

Lane 2 -

Lane 3 -

Lane 4 -

Lane 5 -

Lane 6 -

Lane 7 - pSB primers, biobrick template, 60oC

Lane 8 - pSB primers, biobrick template, 58oC

Lane 9 - pSB primers, biobrick template, 56oC

Lane 10 - LacI primers, Pfu polymerase and pDR11 template. 56oC for 10 cycles then 64oC for 20 cycles.

Lower Gel

Lane 1 - pSB1A2 cut with EcoRI and SpeI

Lane 2 - construct 7 cut with EcoRI and SpeI

Lane 3 - construct 8 cut with EcoRI and SpeI

Lane 4 - construct 9 cut with EcoRI and SpeI

Lane 5 - construct 11 cut with EcoRI and SpeI

Lane 6 - pSB1AK3 cut with EcoRI and SpeI

Lane 7 - CAT insert (cut EcoRI - SpeI)

Lane 8 - Terminator vector (cut EcoRI - XbaI)

Lane 9 - pSB1A2 cut with XbaI and SpeI

Lane 10 - AmyE 5' IS clone (cut XbaI - SpeI)

Lane 11 - Aad9 clone (cut XbaI - SpeI)

Lane 12 - AmyE 3' IS clone (cut XbaI - SpeI)

Lane 13 - pSB1AK3 cut with XbaI and SpeI

The results of this gel were used to calculate the amount of reagents were required for our ligations