Team:Goettingen/NoteBook w10

test of newly competent cells XL1 Blue is ok

many clones on Amp-Xgal plate, no on all control plates

Miniprep of Part1 (nothing grew in the inoculated culture of part8 -> because we used the wrong resistance)

new inoccutaion of part8 from cryo stock in LB-Amp has to be done on monday

Miniprep of green clones:

part6,1 Clone1, Clone 2, Clone 3, clone 4, clone 5, clone 6, clone 7

Nanodrop:

2nd round RD from yesterday

Parts 1-4: SpeI FD

YFP, CFP and Part 8: XbaI FD

Katrin Gunkas Überprotokoll:

                  40µl DNA (all the elution)

                  5µl Enzyme

                  5µl Buffer FD

         = 50µl reaction

-->incubation at 37°C for 1.5h

-->cleanup using the PCR cleanup kit, elution in 30+10µl prewarmed H2O

-->Nanodrop and Gel monday!

Colony PCR from 8.8.13 – Gel run

-  4x 1%-agarose-1xTAE gels

-  Loading of 3 µl 2 log ladder as a marker

-  Loading of 5 µl of Colony-PCR samples

-  Run at 60 – 100 V

-  EtBr staining + destaining with water

-  UV detection

Gel 1:

Gel2:

Gel3:

Gel4:

-> w/o-insert-control clones correspond to part 7 plasmid (Terminator insert; PCR product 129 bp of Terminator + 140 bp (additional bases because of VF2 primer binding site) + 170 bp (additional bases because of VR primer binding site) = 439 bp

-> P3op + pSB1C3 – clones 1, 3 and 4: expected band at ca. 360 bp (ca. 50 bp P3op + 310 bp VR/VF2)

-> Riboswitches + pSB1C3 – clones: expected bands not seen, only bands at lower bp (SeqMan alignment of Riboswitch sequences and VR and VF2 --> the primers were not aligned to the riboswitch sequence --> no unspecific binding of primers somewhere in riboswitch sequence that could explain product shorter than the expected riboswitch products or the terminator product)

(expected bands:

iGEM_40/41: 231/213 bp + 310 bp = 541/523 bp ;

iGEM_40/43: 376/403 bp+ 310 bp = 686/713 bp ;

iGEM_41/42: 318/370 bp + 310 bp = 628/680 bp ;

iGEM_42/43: 463/466 bp + 310 bp = 773/767 bp)

-> DarR + pSB1C3 – clones: expected bands not seen (ca. 630 bp + 310 bp = 940 bp)

-> Cloning with Taq-amplified PCR products (Riboswitches) or PolyA overhang added by Taq after PCR (DarR) seemed not to allow restriction of ends with EcoRI and PstI

-> Cloning using hybridization oligos seemed to work quite well (3 positives out of 4 clones)

-> Repeat cloning of Riboswitches with new primers harboring the overhangs

-> MiniPrep of P3op clones 1, 3, 4 and their re-ligation clone C

MiniPrep of P3op clones

-  P3op clones 1, 3, 4 and re-ligation clone C

-  With Nucleospin kit from Macherey-Nagel

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

-  NanoDrop:

Preparation of P3op + pSB1C3 and P3op + part 6 clones for sequencing

-  Sequencing by SeqLab: in general 600 – 700 ng plasmid DNA in 6 µl + 1 µl Primer in 1:5 dilution of stock (tip: if plasmid concentration is not too high – i.e. 300 or 400 ng/µl, one can simply use 6 µl of plasmid solution with 100 – 200 ng/µl)

-  Part 6.2 C1: 6 µl of 261.5 ng/µl-plasmid solution + 1 µl iGEM 38 (VF2) 1:5 --> tube 1

-  Part 6.2 C2: 6 µl of 226.5 ng/µl-plasmid solution + 1 µl iGEM 38 (VF2) 1:5 --> tube 2

-  Sequencing of P3op C1: 6 µl of 175.7 ng/µl-plasmid solution + 1 µl iGEM 38 (VF2) 1:5 --> tube 3

-  Sequencing of P3op C1: 6 µl of 175.7 ng/µl-plasmid solution + 1 µl iGEM 39 (VR) 1:5 --> tube 4

PCR purification of test Riboswitch PCR reactions from 7.8.13

-  Qiagen PCR purification kit

-  Purification of iGEM_67/70: 4 µl and 8 µl reactions

-  Purification of iGEM_69/68: 4 µl and 8 µl reactions

-  Purification of iGEM_69/70: 2 µl, 4 µl and 8 µl reactions

-  Purification of iGEM_67/68: 4 µl and 8 µl reactions

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

-  Concentration (NanoDrop):

Preparation of CFP and YFP Samples from Katrins Miniprep yesterday for sequencing by SeqLab

 Pipetted ~700ng of DNA + 0.25µl undiluted primer + Water to 7µl as follows:

 1: CFP C1 VF2

2: CFP C2 VR

3: YFP C2 VF2

4: YFP C1 VR

Gel doc of PCR from Katrin yesterday test PCR part7 C1

M/2/4/8/NC

bands are ok so PCR purification was performed

-> marked as rev. terminator (in to do box)

newly prepared competent cells from yesterday (XL1 Blue) are tested with a 1 ng/ µl pbluescript Trafo and a trafo with just water and one without (see journal above)

put to 37°C at 13:00

inoculation of GFP positive cells

inoculation of part 8 and part1 from cryo stocks

First Round RD of the parts for new cloning strategy

Parts 1-4: PstI FD

YFP, CFP and Part 8: EcoRI FD

Katrin Gunkas Überprotokoll:

                  1.5µg DNA

                  4µl Enzyme

                  4µl Buffer FD

                  xµl H2O

         =40µl reaction

-->incubation at 37°C for 1.5h

-->cleanup using the PCR cleanup kit, elution in 30+10µl (40 overall) prewarmed H2O

-->Nanodrop results on eppis (all around 30ng), about 1.2µg DNA retrieved in all samples (Which is highly unlikely and once again should make us think about nanodrop measurements)

Tomorrow 2nd round! Parts 1-4 with SpeI and the others with XbaI!

Transformation from 6.8.13

-   plates: no colonies on 50 µl-plates observed, but on rest-plates

-  Neg. control plates: no colonies

-  w/o DarR insert plate: few colonies

-  w/o Riboswitch insert plate: few colonies

-  w/o Promoter3operator insert plate: few colonies

-  Promoter3operator + pSB1C3: few colonies

-  Riboswitch 40/41 + pSB1C3: several colonies

-  Riboswitch 40/43 + pSB1C3: several colonies

-  Riboswitch 41/42 + pSB1C3: several colonies

-  Riboswitch 42/43 + pSB1C3: many colonies

-  DarR + pSB1C3: many colonies

-  Promoter3operator + part 6: some colonies --> see above (fluorescence microscopy)

-  Plates stored in big fridge at 4 °C

Colony PCR for Trasnformations from 6.8.13

-  according to protocol from ….; preparation of MasterMix for 55 reactions --> 25 µl

-  PCR tube Colour/Number coding of clones:

o   w/o DarR insert plate: 1 colony picked (A; white)

o   w/o Riboswitch insert plate: 1 colony picked (B; white)

o   w/o Promoter3operator insert plate: 1 colony picked (C; white)

o   Promoter3operator + pSB1C3: 4 colonies picked (1, 2, 3, 4; yellow)

o   Riboswitch 40/41 + pSB1C3: 8 colonies (5, 6, 7, 8, 9, 10, 11, 12; orange)

o   Riboswitch 40/43 + pSB1C3: 9 colonies (13, 14, 15, 16, 17, 18, 19, 20, 21; pink)

o   Riboswitch 41/42 + pSB1C3: 7 colonies (22, 23, 24, 25, 26, 27, 28; violet)

o   Riboswitch 42/43 + pSB1C3: 10 colonies (29, 30, 31, 32, 33, 34, 35, 36, 37, 38; blue)

o  DarR + pSB1C3: 10 colonies, but only first 8 inoculated for MiniPrep (not enough medium prepared for 48 and 47); (39, 40, 41, 42, 43, 44, 45, 46, 47, 48; green)

-  PCR taken out, addition of 5 µl 5xLD to each reaction; reactions stored at 4 °C ON --> gel run tomorrow

-  Inoculation of clones 48 and 47 in 4 ml LBCm and incubation ON at 37 °C, ca. 200 - 210 rpm

Primer design for DarRrev for DarR reporter system

-  Sequence of DarR fwd primer + suffix with overhang

-  Sequence of DarR rev primer + prefix with overhang

-> Ordered by Katrin G.

PCR for amplification of different riboswitch inserts: Test PCR for optimal primer concentration

-   Primers iGEM_67/68/69/70

1x reaction (50 µl)

         - preparation of master mix for 20 reactions containing

- addition of template (chrom. DNA/dH2O), primers and a part of the water (indicated in parenthesis in table for composition of each reaction) individually, then addition of 33 µl master mix

Gel run: Riboswitch Test PCR and Test RD of plasmids from YFP and CFP clones

-   1.5 % Agarose-1xTAE gel

-   4 µl PCR reaction + 1 µl 5x DNA Loading Dye

-   5 µl of Test RDs; 1 µl uncut plasmid + 1 µl 5x DNA Loading Dye + 3 µl dH₂O

-   3 µl 2 log ladder

-   Run at ca. 100 V in 1xTAE buffer

-   Staining in EtBr and destaining in water

-    UV detection

Gel 1:

Gel 2:

-> RD: Expected band ca. 750 bp + ca. 2 kb for all digests. The expected bands were obtained for all RDs --> all clones contain plasmids --> prepare the plasmids for sequencing by G2L

