Team:Goettingen/NoteBook w15

RNA extraction from E. coli

with kit: Qiagen RNeasy Mini

-          clean bench and pipets with ethanol

-          prepare RLT buffer with 2-mercaptoethanole (1 ml RLT buffer à 10 µl 2-mercaptoethanol); here: for 15 samples, 17 ml RLT buffer were supplied with 170 µl 2-mercaptoethanol (in other Bacillus-lab in the hood)

-          prepare lysis buffer: 1xTE buffer + 0.5 mg/ml lysozyme in RNase free water (here: 340 µl 10x TE (sterile) + 85 µl 20 mg/ml lysozyme + 2975 ul RNase free waterà 3.4 ml lysis buffer)

-          set one heating block to 70 °C and pre-heat RNase free water for elution (100 µl for elution of 1 sample)

-          set another heating block to 37 °C for lysis

 

a) Lysis: add 200 µl lysis buffer to E. coli pellet, resuspend properly and incubate for 10 min at 37 °C;add 1 ml RLT supplied with 2-mercaptoethanol to the suspension and centrifuge for 5 min at 13 000 rpm, RT

b) removal of gDNA: load supernatant in 700 µl steps on violet columns and centrifuge at 13 000 rpm, RT, 1 min à collect FLOW-THROUG in 2 ml epi tube (column binds gDNA, but NOT RNA à RNA is in FLOW-THROUG!!!)

c) binding of RNA: add more or less equal volume of 70% ethanol (RNA) (here: 1 ml) to flow-through, mix by pipetting and vortexing, then load mixture on pink columns (pink columns bind RNA)

d) washing: à no, one has to discard the flow-through…

d1) load 700 µl RW1 buffer onto column, centrifuge at 13000 rpm, RT, 1 min

d2) load 500 µl RPE1 buffer onto column, centrifuge at 13000 rpm, RT, 1 min

d3) load again 500 µl RPE1 buffer onto column, centrifuge at 13000 rpm, RT, 1 min

e) drying of column: centrifuge column in emptied collection tube 1 min, 13000 rpm, RT

f) elution: place column in fresh epi tube (RNase-free) elute RNA with 50 µl pre-heated (70°C) RNase-free water, incubate tubes at 70 °C for 5 min, then spin down at 13000rpm for 1 min, RT (here: sample “exp. 8” was eluted with 100 µl by mistake…)

g) measure concentration with NanoDrop (set it to RNA!); Blank: HPLC water (no difference to RNase free water…)

stored at -80°C freezer

NanoDrop concentrations from today:

Trafo

Sucessful for:

psB1C3 E+S (phos.) + Promoter 1rev Hyb. (dephos.)

psB1C3 X+P (dephos.) + part 6.4A

→  Colony PCR and Mini-Prep.

→  Send to Sequencing

→  For the clones Promoter 7 and Part 6.4 A 1 the sequence was oky!!! Wohoo

 

Ligation

Repetition of psB1C3 E+S (dephos.) + DarR operator Hyb. (phos.)

 

Plate Reader assay

results saved at iGEM2013->Reporter Team->plate reader->promoter plasmid parts 1234 and part6 test-> characterization of promoter parts_10.09.13

to be analysed and edited into excel sheet after katrin T.

Calculations:

 

Results:

 

Harvesting of cells for qRT-PCR analysis and plate reader assay (promoter clones) of re-trafos from part 1,2,3,4,8 respectively clone 3

-          harvested again cells of over night culture of an OD around between 2-3

 

Promoter characterization: qRT-PCR for RNA isolated from clones 2 (because a mistake happened with the primers – I used primers iGEM_99 and iGEM_100 instead of iGEM_98 and iGEM_99)

-          qRT-PCR with Biorad kit

 

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:

to be added after scanning

pipetting sceme:

 

Results:

results saved in excel sheet iGEM2013->Reporter team->qPCR->Experiment_10.09.2013->experiment 10.09.13 again-> replication_qPCR_Promonter_characterisation_calculations

Plate reader DarR + c-diAMP

Every well was inocculated to an OD of 0.05 from an over day culture.

