Team:Brown/Notebook weekly Logs/Weekly Team2 Notebook

Week 6

Jul 20, 09

Plan for the week: • Run gels: digest OmpC, TetA

       → gel purify

→ ligate Tet into pGEMT-easy→ DH5α transformation → ligate OmpC-TetA standard assembly→ DH5α transformation • OmpR BB: grow cultures tonight→ genomic purification tomorrow • PCR Taz1BB • Amp-Kan-Tet plates • Mutagenic PCR

Tasks accomplished today:

1) Ashley did PCR of Taz1 -BioBrick Primers: Tar BB For (19.3 nm); EnvZ Rev (17.1 nm) -Control Primers: Amp Taz 1 For (23.0 nm); Control Taz1 Rev (26.9 nm) (i) Resuspend dry primers to 100 μM stock (ii) Make 20 μM working stock ( 1μL 100 μM stock + 4 μL H2O= 5 μL total) (iii) Template: Taz1 plasmid (miniprep) Mastermix: 47 μL Primer For + Rev: 1 μL each 3 tubes of BioBrick, 3 tubes of control PCR program: 1) 94°C for 5 min 2) 94°C for 30 sec 3) 57°C* for 30 sec 4) 72°C for 1.5 min 5) GoTo 2, 34 times 6) 72°C for 5 min 7) 4°C forever

2) Gel results for digests: 1% gel L1: 1 kb ladder L 2: Tet Steph (EcoRI, XbaI) L3: Tet MC ( E,X) L4: Tet Ash (E,X) L5: Tet Steph (E,P) L6: Tet MC (E,P) L7: Tet Ash (E,P) L8: Tet old (E,P)

<PHOTO TO GO HERE>

2% gel

L1: 1 kb L2: Omp2 MC L3: Omp1 Steph L4 : Omp 2 Steph L5: 100 bp ladder L6: 100 bp ladder

<PHOTO TO GO HERE>

• Did gel extraction on all samples

3) Making competent RU1012 (i) Plated RU1012 agar stab on LB-Kan plate (ii) 11 am: started liquid culture (iii) 6 pm: inoculate into SOB broth (2 mL, 4 mL, and 10 mL samples) (iv) check OD600 with 3 μL sample on nanodrop 11:40 am: 2 mL: 0.356 4 mL: 0.380 10 mL; 0.388 4) Testing transformation efficiency of DH5α (i) add 50 μL competent cells + 1 μL plasmid (OmpR miniprep 7a) (ii) incubate 30 mins on ice (iii) heat shock 30 sec at 42 °C (iv) incubate on ice for 2 min (v) 200 μL LB media (vi) incubate 20 min at 37°C (vii) plate different densities: 100 μL, 10 μL, 1 μL, incubate at 37°C at 4:30 pm (viii) calculate CFU

5) Gel for Taz1 Lane 1: 1 kb ladder Lane 2: 100 bp ladder Lane 3: Taz 1 Lane 4: Taz 2 Insert picture here

July 21, 09

Tasks for today: • Run digest again, test E and S on tet • Genomic purification of DH5α then PCR OmpR, Run gel • PCR Taz1, Run gels • Calculate competency of gels

1) Digest of Tet (from 7/16 minipreps) with EcoRI and SpeI Concentration (ng/μL ) μL DNA μL water Tet Steph 129.3 7.737 8.27 Tet Ash 126.1 7.93 8.07 Tet Michael 71.8 13.93 2.07

Incubate till 12:00 pm (1.5 hours) Insert gel here Lane1: 1 kb ladder Lane 2: Ash Lane 3: Ash 2 Lane 4: 1 kb ladder Lane 5: Steph Lane 6: Steph 2 Lane 7: Michael Lane 8: 1kb

E, S cut successfully for Tet→ enzymes are ok

2) Genomic purification performed. Primers: OmpR BB Reverse, OmpR BB Forward Mastermix: 47 μL; Primers: 1 each, DNA: 1 μL Insert gel here Lane1: 1 kb ladder Lane 2: 1 kb ladder Lane 3: Tube 1 Lane 4: Tube 2 Lane 5: Tube 3 Lane 6: Tube 4 Lane 7: 100 bp ladder Lane 8: 1kb

