Team:Goettingen/NoteBook w9

Pooling of the PCR products from yesterdays riboswitch and new NanoDrop measurement

Gel doc:

42/41, 42/43, 40/41, 40/43

Restriction digestion of these riboswitch PCR products:

EcoRI +PstI

for 42/43, 40/41, 40/43

30µl PCR product

3µl EcoRI

3µl PstI

4µl FD Buffer

7µl H2O

for 42/41:

37µl PCR product

3µl EcoRI

3µl PstI

4µl FD Buffer

7µl H2O

EcoRI +SpeI

for 42/43, 40/41, 40/43

30µl PCR product

3µl EcoRI

3µl PstI

4µl FD Buffer

7µl H2O

for 42/41:

37µl PCR product

3µl EcoRI

3µl PstI

4µl FD Buffer

7µl H2O

digested for 30min at 37° (or 1h?)

Gel run:

Pouring of 1% agarose gel

Loading of 4 µl supplied with1 µl 5x LD of each digest

Loading of < 4 µl of the remaining uncut purified PCR products supplied with 1 µl 5xLD (just 40/41, 40/43 and 43/42 left…)

Loading of 3 µl log ladder

Run at 100 V

EtBr staining and de-staining

UV detection

Gel:

->  Strangely, the relationship between the length of the different inserts fits on their own. However, they are running at a much lower height than expected (expected 200 – 500; observed: 100 – 200…) -> something strange happened during the PCR, because already on the gel to see if the PCR worked, the bands are too low… -> Wait for Colony PCR (see below); if all clones negative, repeat PCR

->  Reactions stored at – 20 °C in one of our pink boxes

Preparation of LB plates

1x 500 ml LBChl

Competent Cells-Test

-          No colonies on LBAmp+X-Gal, LBAmp, LBCm

-          > 50 colonies on 50 µl plate of 1 ng pBluescript transformation (expected contamination was not observed); on Rest-plate: many more colonies; colonies appeared blue, but some seemed to be white (these colonies could have produced too low amounts of indigo dye – there are always differencens in the strength of the blue colour of different colonies; sometimes it’s hard to distinguish if these colonies are white or just light blue -> one can put the plates to 4 °C for some time, then the blue colour of the really blue colonies gets more intense), or they contain a somehow mutated plasmid…)

-          Neg. control (see pBluescrpit cloning) showed no colonies

-> 3 blue clones from 50 µl plate of pBluescript transformation were picked and streaked out on LBAmp; Clone 1 was inoculated in 4 ml LBAmp for MiniPrep and used as a control for Colony PCR (see below)

Transformation of pBluescript and pSB1C3 ligations from 31.7.13

-          No colonies on neg. control plates

-          Blue and white colonies on all other plates

-          Counting of the white colonies on 50 µl plates for w/o-insert-control, DarR and Riboswitch transformations

-> There were more white clones on the ligation plates than on w/o insert control (white clones might be derived from relegated and mutated vector (eg. Frame shift))

-> Colony PCR incl. Master Plate and inoculation of 4 ml LBAmp cultures for MiniPrep (3 white clones from w/o insert control, 5 white clones from each of the other plates)

pSB1C3:

-          No colonies on neg. control plates and on ligation plates

-> Further incubation of all plates

Colony PCR for pBluescript cloning on 31.7.13

-          Reactions:

-                    1x pBluescript plasmid 1:10 diluted (white epi A)

-                    1x C1 from transformation of 1 ng pBluescript plasmid (see above) (white epi B)

-                    (white) C1, C2 and C3 from w/o insert control plate (white epis C, D and E)

-                    (white) C1 – C5 from DarR plate (white epis 1 – 5)

-                    (white) C1 – C5 from Ribo 40/41 plate (yellow epis 1 – 5)

-                    (white) C1 – C5 from Ribo 40/43 plate (orange epis 1 – 5)

-                    (white) C1 – C5 from Ribo 42/41 plate (violet epis 1 – 5)

-                    (white) C1 – C5 from Ribo 42/43 plate (blue epis 1 – 5)

-          Colony PCR was performed according to protocol from 10.7.13 with some modifications:

a) Inoculation of liquid culture for MiniPrep, as well

b) Primers M13-PUC-fwd and M13-PUC-rev diluted 1:20 in HPLC water and used for Colony PCR (now as well in 1:20 dilution in our box with all other primers) -> I changed the Annealing Temperature in one of the cyclers in which the protocols are saved!!! It’s 52 °C, not 54°C as required for VF2/VR PCR.

