7 September 2008

Dry Lab: Motility Analysis

Monday

• Track 20 cells
• Determine the segmentation of the data:
  • plotting velocity to exp(i*theta)
  • where theta is the difference between the angle of rotation of the cell's tip to its centroid and the angle of the centroid displacement

Tuesday

• Track 20 cells
• Prepare for meeting with supervisors

Wednesday

• Acquire longer microscope videos of swimming B. Subtilis
• Track at least 20 cells manually
• Adjust matlab code according to the segmentation of the data

Thursday

• Hopefully extract run_time, tumble_time,run velocity and tumble angle using our matlab code
• Plot their distributions and compare to model

Friday

• Acquire more data by tracking cells manually
• In theory we would need to track about 100 cells to 150 cells

Wetlab

B.subtilis

Monday

• Prepare Lb agar plates and LB media for the week ahead.
• Check PCR from friday on a 1% agarose gel.
• Innoculate 20ml of LB agar for overnight culture

Tuesday

• Carry out the final protocol for the O.D.₆₀₀ vs cell number. From previous trials it is clear that it is better to base the data collection on the O.D.₆₀₀ as oppose to regular time intervals. In addition it is clear a greater spread of dilutions are required for an increased data collection.
• Innoculate 20ml of LB agar for overnight culture

Wednesday

• Microscope from 11-2 to collect wild-type movement of B.subtilis
• Count the cells from the O.D.₆₀₀ vs cell number experiment.

Thursday

• Innoculate 20ml of LB agar for overnight culture

Friday

• Microscope from 11-2 to collect wild-type movement of B.subtilis

Cloning

Ongoing/Where there is spare time

• PCR trial all genomic sequences to determine optimal conditions with Taq then produce clones with Pfu
• Digest with XbaI and SpeI and incorporate into biobricks
• Follow up by assaying orientation (either with sequence internal restriction site or by using XbaI and SpeI, as they should only be able to cut inserts in the correct orientation)

Monday

• GeneArt constructs 7, 8, 9 and 11 (SacB-EAK16, LipA-EAK16, P43-RBSgsiB and Pveg-RBSgsiB) to be ligated into Biobrick vector pSB1A2 or pSB1AK3
• CAT to be ligated togethre with the terminator (B0015) to form CAT-Terminator
• AmyE 5' and 3' integration sequences and Aad9 (Spectinomycin resistance gene) to be integrated into pSB1A2 or pSB1AK3
• GeneArt constructs 3, 6, 10 and 12 (EpsE gene and PgsiB-RBSspoVG in construct 3, LipA-Elastin and PgsiB-RBSgsiB in construct 6, P43-RBSspoVG and Pveg-RBSspoVG), along with all above biobricks will be transformed into Xl1-Blue E.coli
• Depending on results, LacI PCR to be digested and purified ready for ligation into pSB1A2/pSB1AK3

Tuesday

• E.coli containing GeneArt Constructs 7, 8, 9, 11 and CAT-Terminator to be grown up for midi-prepping Wednesdays
• E.coli containing AmyE 5' and 3' integration sequences and Aad9 to be grown for mini-prep on Wednesday
• GFP-Terminator to be cut ready for building
• Ligate LacI into biobrick and transform into XL1-Blue

Wednesday

• Constructs 7, 8, 9, 11 and CAT-Terminator to be midi-prepped and digested ready for building of the phase 1 constructs
• E.coli containing constructs 10 and 12 to be grown up overnight for midi-prepping
• Aad9 and the AmyE integration sequences to be miniprepped and digested to check insert orientation
• Correctly orientated biobricks to be grown up overnight for midi-prepping Thursday
• Grow E.coli containing the new LacI biobrick
• Time permitting (If not will be carried out Thursday):
  • CAT-Terminator to be ligated to P43-RBSgsiB (9) and Pveg-RBSgsiB (11) separately OR AmyE 3' integration sequence tomorrow (depends on status of AmyE 3' sequence) and transformed into XL1-BlueE.coli
  • GFP-Terminator to be ligated to P43-RBSgsiB (9) and Pveg-RBSgsiB (11) separately and transformed into XL1-BlueE.coli(depending on CAT-Terminator ligation petner)

Thursday

• Constructs 10, 12 the AmyE integration sequences and Aad9 to be midiprepped
• AmyE integration sequences and Aad9 to be digested ready for builiding of phase 1 construct
• Constructs 10 and 12 to be digested and gel-purified ready to be incorporated into biobricks
• E.coli containing constructs 3 and 6 to be grown up overnight for midi-prepping
• Mini-prep the LacI biobrick and check for correct orientation. Grow a culture with the LacI gene in the correct orientation for midi-prep tomorrow
• Time permitting (If not may be carried out Friday):
  • Aad9 to be ligated to the P43-RBSgsiB and Pveg-RBSgsiB separately and transformed into XL1-BlueE.coli
  • Terminator to be ligated AmyE integration sequeces (separately) and transformed into XL1-BlueE.coli
  • Constructs 10 and 12 to be ligated into biobricks

Friday

• Constructs 3 and 6 to be midiprepped
• Constructs 3 and 6 digested and run on a gel to separate components by gel purification
• Midi-prep LacI
• Finish any unfinished jobs from earlier in week