→ Do not use tubes 2 and 3 of genomic DNA 3) PCR of Taz 1 Insert gel pictures here A: amplification. B: Biobrick; Numbers correspond to Taz samples from minipreps of 7/17) Gel 1 Lane 1: 1kb Lane 2: 1A Lane 3: 1B Lane 4: 2A Lane 5: 2B Lane 6: 3A Lane 7: 3B Lane 8: 1 kb

Gel 2: Lane 1: 1kb Lane 2: 4A Lane 3: 4B Lane 4: 5A Lane 5: 5B Lane 6: 6A Lane 7: 6B Lane 8: 1 kb

July 22, 09 1) OmpC: Grow liquid cultures, make glycerol stocks, digest with EcoRI and SpeI, run 20 μL on 3% gel with minimal loading dye

2) Sequencing: OmpR, Registry (purified plasmid form), purifying PCR product

3) Sequencing, Registry Taz1→ miniprep transformation in DH5α; make glycerol stocks; purify PCR

Purification of OmpR1, Taz 2B, Taz 4A Nanodrop concentrations: OmpR1: 28.1 ng/μL Taz 2B: 16.6 ng/μL Taz 4A: 16 ng/μL

Sent in for sequencing

4) tetR: ligation into pGEM T easy (EcoRI, PstI)→ ligation into appropriate expression vector

Digest: 8μL pGEM, 8 μL water, 2 μL multicore buffer, 1 μL EcoRI, 1 μL PstI Incubate at 37°C Ligation of TetA into pGEM T-easy 10x buffer 2μL ligase 1 μL vector 1μL DNA 10 μL Water 6 μL Incubate overnight at 4°C

July 23, 09 1) 9am: stop inoculation of ompC, miniprep, nanodrop, sequencing, glycerol stocks 2) Find expression vector for tetR and ligate (from pGEM T-easy-tetR ligation) 3) Design primers for ompC 4) Make RU1012 competent Taking concentrations of RU1012 00 λ600 10:30 am 0.01 11:30 am 0.03 12:30 pm 0.05 1:10 pm 0.09 2:00 pm 0.12 2:40 pm 0.15 3:10 pm 0.2 3:50 pm 0.26 4:20 pm 0.33 4:50 pm 0.38 5:20 pm 0.50

Completed Inouye protocol, stored at -80°C

July 24, 09 1) Inoculated tet-pGEM T-easy in liquid cultures→ miniprep, nanodrop, digest→ expression vector

2) Run gel: Omp: cut with S, P Tet: cut with X,P Plasmid: 2079 bp Tet: 1191 bp OmpC: 108 bp

Supercoiled v.s Linear v.s Circular plasmids

Supercoiled: naturally produced by E.coli (i.e. miniprep) runs faster than linear plasmid Linear: plasmid fom restriction digest Circular: covalently closed plasmid (i.e ligation) runs slower than linear plasmid

July 25, 09 1) Standard assembly 10 μL Ligation mix 2) Gel Extraction 3) Nanodrop Concentrations: OmpC1: 1.8 ng/μL OmpC2: 3.9 ng/μL Tet3: 12.3 ng/μL Tet1: 4.6 ng/μL 4) Digest OmpC with SpeI and PstI

         		Nanodrop concentration:

Omp1 MC: 30.8 ng/μL Omp2 MC: 31.1 ng/μL 5) Ligate Tet3 into OmpC digest 6) Transformation of OmpC-TetA into DH5α and RU1012 Syzmanski Protocol - 50 μL cells and 1 μLDNA→ ice for 30 mins - Heat shock 42°C for 30 sec - 2 min on ice - 200 μL LB - incubate 20 min at 37°C - plate on Amp plates

July 26, 09 1) Inoculated ompC-tetA ligations @ 9:30 am→ miniprep liquid cultures→ glycerol stocks