Inoculation/Plating was done in the following order:

1. Picking of clone and streak-out on Master Plate

2. Inoculation of 4 ml LBAmp  liquid culture ON for MiniPrep

3. Inoculation of PCR reaction

-> Incubation of liquid cultures and plates ON at 37 °C

-> After PCR, the PCR reactions were stored at -20°C in yellow-tip-box-rags sealed with parafilm

Transformation of parts BBa_E0030 and BBa_E0020

-          Solved parts in 10 µl HPLC water

-          Transformation of E. coli DH5α according to methods folder

-          Plating on LBCm plates + Incubation ON at 37 °C

->  For some of the Riboswitches, we need a Reporter without RBS. But The GFP of part 6 already contains a terminator and an RBS. So, I searched in the registry for a GFP reporter (or something similar) without RBS (and maybe with terminator). However, the transformation of these parts is just a try, because previous transformations of distribution kit plasmids did not work at all (including the GFP in part 6 without any regulatory sequences). If the transformation fails again, we will order the forward primer for amplifying GFP from part 6 (see below)

Primer Design

-          Design of forward primer for amplification of GFP from Part 6 (as a reverse primer, we can use VR. Then we directly amplify the terminator with the GFP)

-          I introduced a point mutation in the primer for the third base of the second codon after the ATG (AAA -> AAG; both encodes for Lys), because then, the primer won’t form secondary structures (without this mutation, it forms strong secondary structures)… but now, I’m unsure, if we can do that. Because it should actually be the same sequence we clone into our other plasmids…

Primer not yet ordered

Restriction digestion of Part1,3,4 with SpeI and PstI:

Gel doc:

part M/1/ 1/3/3/4/4

-> only partial restriction -> we’ll do an overnight restriction

New Riboswitch PCR

(as performed on 24.6.)

Tm (iGEM_40) = 65.7 °C

Tm (iGEM_41) = 65. 7 °C

Tm (iGEM_42) = 56.7 °C

Tm (iGEM_43) = 57.3 °C

PCR reaction and protocol as usual (50µl) (template: B. subtilis chrom.DNA)

Concentration of primer iGEM_43 doubled (4µl per reaction)

Reactions stored at -20°C in Falcon Tube

Gel doc:

iGEM_42/43, 42/43, 42/43; 42/41, 42/41, 42/41; 40/41, 40/41, 40/41; 40/43, 40/43, 40/43

after PCR purification following values with Nanodrop:

Restriction digestion of Part 7 with EcoR1 and PstI

-          2x 40 µl digestion setup with ~1,5 µg template

-          1h at 37°C

-          Run on gel for gelextraction

Gelextraction of Part 1,3,4,7

(planned ON digest of parts 1,3,4 was not done…???)

-          Whole volume of the digestion setups run on gel, 85 V for ~45 min

-          stained in EtBr for 15 min, washed in H20 for 10 min

-          1 LogLadder; 2-6 Part 1; 7-14 Part 3;   1 LogLadder; 2-7 Part 4; 8-16 Part 7

-          Thick band in the middle was cut out, same templates pooled. For part 7, upper band cut out and pooled in the gel extraction process

-          Standard protocol for gel extraction was used. Eluation in HPLC H2O, 2 Steps with 15 µl each (prewarmed 80°C)

Nanodrop measurements:

