Team:Goettingen/NoteBook 16

Platereader assay of RiboA with c-di-AMP and Polyamine

RiboA 5.8.I and CFP CI in BL-21 and a BL-21 wt were inoculated in an ON culture. At 10am a synchronization culture for each was set up with an StartOD of 0.5.

At 11:30am each well of the 96 well plate was inoculated with a start OD of 0.05.

Setup:

Application of some qRTPCR probes onto a gel

in order to see weather different melting temperatures in the qRTPCR make a difference

following probes were used:

83°C: B3, C3, E5

83,5°C: B5, D5, E7

Gel doc:

wells: B3, C3, E5, B5, D5, E7

Repeat of Platereader with DarR + c-di-AMP

Same setup as on the 12.9. Started at 12 o'clock

Click to view full resolution

Readied parts for shipment

Ribo A-D, Ribo A 5.8.1, A1, 6.1 and P7 minipreps were diluted to a concentration of 25ng/µl and a volume of 10 µl.

Stored in a Eppi-rack at -20°C in the top shelf of the freezer. To be marked for shipment

qRT-PCR together with Katrin G. according to the method collection protocol

10 µl 2X SYBR Green reaction mix (light sensitive)

0.4 µl Reverse Transcriptase

x µl RNA (100ng/20 µl)

x µl H2O

Total 17.6 µl

-          Final reaction (total volume: 20 µl) should contain 100 ng RNA, 10 µl 2x buffer, 0.4 µl enzyme and 1.2 µl of each primer (1:20 diluted in RNase free water)

-          Mastermixes for each RNA sample and a neg. control were prepared. They consisted of2x buffer (light-sensitive – contains SYBRgreen!), RNA/RNase free water for no-template-controls, RNase free water, and enzyme for 3.5 reactions.

-          Mastermixes consisting of 1.2 µl Primer fwd and 1.2 µl Primer reverse per reaction were prepared and then added to 2.4 µl primer mix prepared in the wells of the qRT-PCR plate

-          17.6 µl of the buffer/template/water/enzyme mastermix were added to the wells

-          qRT-PCR run using standard protocol

RFP primers (iGEM_98 and iGEM_99) and rpoA primers (iGEM_102 and iGEM_103) were used

Calculations:

Pipetting sceme:

Results:

Competition/Feeding experiment

OD measurements of ON culture

All cultures were synchronised to OD 0.5. After 2,5 h, fresh media was inocculated with a single strain or mixed. For each, one doublicate with IPTG was set up.

Cultures were inocculated to an OD of 0.1. For the mixed cultures, each culture was added to an OD of 0.05. After 3h those ODs were meassured.

From these cultures, a dilution series of 1 cell/µl was set up.

These dilutions were plated onto +/- IPTG + XGal LB plates with 50 cells per half. On one half of the according plate, the premixed cultures were streaked. On the other half, the same mixes were plated from the isolated grown cultures with 25µl (25 cells) per culture. Every single culture was plated seperate as well.

Plates were cultivated at 37°C in the dark over night.

Pictures taken next day

DNAse digest

stored in heat block 30 min at 37°C

then EDTA was added 2,5 µl per probe and put into thermo cycler at 65°C for 10 min

Control PCR

-          final reaction composition:

in thermo cycler protocol: Ivonne -> PhuS

Gel doc:

wells: Part1C3exp, part2C3exp, part3C3exp, part4C3exp, part8C3exp, Part1C3stat, part2C3stat, part3C3stat, part4C3stat, part8C3stat; Part1C3ON, part2C3ON, part3C3ON, part4C3ON, part8C3ON, gDNA

Nanodrop:

Inocculation of ON cultures

Ribo A 5.8.1, CFP religant, DAC and DAC control inocculated 4ml each for feeding/competition experiment