Week 7

July 27, 09 1) Digests: RBS (SpeI, PstI), TetA (XbaI, PstI), OmpC (SpeI,PstI), TetA-pGEM (XbaI, PstI) 2) Ligation with Digested Tet from July 24th 3) Transform ligation: RBS-tetA→ inoculate ligation→miniprep, glycerol stocks 4) Ran gel: tet-pGEM (digest with X< P); should see 2 bands at 3000 and 1000 bp→ gel purify tetA 5) Digest: pBluscript (160.7 ng/μL)--? Transform into DH5α→ tet plates

July 29, 09 1) Digest SK with X, P 2) Obtain Tet X,P→ ligate into SK

→ sequencing

→ ligation with RBS (done with optimized protocol and standard protocol, incubated at 4°C overnight 3) Make amp-tet plates 4) Transform RU1012 with Taz1 (incubation started at 4:40 pm)→ run SDS page gel

Aug 2, 09 1) IPTG induction of Taz1 from RU1012 8:30 pm: started liquid cultures (5 mL LB, 5 μL Amp, colony of RU1012+Taz 10:30 am: move 0.5 mL liquid culture to 5 mL new liquid culture IPTG volumes: 5 μL, 6 μL, 7.5 μL, 10 μL 12:30 pm: spun 1 mL aliquot of 6 μL and 7.5 μL liquid cultures spun 5 mL aliquot of 5 and 10 μL liquid cultures→ freezer 1mL cultures -resuspend in 200 μL dH2O - take 20 μL of dH2O and move to another tube - add 2x sample buffer - incubate 5 min at 95 °C, vortex, lyse - spin at high speed, load supernatant

Week 8

Aug 4, 09 1) Tested Mutagenic Primers 2) SDS Page of Taz1→ run for 90 min at 121 V, stain overnight 3) DNA purification of PCR TazMut 4) Ligation of purified PCR product: mut Taz into pGEM

Aug 5, 09 1) Ligation of OmpR BB and Taz1 BB into pGEM T-easy 2) Transformation into DH5α, plate on Amp plates → ligation was unsuccessful→ redo

Aug 6, 09 1) Redo transformations Control: weird, clear colonies Taz MutC: 2 colonies Taz MutE: some clear colonies Taz MutD: good plate Taz MutA: none Taz Mut B: some Taz BB: some OmpR BB: good plate 2) liquid cultures of Taz B,C,D,E, Taz BB and OmpRBB 3) miniprepped all but TazE (nothing grew) 4) Sent in mutagenic products for sequencing

Week 9

Aug 10,09 1) Digests a. pGEm-OmpR with E,P→ insert b. pGEM-Taz with E,P-→ insert c. July 11 miniprep samples of OmpC to get BB ector with E,P cut sites→ vector backbone d. Ran a gel→ gel extraction e. Revived glycerol stocks of Taz1 f. Inoculated July 27 Taz1-DH5α transformation colonies in liquid cultures

Aug 11, 09 1) Round the Horn PCR! Primers: (i) P (phosphorylated)-For Mut R1 Taz 1+ Rev Mut R1Taz1-P (ii) P-For Mut R2 Tot Taz1+ Rev Mut R2 Tot Taz1-P (iii) For Mut R2 A Taz1+ Rev Mut R2 Tot Taz1-P (iv) P-For Mut R2B Taz1+ Rev Mut R2B Taz1-P (v) ForMut R1 Taz1+ Rev Mut R1 Taz1 (linear) (vi) For Mut R2B Taz1+ Rev Mut R 2B Taz1 (linear)

                   Primer Phosphorylation

(i) 37 μL H2O (ii) 5 μL PNK buffer (iii) 1 μL 50mM MgSO4 (iv) 5 μL primers (100 μM) (v) incubate at 37°C (vi) kill PNK- heat mixture at 95°C for 5 min

Aug 12, 09 1) started PCR PCR mixture 39 μL dH2O 5 μL 10x polymerase buffer 1.5 μL forward primer 1.5 μL backward primer 1 μL DNTPs 1 μL template=miniprep Taz1 1 μL Pfx platinum polymerase