-> PCR: for expected products: add ca. 2x 20 bp to sizes indicated above. The expected bands were obtained. However, for iGEM_67/68, iGEM_69/68 and iGEM_67/70, a slight band at approx. the bp of 2x expected was observed for 2 µl reaction (“product dimmers?”), this seemed to decrease with increasing primer amount. Hig iGEM_69/70 primer amounts seemed to interfere with PCR. The negative control only shows primer clouds at bottom of lanes. The optimal primer concentrations for the different PCR reactions seem to be:

Primers for generating reverse terminator insert

    -> Primers arrived: dissolved in HPLC water and diluted 1:20

-> Primers tend to form secondary structures --> Test PCR for optimal primer concentration

-> Annealing temperature: TA = 0.5 * [Tm(iGEM_71) + Tm(iGEM_72)] – 6 °C = 0.5 *[60 °C + 59.1] – 6 °C =53.55 °C

TEST PCR

1x reaction (50 µl) using part 7 C1 plasmid as template

Plasmid concentration when used as a template: 10 – 20 ng/ reaction

Part 7 C1 purified on 11.7.13; concentration = 120.5 ng/µl --> 1:10 dilution in water --> ca. 12 ng/µl

-  preparation of master mix for 5 reactions containing

-   addition of template (chrom. DNA/dH2O), primers and a part of the water (indicated in parenthesis in table for composition of each reaction) individually, then addition of 33 µl master mix

PCR protocol

-> run ON

Restriction digest of DarR (polyadenylated PCR product)

-> Volume: 12 µl of unpurified DarR polyA reaction

- Preparation of the digest:

-> 1 h at 37 °C in ThermoCycler

-  Purification of RD reaction using PCR clean up kit (Qiagen): 500 µl PB buffer, 1x elution with 22 µl pre-warmed HPLC water (incubation for ca. 4 min at RT, ca. 1 min at 50 °C)

-  Concentration (NanoDrop)

Purification of pBluescript(RD with EcoRI) from gel

-  1.1088 g (2 ml epi with gel) – 1.0376 g (another empty 2ml epi) = 0.0712 g --> ca. 70 mg gel --> 210 µl QG buffer were used

-  Elution with 2x 22 µl pre-warmed HPLC water

-  Concentration (NanoDrop)

Ligation of DarR/hybridization oligo inserts with pSB1C3/part6

-  Ligation reactions

A) DarR insert (from polyA product) + pSB1C3 E+P (from part 7; 31.7.13)

B) hybridization oligo Promoter 3-DarRoperator + pSB1C3 E+S (from part 7)

C) hybridization oligo Promoter 3-DarRoperator + part 6 E+ X

For DarR ligation: ratio 1:3 was kept --> ca. 50 ng vector (2070 bp) + ca. 43 – 51 ng insert (600-700 bp)

For Hyp oligo: 20- 30 ng vector + half amount of reaction mix from

-> Incubation at 16 °C for several hours (started at 11:30)

Gel run of DarR insert and pBluescript+EcoRI

-   1 % agarose-1xTAE gel

-    Loading of 3 µl 2 log ladder

-    Loading of 1 µl pBluescript (2.8.13; 342.0 ng/µl) + 3 µl dH₂O + 1 µl 5xLD

-    Loading of 4 µl pBluescript purified EcoRI RD + 1 µl 5xLD

-    Run at 100 V

-    EtBr staining + destaining in water

-    UV detection

Gel:

-> EcoRI-linearzied pBluescript seems to be pure. Still, shortly below the linearized-plasmid-band, some additional weak bands were seen --> possibly unspecific side products? But the strong band of the supercoil-plasmid is gone --> further digest with PstI

-> For DarR insert, an additional band at 0.1 kb was obtained --> polyA’s that were cut off by the enzymes??? Or a non-specific side product from PCR? --> if this fragment can be cloned, we will notice it when the transformants are tested in Colony PCR…

Digest of pBluescript-EcoRI with PstI

Ca. 33 µl pBluescript left

-> Addition of 4 µl FD buffer and 4 µl PstI FD; incubation for 1 h at 37 °C

Design of oligos for self-hybridization

-> These oligos contain the reverse complement sequence of the parts indicated below, since they are designed for reverse integration into pSB1C3 (DarR transcription unit)

-> Oligos hybridization leads to EcoRI and SpeI overhangs

-  For strong RBS (part 8)

-  For promoters parts 1,2,3,4 (an additional sequence of ca. 60 bp (reverse translation of “cyclicdiampacterim”) was added in front of the promoter so that binding of RNA-Pol does not interfere with binding of RNA-Pol to promoter of GFP transcription unit)

Transformation of parts from distribution kit 2013 (CFP, YFP)

Inoculation of 3 clones of each transformation in LBcm for miniprep

Preparation of back-up plates

-> Forgotten in fridge (4 °C) on 5.8.13

-> Put to 37 °C by Dominik today