LB Cm	LB Cm + 5nnmol cdiamp	LB Cm + 10nnmol cdiamp	LB Cm + 15nnmol cdiamp	LB Cm + 30nnmol cdiamp	LB Cm + 50nnmol cdiamp	LB Cm + 100nnmol cdiamp	LB + Amp DH5 strain		DarR Konstrukt A1
LB Cm	LB Cm + 5nnmol cdiamp	LB Cm + 10nnmol cdiamp	LB Cm + 15nnmol cdiamp	LB Cm + 30nnmol cdiamp	LB Cm + 50nnmol cdiamp	LB Cm + 100nnmol cdiamp	LB + Amp DH5 strain
LB Cm	LB Cm + 5nnmol cdiamp	LB Cm + 10nnmol cdiamp	LB Cm + 15nnmol cdiamp	LB Cm + 30nnmol cdiamp	LB Cm + 50nnmol cdiamp	LB Cm + 100nnmol cdiamp	LB + Amp DH5 strain
LB Cm	LB Cm + 5nnmol cdiamp	LB Cm + 10nnmol cdiamp	LB Cm + 15nnmol cdiamp	LB Cm + 30nnmol cdiamp	LB Cm + 50nnmol cdiamp	LB Cm + 100nnmol cdiamp	Blank LB		Kontrolle 6.2
LB Cm	LB Cm + 5nnmol cdiamp	LB Cm + 10nnmol cdiamp	LB Cm + 15nnmol cdiamp	LB Cm + 30nnmol cdiamp	LB Cm + 50nnmol cdiamp	LB Cm + 100nnmol cdiamp
LB Cm	LB Cm + 5nnmol cdiamp	LB Cm + 10nnmol cdiamp	LB Cm + 15nnmol cdiamp	LB Cm + 30nnmol cdiamp	LB Cm + 50nnmol cdiamp	LB Cm + 100nnmol cdiamp

Resultsgraphs to be added

Summary: DarR inhibits any GFP production, eventhough no c-di-AMP is there. LB Blank was contaminated. Repeated at 18.9

DarR streak out on 6-well agar plate

Each well was filled with 4 ml of LB Agar and Cm

NO c-di-AMP	10 µg/ml	30 µg/ml	<- DarR
NO c-di-AMP	10 µg/ml	30 µg/ml	<- 6.2 Ctrl

Grown at 37°C overnight.

Pictures showed no fluorescens in the DarR lane. The control showed GFP on all plates. Same result as the plate reader experiment

No fluorescence in any of the  samples.

Trafo in XL1 blue

psB1C3 E+S (phos.) + Promoter 1rev Hyb. (dephos.)

part7 E+S (dephos.) + DarR operator Hyb. (phos.)

psB1C3 X+P (dephos.) + part 6.4A

Harvesting of cells for qRT-PCR analysis and plate reader assay (promoter clones) of re-trafos from part 1,2,3,4,8 respectively clone 3

-          measuring of OD600nm of ON cultures in 1:10 dilution of LB and calculation of actual OD600nmà OD600nm in ON cultures varied from 3.93 to 4,52 nmà inoculation of synchronization pre-culture: 4 ml LBAmp + 50 μl ON culture, incubation at 37 °C at ca. 200 rpm for > 2 h (the OD600nm was measured and the cells kept on bench)

-          inoculation of an over day culture 15 ml LB Amp + around 2 ml from synchronization culture

-          measuring of OD600nm in 1:10 dilution of LB and calculation of actual OD600nm after 30 min, 1h, 1,15h, 1,30h, 1,45h, 2h, 2,15h, 2,30h, 2,30h, 2,45h, 3h, 3,30h, 4h, 4,30h, 5h

-          (Exel table of values to be done tomorrow)

-          harvested cells of OD around 0,6 and between 2-3 stored in green cryo boy at -80°C

 

a) OD600nm ON culture

Strain and Clone	OD600nm (1:10 dilution in LB)	OD600nm (1:1 calculated)	exact ON culture volume for synchronization culture in µl*
P1 C3	0,452	4,52	44
P2 C3	0,400	4,00	50
P3 C3	0,393	3,93	51
P4 C3	0,435	4,35	46
P8 C3	0,430	4,30	47

b) measurement of synchronization culture before main culture and titer plate preparation

Strain and Clone	OD600nm (1:10 dilution in LB)	OD600nm (1:1 calculated)	synchronization culture volume for main cultures (15 ml) in ml *
P1 C3	0,04	0,4	1,88
P2 C3	0,037	0,37	2,03
P3 C3	0,031	0,31	2,42
P4 C3	0,036	0,36	2,08
P8 C3	0,036	0,36	2,08

 

Plate Reader assay

-          measuring of OD600nm in 1:10 dilution of LB and calculation of actual OD600nmàinoculation of plate reader wells: 170 μl LBAmp, OD600nm = 0.05 (approx)

(preparation of plate reade titer plate (170 µl culture) of part 1,2,3,4,8 respectively clone 2 and part 1,2,3,4,8 respectively clone 3and both cultures: OD600nm = 0.05

for plate reader test of OD and fluorescence)

C2 culture volume for titer plate (170 µl) in µl *	C3 culture volume for titer plate (170 µl) in µl *
2	1,9
2	2,1
1,7	2,2
1,8	2
1,9	2

  incubation at 37 °C, strong shaking

-          measuring of OD600nm against LBAmp as blank

-          measuring of RFP fluorescence (excitation: 555 nm; emission: 583 nm)

-          run over day and ON

-          pipetting scheme

Pipe:

Competent cells BL-21 and DH5α

4ml of BL-21 cells made competent for transformation of new DarR

250ml of DH5α made competent and frozen at -70°C. Still need testing by Katrin Gunka

Miniprep of Ribo A-D and Ribo A C5.8.1

2x 4ml each pooled and prepped.

