Team:Goettingen/NoteBook w5

Transformation of Ligation with GFP-Terminator insert and part 8 backbone worked!!!

No clones on negative control, but very few clones on without insert control. à possibly again incomplete digestion of part 8 (just 20 min) à use 1 h for dig, since reaction for gel ex yesterday seemed to be more completely digested….

Stored at 4 °C

No colonies on LB^Cm plates

è Further incubation till next morning

Part 7:

15 µl part 7 plasmid DNA (1 µg)

10 µl NEB buffer 2.1

2 µl EcoRI

2 µl PstI

71 µl H₂O

 

GFP-Term:

5 µl GFP-Term C1 plasmid DNA (1 µg)

10 µl NEB buffer 2.1

2 µl XbaI

2 µl PstI

81 µl H₂O

Both reactions were split into 2x 50µl (for more thorough reaction)

Both incubated for 30 min at 37°C, then heat inactivation for 20 min at 80°C

Gel-Extraction of pSB1C3 from the restriction digest of part 7 (EcoRI+PstI) for future ligations

Gel:

Purification of DNA from gel using Qiagen PCR purification kit:

-          like Katrin 25.06.2013

Elution with 30 µl HPLC water (pre-warmed)

NanoDrop:

pSB1C3 clone	Concentration (ng/µl)	A260nm/A280nm	A260nm/A230nm
1	6.2	1.35	0.12
2	11.3	1.27	0.21
3	10.2	1.71	0.06

 

 

è DNA frozen away in one of the red boxes with today´s date on the vial.

Clonation of the RBS-GFP-Terminator Construct:

Gel-Extraction of the GFP-Terminator construct from the restriction digest (XbaI+PstI), ligation into opened RBS-Vector (Part 8) and transformation

Gel:

Purification of DNA from gel using Qiagen PCR purification kit:

-          like Katrin 25.06.2013

-          Elution with 30 µl HPLC water (pre-warmed)

NanoDrop:

GFP-Term clone	Concentration (ng/µl)	A260nm/A280nm	A260nm/A230nm
1	5.4	1.75	0.07
2	19.5	1.62	0.19
3	6.2	1.32	0.10

 

 

è DNA frozen away in one of the red boxes with today´s date on the vial.

Ligation

-Clone 2 was used for ligation to the opened RBS-Vector (also known as part 8 –see 1.7.13)

-ligation was pipetted as follows:

13.5µl HPLC H2O

2µl Ligase buffer

2.5µl RBS-Vector DNA (~20ng)

1µl GFP-Term Clone 2 DNA (~20ng)

1µl Ligase

-For the control, the GFP-Term.-Insert was substituted with HPLC H2O

-Ligation was performed at RT for 10 minutes

-Heat-inactivation was performed at 80°C for 20 minutes

Transformation

-The whole ligation was used for the transformation.

-Transformation performed according to the methods-folder

Extraction of Terminator and RBS from cultures prepared on 03.07.

 

-       extraction of plasmids via NucleoSpin^RPlasmid kit

-       concentration measurement on Nanodrop

Sample	Concentration
T1	69 ng/µl
T2	80 ng/µl
T3	118 ng/µl
T4	95 ng/µl
RBS1	too low
RBS2	52 ng/µl
RBS1	too low
RBS1	too low

 

Digestion of extracted plasmids containing Terminator and RBS

è followed protocol for digestion as written in BioBrick^RAssembly Kit

 

Terminator:	Volume
plasmid DNA (pSB1C3)	500ng
PstI	1µl
EcoRI	1µl
NEB cutter buffer (10x)	5µl
H2O	to 50µl

è incubation for 10min at 37°C

è heat inactivation for 20min at 80°C

è store at -20°C

 

M| T1| T2| T3| T4

è Digestion failed   è set up digestion again è add 1µl of PstI and 1µl of EcoRI-HF è incubation for 45min at 37°C

RBS:

Components	Volume
plasmid DNA (pSB1C3)	10µl
PstI	1µl
EcoRI	1µl
NEB cutter buffer (10x)	5µl
H2O	33µl

è incubation for 30min at 37°C

è heat inactivation for 20min at 80°C

è store at -20°C

M |R2 |T1 |T2| T3| T4

 

-          0.5 µl uncut plasmid + 3.5 µl dH₂O + 1 µl LD 5x

-          Ca. 40 ng of R.D. of plasmids (= 4 µl) supplied with 1 µl of LD 5x

-          Run at 100 V on 4 %-agarose-1xTAE gel; marker: log ladder (3 µl)

-          EtBr staining, destaining, UV detection

Uncut plasmids visible (between 3 and 8kb, although about 2.5kb big), linearized plasmids visible (between 8 and 10 kb…), promoter fragments not visible at all (should be 30-120bps). Will add picture later.

