Team:HKU-HKBU/29 June 2009

1, test E.coli BL21 swimming ability   (QQ)

2, search E.coli strains with complete LPS: MG1655   (Samuel)

3. transfer plasmid pET-sp-strp-AIDA    (Alex and Brooks) -tranfer to BL21 -run gel -Western blotting, anti-gfp -Fluorescence microscope

4. pET-sp-strp-AIDA: remove gfp     (James and Amy) Biotin-labeled antibody: immunostaining

5. Speed control: control cheZ   (QQ and Nova) cheZ under promoter of arabinose or IPTG -transfer plasmid into MGS (cheZ-) -Western blot different IPTG conc. to see cheZ expression level -swimming speed assay under different IPTG conc.

6. Particle: biotin-labeled   (Bosco)

7. Biobricks AIDA  strep  cheZ   cheYp

Promoter---rbs---cheZ---double terminator PET---sp---gfp---strep [PT7rbs][spgfp---strep]---[terminator] (fusion protein)

Backbone: PSB1A2, using PMB1 as origin

29/6/09 Minutes

7:20pm Yinan started his presentation on the potential bacteria for our project. The bacteria cannot be isolated from the culture. 7:30 Yinan ended his presentation

Amy started her presentation on speed control. 7:55 Amy stopped. 8:00 pm Bosco started his ppt. 8:20 stopped 8:30

Yinan : fast speed strain choice (x) Strain? 1. Polar expression 2.non-tumbling speed-controlable

Genes: AidA LPS-complete strains Express E. coli BL21 LPS-incomplete-strain (x) Express all over the membrane

For non-tumbling and speed control We need CheZ If it is out knocked out, the bacteria will not swim. CheZ-knocked out strain, it has been made. For E.coli MG1655- don’t know if it is LPS complete.

CheY We got a strain derived from MG1655 without the CheY gene.

Another problem, E.coli BL21, it is LPS complete, but we don’t know whether it can swim. We may need to find another strain that is LPS complete and can swim.

Things to check: 1.	swimming ability 2.	LPS completion Member assignment to help resolve the problems listed above.

Another problem : Speed control Amy talked about this but there is no clear choice to control the swimming speed, either chemical or through genetics.

The speed control test must be continued. Don’t know how to do yet.

1 possible solution by Yu bin: control CheZ and make the bacteria swim and stop. 2 Amy’s solution : over-expression of the stator genes, external environment. 3.	Copper ions and EDTA (fastest)

3rd problem : the particles that the bacteria are going to push (talked by Bosco) Whether the method works and whether biotin can bind

4. Biobrick

The plan of next week: 1.Strain a. Test E. coli BL21 swimming ability : QQ b.search E.coli strains with complete LPS search MG1655 first Mg1655wild type from E.coli K12 LPS complete or not? MG3 Chez- MG4 CheY-

2 seatch E.coli strains with complete LPS Surface polar display Bl21 AIDS Pet-23a promotor from bacteriphage T7 People introduce t7 polymerase gene in the BL21 chromosome Insert the gene into the plasmids at the pl5A origin

PET23-A has a COLE-1 origin

Signal peptide It will bind to the inner membrane and its tail will be transported upward. The signal peptide will then be cleaved. AIDAC will stay at the outer membrane and become a domain with a barrel shape. Streptavidin will go to the external membrane through the barrel.

Transform the plasmid into BL21.

3.PET-sp-gfp-strp-AIDA 1.transform BL21 2.SDS-PAGE 3.Western blot anti gfp 4.fluorescense microscope

4.PET-sp-STRP-AIDA

biotin-labeled antibody immuno-staining. FITC-AB 2ND ANTIBODY

5.Speed-control control CheZ To test the swimming speed under different IPTG concentrations.

IPTG induces the promoter.

6.Particle Biotin-label

7.biobrick

PET-sp-gpf-strep

Pipette tips

Protocols available at www.openwetware.org

Write down our own protocols. Record our mistakes. Write down the protocols in our notebooks