-          Part1: 6,7 ng/ µl

-          Part3: 14,4 ng/ µl

-          Part4: 15,8 ng/ µl

Part7: 56,6 ng/ µl

Transformation of ON ligations from 30.7.13 and Test of DH5α competent cells

-          Strain: DH5α

Transformation A1: pBluescript cloning

-          ON ligation reactions for cloning of DarR and Riboswitches 40/41, 40/43, 41/42, 42/43 into pBluescript -> addition of whole ligation reactions to comp. cells-> transformation -> plated on LBAmp+X-Gal plates

-          neg. control: addition of 10 µl dH2O-> transformation -> plated on LBAmp+X-Gal plates

Transformation A2: competent cells-testing

-     pBluescript (272 ng/µl) -> preparation of 1:272 dilution in dH2O (= 1 ng/µl) -> addition of 1 µl of the dilution to comp. cells -> transformation -> plated on LBAmp+X-Gal plates

-     1 tube with comp. cells was left untransformed (no DNA, no water) ->cells had to endure same transformation procedure as all other cells -> 150 µl were plated on LBAmp, LBCm or LBAmp+X-Gal plates (no rest plates, no 50 µl plates)

-    The negative control with water from Transformation A1 is considered to be a negative control for comp.cell-test, as well

Transformation B: pSB1C3 cloning

-    ON ligation reactions for cloning of DarR and Riboswitches 40/41, 41/42, 42/43 into pSB1C3-> transformation -> plated on LBCmplates

-     neg. control: addition of 10 µl dH2O-> transformation -> plated on LBCmplates

->  transformation was performed according to the methods folder with the following silly mistakes

1. heat shock took longer than 90 sec., probably 100 sec?

2. while all reactions where heat-shocked, the pSB1C3 neg. control was still on ice -> heat-shock done with slight time shift to all other samples…; heat shock for pSB1C3-NC took 100 sec precisely

3. I contaminated at least 2 plates during plating…

4. Since X-Gal is light-sensitive, the plates were incubated in a paper box at 37 °C

->  Conclusion: This transformation will work. Because I DON’T want it to work.

Preparation of Plates

1x 500 ml LBCm

1x 250 ml LBAmp+X-Gal

-> plates started already to solidify -> clumps were in the agar, while I poured them… I’m not sure, if we should use them…

RD of Parts 1-4 and Part7 (see yesterday)

 a 1%gel was run to determine the quality of the digest

Gel:

M|Part1|Part2|Part3|Part4|Part7|M|Oligo Hybridization iGEM63+64 (see yesterday)

Parts 1, 3 and 4 seem to be pretty much completely digested. Part 2 shows strange bands, the tube was discarded. The Primer hybridization shows no telling bands but a normal primer cloud. Hopefully it can be ligated.

->Parts 1, 3 and 3 were purified using the PCR clean up kit and eluted with 50µl pre-warmed H20 (mistake)

->2nd round of the RD with PstI (NOT FD because empty new stuff ordered). Due to the 50µl elutions, I distributed 25µl into a new vial and then pippetted two digestion reactions per construct. This should have the beneficial side effect of an even more complete RD.

           2x        4µl PstI

                  4µl Buffer 0

                  25µl DNA (everything)

                  7µl H2O

                  40µl reaction

->Incubation at 37°C for 6,5h

->frozen away, gel and clean-up tomorrow

DarR and Riboswitch Clonation from last week

The plates used for DarR in pSB1C3, Riboswitch in pSB1C3 and E0840 turned out to be of the wrong antibiotic recepee (5µg/ml instead of the right one: 35µg/ml).

->The inoculated cultures from yesterdayof course didn´t grow therefore

->The plates from last week were washed with LB media and plated on new, right 35µg/ml plates to see if they contain correctly ligated clones.