Discussion:

The thick gel might have unrepresentative effects on large fragments (e.g. plasmids). This thesis is supported by the unreadable marker bands between 3 and 8 kb.

The promoter fragments might just be too small to be stained properly or they are not in the constructs. Only sequencing can solve this riddle…

 

Wells used as follows:

M_Part1uncut_Part1RD_Part2uncut_Part2RD_Part3uncut_Part3RD_Part4uncut_part4RD_M

used Marker: NEB Quick-Load 2-Log DNA Ladder (0.1-10kb)

Restriction digestion of isolated plasmids from yesterday run on 1% gel (for 1.5h)

can not open scn file. Saved under Reporter team gel doc in folder with this date (gel doc 2013-07-03 15hr 29min, control = 14hr 34 min)

 

 Wells as following:

RiboA (C1,C2,C3), RiboB (C1, C2, C3), RiboC (C1), RiboD (C1, C2, C3), lin part7 (C1, C2, C3), DarR (C1, C2, C4), GFP (C1,C2,C4), pSB1C3 (C1, C2, C3)

same order of wells for control

Control:

 

Digested:

è GFP-Terminator construct could be cloned successfully (fragment of slightly > 1 kb corresponding likely to GFP (ca. 900 bp) and terminator (ca. 170 bp) visible)

è Clones derived from transformation of linearzised part 7 contain apparently plasmid with part 7 (terminator) à ca. 200 bp fragment visible after digest, corresponding presumably to terminator à clones result possibly from transformation of not linearized plasmids present after incomplete digest of part 7

è For C2 of RiboB cloning, a weak band at ca. 200 bp could be detected, that does not fit with the expected insert of 370 bp à something else got cloned…

è For transformations of ligation reactions containing linearized pSB1C3 as a vector, no transformants containing the desired inserts could be obtained à plasmid that was transformed could be one of the residual template plasmids containing Promoter-RBS-RFP-Terminator (ca. 1.5 kb): after R.D., bands at 1.5 kb and 2 kb corresponding to the expected fragments could be detected. However, band at 1.5 kb is very weak, and for uncut plasmids, a strong band at this height representing the supercoiled plasmid could be detected à it’s also possible that pSB1C3 can’t be restricted properly… anyway, linearized pSB1C3 seems not to work for cloning à cut out terminator from part 7, extract backbone (= linearized pSB1C3) and use it for cloning of Ribo- and DarR biobricks instead…

Sequencing data arrived!

àNo biobrick in the DarR operator construct!

Transformation of T7 promoter

 

-       use T7 promoter BBa_1712074 in pSB1AK8 backbone (plate 5, 6N)

-       resuspend in 10µl H₂O

-       incubation at RT for 5min.

-       transformation of 2µl T7 promoter with 200µl of comp. E.coli cells and incubation for 30min on ice

-       90sec heat shock at 42°C

-       5min on ice

-       add 800µl LB medium and incubate for 1h at 37°C with agitation

-       plate out 100µl of transformation mixture on LB medium containing amikacin

-       centrifuge tubes for 1min, discard 800µl and plate out the rest on new plates

-       incubate plates for 2 days at 37°C

 

Cultivation of E.coli containing Terminator and RBS

-       reporter team cloned terminator (BBa_B0015; pSB1C3 backbone; Cm-resistance) and RBS (BBa_0034; pBS1A2; Amp-resistance) into E.coli

-       inoculation of each 4 liquid cultures in LB containing Cm and Amp

-       incubation at 37°C o/n with agitation

 

Back-up plates

-          for all inoculated clones (except for clones containing plasmids for parts 1 – 4)

-          incubation over day at 37 °C

-          stored at 4 °C

Plasmid Mini Preparation

-          for all inoculated clones

-          kit from Macherey-Nagel

-          elution with pre-warmed 30 µl HPLC water

-          DarR biobrick clone 3: almost all lysate spilled…

-          Concentration measurement on NanoDrop

Concentrations:

è DarR biobrick clone 3 has strangely high concentration

Test restriction digest of isolated plasmids

è Cut out inserts with EcoRI and PstI (Fermentas/ThermoScientific enzymes, not distribution kit!)

