Team:Goettingen/NoteBook w8

Miniprep of the Promoter-GFP Clones

Minipreps performed with the Plasmid purification kit, elution in 30µl pre-warmed water, AW buffer step included

Nanodrop:

Plates with clones of Riboswitch, DarR and E0840 Clonation put into fridge for further processing on monday

Growing and harvesting of the cells

Main over day culture in CSE 0,5% Glc + 1% xylose at 37°C

·         wt

o   inoculation at 7.35h,

o   harvest wt1 and wt3 at 11.00h with an OD₆₀₀ = 1,1 and 0,96
harvest wt 2 at 11.12 with an OD of 0,98

·         1344

o   inoculation at 7.40h

o   harvest 1344.1 at 15.15h with an OD of 0,95
harvest 1344.2 at 16.00h with an OD of 1,14
harvest 1344.3 at 17.00h with an OD of 0,9

·         1346

o   inoculation at 7.50h

o   harvest 1346.2 at 13.05h with an OD of 0,944

o   harvest 1346.1 and 1346.3 at 13.25h with an OD of 0,98 and 0,95

Of each sample, 25 ml were harvested and collected in a falcon tube with previously filled 15 ml killing buffer, which was frozen over night with a big surface. The falcon was subsequently mixed by hand until the killing buffer thawed completely. Then, 5 min 8500 rpm 4°C, discard supernatant and freeze in liquid nitrogen. Store at -80°C. These are the samples used for the microarray.

For additional c-di-AMP quantification also harvest 10 ml of each sample in a 15 ml falcon tube. Snap freeze and store at -80°C.

For exact protein determination harvest also 2x1 ml of each sample in a 2 ml eppi. Centrifuge, discard supernatant and store at -20°C.

Promoter-GFP Clonation

Photos of the plates with Promoter-GFP in pSB1C3:

GFP:

Through light:

a lot of positive clones, but also negative ones.

-> 8 positive clones were picked and inoculated into 4ml Cam cultures for minipreps tomorrow

-> Since there was no religation control on any plates made by Bingyao, I could not check the vector without the Promoter for GFP. The green cultures could therefore also be religated vectors expressing GFP without the Promoter. Sequencing will show us what is in there.

->KEEP IN MIND: ALLWAYS DO A RELIGATION CONTROL AND TRANSFORM IT WITH THE OTHER LIGATIONS!

Growing of cells for microarray in Groningen

·         over day cultures in LB medium

o   wt at 28°C

o   1344 (kan, spec) at 37°C

o   1346 (E/L, tet, cat) at 42°C

·         over night culture in CSE 0,5% Glc

o   wt at 30 °C

§  inoculation: 0.5, 1, 2, 5, 10, 15, 20, 25, 30, 50 (µl)

o   1344 at 30 °C

§  inoculation: 0.5, 1, 2, 5, 10, 20, 30, 50, 100, 150

o   1346 at 30°C

§  inoculation: 100, 200, 300

o   1346 at 37°C

§  inoculation: 5, 10, 20, 50, 100, 150, 200

BOLD = used for inoculation of main over day culture

Minipreps Part 1-4, Part 7

eluted in HPLC water(30ul), measured by Nanodrop, (ng/ul)

Gel of today´s PCR for the DarR biobrick:

M|2µlPrimer|4µlPrimer

Gel here:

->Cleanup of today´s PCR and yesterdays with the PCR purification kit, elution in 30µl prewarmed water

Nanodrop:

 ->Stored in the “white sticker Ute” Box

DarR PCR

-          DarR fusion PCR

-          After PCR cleanup, yield way to low. maybe mistake somewhere. Repeated on 24.7

Ligation Part 6 + Part 3

-          Setup: 5 µl Part 6 (9,8 µl/ng), 10 µl Part 3 (23,5 µl/ng), 2 µl Buffer, 1 µl T4 Ligase, 2 µl H2O

-          Overnight ligation 16°C

→transformation done by Bingyao next day

Ligation of Part 7 + 40/41 Riboswitch and Part 7 + DarR

-          Due to low concentrations of Backbone and insert, ligation was set up in 20 µl and with only 20 ng of BB.

-          both ligations with 2 setups. 5 µl and 10 µl of insert

-          re-ligation done

-          overnight ligation 16°C

→transformation done by Bingyao next day

Inocculation of Cryostocks

-          Parts 1-4 and Part 7 inocculated in 4x4ml LB + Antibiotics overnight

Continue the Restriction yesterday-Part 8 with PstI

Add 2 additional SpeI into the system and digest another 1 hour, most DNA is cut. <--continue the uncomplete first round digestion(SpeI)

The second round of part8 digestion with fast PstI:

Add 4ul PstI into the system

Digest 2hours.

Dephosphorylation

    Add 2ul of fast AP into the system(fast AP is compatible with FD buffer)

    Reaction on 37 degree, 30min

Purification with PCR clean up kit

    Final concentration : on the tube, about 28ng/ul;

Innoculate ON culture for plasmid mini-prep

Part7 C1

RBS-GFP-Term construct C1,C4,C5

Test restriction of plasmid RBS-GFP-Term:

C2 as control, C1,C4,C5

 --->the Part6 already has the RBS and Term.

 [that simply makes the work above meaningless]

For the construct of  RBS-DarR ORF-Term construct (expression construct)

DarR ORF into Part8(PBS plasmid)

     suffix insertion { cut part8 with SpeI and PstI, and the DarR ORF with XbalI and PstI }

RBS-DarR ORF into part7(Term plasmid)

     Prefix insertion {cut part7 with EcoRI and XbaI, the RBS-DarR ORF with EcoRI and SpeI)

The first round of part8 digestion with SpeI: to have linearized structure for DarR ORF into Part8

        Fast SpeI: 4ul

        FD buffer : 4ul

        part8 DNA: 1.5ug

        Water: fill up to 40ul

Digestion:1.5 hour, not fully digested

        Stored at -20 overnight

          tbc

Restriction digestion of part 7

-          template from stock

-          restriction with EcoRI and PstI FD, gel extraction to remove terminator out of the construct

-          Nanodrop meassurment 5,3 ng/µl

-          Inocculation from cryostock. 5h, only 2h on the shaker (stupid me). Yield after miniprep way to low (10x 50 µl with 1-3 ng/µl)

-          →redone on 23.7 overnight inocculation

(pic to be added)