Nanodrop measurement:

Sample	Ribo A C5	Ribo B C5	Ribo C C5	Ribo D C2	Ribo A 5.8.1 +CFP
ng/ µl	229,7	77,4	81,9	89,5	256,1

Test R.D of Ribo A-D from masterplate on Thursday, 12.9

Preparation of cryostocks of DarR rev Term rev C3 and DarR reporter system C, Part 1 retrafo 3, Part2 retrafo3, Part3 retrafo3, Part4 retrafo3 and Part8 retrafo3

stored at -80°C freezer in green box

 

Promoter characterization: qRT-PCR for RNA isolated from clones 2

-          qRT-PCR with Biorad kit

-           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_99 and iGEM_100) and rpoA primers (iGEM_102 and iGEM_103) were used

Calculations:

pipetting sceme:

Results:

 

results saved in excel sheet iGEM2013->Reporter team->qPCR->Experiment_10.09.2013->replication_qPCR_Promonter_characterisation_calculations

 

Inocculation of Part 1 retrafoC 3, Part2 retrafoC3, Part3 retrafoC3, Part4 retrafoC3 and Part8 retrafoC3 in LB medium with Amp for OD measurements and plate reader for tomorrow

Inocculation of Part 1 retrafo C2, Part2 retrafoC2, Part3 retrafoC2, Part4 retrafoC2 and Part8 retrafoC2 in LB medium with Amp for plate reader 

 

Primer designed for RTPCR CFP

Assisted Katrin Gunka in designing new primers for CFP. No good alignment with the GFP primers.

New primers ordered today: iGEM_106 and iGEM_107

Ribos A-D and RiboA+CFP for shipping

Ribo A-D picked from masterplate into 2x 4ml LB+Cm each (just to be sure)

Ribo A+CFP from Cryo Stock (RiboA C5.8.1) into 2x 4ml LB + Cm

ON at 37°C for Miniprep on Wednesday

Inoculation of E.coli for competent cells (cloning strain DH5a)

4ml LB inoculated as a step between to keep culture alive till 3pm.

2x 250 ml SOB + Mg inoculated with ON culture.

One flask with 3ml, 1 with 6ml. Put into 18°C shaker at 3pm.

Plates for interaction essay

12 plates in total, all with 80 μg/ml X-Gal

6 with 1mM IPTG, 6 without

Streak out on Thursday, when new DarR is transformed into BL-21

Inoculation of BL-21 strain for competent cells

4ml LB inoculated from cryostock

Oligo Hybridisation

Promoter 1rev: 10 µl iGEM_74 + 10 µl iGEM_79 à 10 min at 98 °C, let it cool down until 37°C

DarR Operator: 10 µl iGEM_104 + 10 µl iGEM_105à 10 min at 80 °C, let it cool down until 37°C

Digestion of vector psB1C3 with Xbal and PstI and dephosphorylation

Test gels looked fine!

Ligation

psB1C3 E+S (phos.) + Promoter 1rev Hyb. (dephos.)

part7 E+S (dephos.) + DarR operator Hyb. (phos.)

psB1C3 X+P (dephos.) + part 6.4A

Fotos of Promoter Parts Plates from 2.9.13

-          fotos were taken after ca. 1 week of keeping plates in fridge( 4°C), so that clones might get more pink

-          however, its hard to see that part 2 C2 and C3 are slightly pink compared to Part 2 C1 and part 1 clones

Inoculation of clones for cyrostocks 

-          inoculation of clone 3 of each re-trafo part 1 – 4 and part 8 in 4 ml LBAmp, incubation ON, 37 °C, ca. 200 rpm

Inocculation of DarR rev Term rev C3 and DarR reporter system C1 in LB medium with Cm

for preparation of cryostocks tomorrow

Cloning procedure delivered to Sam

Repeat of Fluorescence Assay of our RiboswitchA CFP C5.8.1+2 and the CFP Religate C1+2 as control (Fluorescence characterization of RiboA) 

ON culture was grown up around OD 2.0

At 9:45 fresh LB was inoculated with an OD of 0.5

At 11:15 all cultures had grown to around 1.05

C5.8.1	C5.8.2	CFP C1	CFP C2
1.05	1.05	1.01	1.07

1mL for each culture was inoculated with a start OD of 0.05. No AB was used

Each culture was pipeted as triplets onto a 96 well plate with the same scheme as before.

The plate reader essay started at 11:45

Cryostocks of C5.8.I, C5.8.II and CFP C5.I, CFP C5.II

900µl of ON cultures (OD between 1 and 1.8) were mixed with 100 µl 100% DMSO in screw-lid tubes and put into one of the green boxes in the -80°C freezer.

LB-Cm plates 

2x 500ml LB Agar + 35 µg/ml Cm poured to plates

500ml LB Agar left over for feeding experiments

Inoculation of E.coli for competent cells (cloning strain DH5a)

2x 4 ml LB inoculated and grown for ca. 20h at 28°C

500ml SOB-Mg medium autoclaved