è Only 3 clones tested, if there are 5 to waste less enzyme…

For 1 reaction (10 µl total volume)

0.5 µl PstI

0.5 µl EcoRI

1 µl buffer 0 (both enzymes have 100 % activity and no star activity in this buffer)

3 µl plasmid DNA mix containing ca. 200 – 300 ng plasmid (see picture of NanoDrop table above, rightmost + smallest part of table)

5 µl dH₂O

è Master Mix consisting of

12.5 µl PstI

12.5 µl EcoRI

25 µl buffer 0

125 µl dH₂O

è In total: 250 µl

è Add 7 µl to each plasmid DNA mix

Incubation for ca. 1 h at 37 °C

Freezing of samples at -20°C

 

Digestion of plasmid pSB1C3 with EcoRI and PstI from linear precursor

 

Components:	Volume
plasmid DNA (pSB1C3)	20µl
PstI	1µl
EcoRI	1µl
NEB cutter buffer (10x)	5µl
H2O	23µl

 

è incubation for 30min at 37°C

è heat inactivation for 20min at 65°C

è store at -20°C

 

Dephosphorylation of vector pSB1C3

Components:	Volume
digested pSB1C3	50µl
antarch’s buffer (10x)	5µl
antarch’s phosphatase	1µl

 

Measurement of DNA concentration

Sample	Concentration
pSB1C3 (digested/dephosphorylated)	5,4ng/µl
Strep-Tag (digested)	126ng/µl
S. areus DAC domain (digested)	181ng/µl

 

Ligation of Strep-Tag; S. aureus DAC domain purified and S.aureus non purified with pSB1C3

Strep-Tag - components:	Volume
insert (Strep-Tag)	1µl
plasmid DNA (pSB1C3)	2µl
T4 ligase	1µl
T4 ligase buffer (10x)	2µl
H2O	14µl

 

S.aureus purified and non purified - components:	Volume
insert (S. aureus DAC domain)	1µl
plasmid DNA (pSB1C3)	2µl
T4 ligase	1µl
T4 ligase buffer (10x)	2µl
H2O	14µl

 

è incubation for 1h at room temperature

Transformation of ligated samples with competent E.coli cells

-       add ligation sample to 200µl of comp. cells and incubate for 30min on ice

è each 2µl and 10µl of ligated Strep-Tag, DAC domain S.aureus and relegation control used

-       90sec heat shock at 42°C

-       5min on ice

-       add 800µl LB medium and incubate for 1h at 37°C with agitation

-       plate out 100µl of transformation mixture on LB medium containing 35µg/ml chloamph.

-       centrifuge tubes for 1min, discard 800µl and plate out the rest on new plates

-       incubate plates for 2 days at 37°C

 

è Transformation failed

 

Reactions according to manual of Distribution kit:

-          Parts 1 – 3: restriction of 1 µg plasmid DNA with SpeI and EcoRI-HF; 100 µl in total (DNA, enzyme amounts and buffer amount doubled compared to common 50 µl reaction; but distributed to 2 epis with 50 µl each à more complete reaction)

-          Part 4: restriction of ca. 250 ng plasmid DNA (8.7 µl from 29.5 ng/µl solution) with SpeI and EcoRI-HF

-          Part 8: restriction of 500 ng plasmid DNA with SpeI and PstI

-          For parts 1,2, and 3: MasterMix consisting of both enzymes and buffer and sufficient for 4 reactions was used

 

è 20 min at 37 °C digestion

è 20 min at 80 °C heat-kill

 

-          1xTAE-1%agarose gel

-          Load 3 µl of each digest (ca. 30 ng) and 1 µl of plasmid as a control, supplied with 5xDLD

-          3 µl of log ladder as marker

-          Run at 100 V

-          EtBr staining + destaining

Gel:

 

è Still, R.D. is partial à next time: try 30 min incubation

è One can conclude linearization of all plasmids, but promoter fragments are not visible…. Try higher concentrated gel and load higher amounts of DNA

è Reactions stored at – 20 °C

 

Transformation from 27.6.13

-          Colonies grew on all plates except for negative controls and those of pSB1C3 transformation

-          Colonies on without-insert-control of linearized pSB1C3 look slightly pink à remaining template from PCR transformed = pSB1C3 plasmid?

-          Colonies on without-insert-control of linearized part 7 vector might result from not digested plasmid (partial digestion, as usual….)

-          Just 1 clone for Riboswitch biobrick C…

-          Inoculation of 5 clones for each (except for Ribo C) transformation; for without-insert-controls, 3 clones each were inoculated; 4 ml LB+Cm (35 µg/ml)

-          Incubation ON

 

New plasmid preparation for parts 1 – 4

-          Inoculation of parts 1, 3, 4 C1 and part 2 C2 in 4 ml LB+Amp

-          Incubation ON

Re-streak clones for parts 6 and 7 on 35 µg/ml Cm plates + incubation on

Purification of part 8 (RD) products from today

PCR clean-up with Qiagen kit

 

Elution with 50 µl HPLC water (pre-warmed)

NanoDrop:

	Concentration (ng/µl)	A260nm/A280nm	A260nm/A230nm
part 8 purification	7.5	1.34	1.39

 

 

 

Either just low amounts of DNA in the sample or the purification was not very efficient. Eluded a second time

with 20µl, no DNA found.