Gel extraction of Part 7

-          Gel Ex using the standard protocol. Eluation in 2 steps: 40 µl and 10 µl, prewarmed H2O

-          2 epis 50 µl each, stored in -20°C

-          Nanodrop meassurment: 6,4 and 6,3 ng/ µl. Also written on the tube

Ligation of DarR, the Riboswitch constructs (40/41, 42/41, 42/43)

Ligation of DarR and Riboswitch inserts with pSB1C3 backbone purified from part 7

-          Reaction of 20 µl total volume (1 µl ThermoScientific T4 Ligase + 2 µl 10x buffer + 17 µl insert-vector-water mix)

-          Vector: gel ex + purification of pSB1C3 backbone from part 7 by Dominik and Jonathan -> there was not enough vector left for the cloning of all riboswitches -> therefore, Riboswitch iGEM_40/43 was left out

-          Inserts: from 15.7.13

-          Calculation of approximate insert amounts for vector:insert ratio = 1:3 using online ligation calculator:

Size of vector ca. 2070 bp

Size of DarR and Riboswitch inserts -> uncut PCR product size used

Reactions

-          Preparation of Master Mix for 6 reactions in total (Actually, it’s more clever to put the vector and 1 µl water/reaction more into the master mix, but I prepared the DNA mixtures first. Because of a pipetting error, I had to do the master mix this way. Otherwise the buffer and ligase concentration between the reactions would have varied… So, don’t wonder if this protocol appears a bit… linky?...)

-          Preparation of DNA mixtures (14 µl in total; ca. 20 ng of vector for cloning of riboswitches and w/o insert control)

-          Addition of 6 µl Master Mix (it could be, that – when I was pipetting the riboswitch reactions – I did not change the tip… but I’m not sure about that. It was just a sudden thougth…)

-          Incubation on at 16 °C

Ligation of DarR, the Riboswitch constructs (40/41, 40/43, 42/41, 42/43) with pBluescript

Ligation of DarR and Riboswitch inserts with pBluescript vector digested with PstI and EcoRI

-          Reaction of 10 µl total volume (1 µl ThermoScientific T4 Ligase + 1 µl 10x buffer + 8 µl insert-water mix)

-          Vector: digested and purified by DAC team; Amp resistance + MCS in lacZ gene -> plate transformation reactions on LBAmp+X-Gal

-          Inserts: from 15.7.13

-          Calculation of approximate inserts amounts for vector:insert ratio = 1:3 using online ligation calculator:

Size of digested vector ca. 2616 bp

Size of DarR and Riboswitch inserts -> uncut PCR product size used

Reactions

-          Preparation of Master Mix for 7 reactions in total (ca. 20 ng of vector for cloning of riboswitches and w/o insert control)

-          Preparation of DNA mixtures

-          Addition of 5 µl Master Mix

-          Incubation on at 16 °C

Attention: DarR and Riboswitch iGEM_41/42 inserts are almost empty! I marked the tubes with a green dot. We have to prepare new ones! -> Whenever Spe and Pst FD arrive:

Inoculation of 8 Clones from each, Riboswitch, DarR and E0840 (were put in the fridge on friday, plated 23. and 24.7.)

 ->E0840 heavily contaminated with something that looks like fungus. Plates discarded.

->This leaves DarR and the Riboswitch for inoculation in 4ml Cam cultures for a miniprep tomorrow.

->in addition, a masterplate was plated.

Hybridization of iGEM_63 and iGEM_64 to generate the Promoter(part3)-Operator construct

The primers were first diluted in HPLC-H2O according to the QC-sheet (100µM). Then a 1:20 dilution stock was pipetted. For this experiment, the 1:20 dilution stocks were used.

Protocol:

10µl iGEM_63

+         10µl iGEM_64

           20µl reaction

->80°C for 10 minutes (heatblock)

->after 10 minutes, turn the heatblock off and let the sample slowly cool down in it

-> The ends of the oligos are “digested” with EcoRI and SpeI and phosphorylated. The product of this experiment can directly be ligated in front of the GFP-part!

See the gel tomorrow

Restriction digestion of Part 1-4 and Part 7

-          Part 1-4 digested with SpeI FD

-          Part 7 digested with EcoR1+SpeI FD + Phosphatase treatment (last 20 minutes of digestion)

-          40 µl setups, standard assay.

->for part 1-4, restriction with PstI (not FD, because empty) on 30.7.