PCR program (i) 95°C 1 min (ii) 93 °C 30 sec (iii) 48 °C 30 sec (iv) 72 °C 18 sec (v) Go to 2 25 times (vi) 4 °C hold

2) Loren Looger’s suggestions for changing Tar to become histidine receptor R69E, R69D, R69Q, R69N R73E, R73D, R73Q, R73N

Aug 13, 09 -Ligation of PCR products

Aug 14, 09 1) PCR amplification of Taz with new primers 2) Gel visualization (bright bands!) 3) Gel extraction (Qiagen) 4) Ligation

Aug 15, 09 1) Transformation results: no colonies on all except Taz1 PCR (150 μL ) and Taz Gel (50 μL )→ liquid cultures 2) Ligation again with different optimized combinations→ good results

Aug 16, 09 1) miniprep of Taz-pGEM ligations 2) Transformation of Taz-pGEm ligations

Week 10

Aug 17, 09 1) PCR Biobrick Taz→ run gel→ extract→ ligation into pGEM 20μL H2O 25 μL mastermix 1 μL template Taz miniprep 5 from 8/11 2 μL forward BB primers 2 μL reverse BB primers

PCR program: (i) 94°C 5 mins (ii) 94 °C 30 sec (iii) 53.5 °C 30 sec (iv) 72 °C 90 sec (v) Repeat 2-5 34x (vi) 72 °C 5 mins (vii) Hold 4 °C

Nanodrop concentrations of gel extractions of Taz BB PCR: Taz BB PCR1: 57.2 ng/μL Taz BB PCR2: 66.7 ng/μL

2) Digest Taz 1 minipreps (pGEM) wih Bam and Eco→ gel extract

Taz 1 miniprep (8/16 samples): Nanodrop concentrations (ng/μL) (1) :95.70 (2): 89.64 (3): 164.52 (4): 150.93 (5): 198.37 (6): 76.46 (7): 86.61 (8): 99.79 Digest at 37°C for two hours. 11:40 am-1:40 pm

3) Ligate Taz 1 into pGEm

Use Team 1’s gel extraction of pGem backbone(17.5 ng/μL), digested with B, E Ligate digested Taz1 into pGem: 3 μL insert, 1 μL vector 1 μL ligation buffer (10x) 1 μL ligase

Use optimized gel extraction protocol: load 2 20 μL samples in separate wells Cut as close as possible for each band→ combine in 1 tube QiaQuick Protocol notes: no unnecessary steps

4) Liquid culture at 5 pm of Taz1-pGem ligations 5) Geneart order

Aug 18, 09 1) Miniprep of Taz-pGEM 2) Run gel of digest of Bam-Eco on Taz-pGEM→gel extract→ ligation into pNoTat

Gel picture of Taz 1-pGEM digest (E+B)

Lane 1: 1 kb ladder Lane 2: digest #1 Lane 3: #2 Lane 4: - Lane 5: #3 Lane 6: #4 (mixture wasn’t 20 μL) Lane 7: - Lane 8: #5

Lane 1: 1 kb ladder Lane 2: - Lane 3: #6 Lane 4: #7 Lane 5: #8 Lane 6: - Lane 7: 1 kb ladder Lane 8: -

Refer to Taz Bam Eco Digest 8/18.tif for images Digests 1,2,3,6,7,8, came out nicely

Gel extraction: Mass (g) Mass+ gel (g) Gel (g) QG buffer (A) Tube 1-2 1.0201 1.1442 0.1241 372.3 (B) Tube 3 1.0106 1.0767 0.0661 198.3 (C) Tube 6-8 1.0213 1.2399 0.2186 655.8

Nanodrop concentrations: ng/μL A: 8.9 B: 8.7 C: 15.9

3) Transformation of Taz BB pGEM 4) Stratagene

Aug 19, 09 Ligation of TazBB-pGEM (10:30 am -10:45 pm) 1) 6.19 μL H2O 2) 1 μL 10x buffer 3) 1 μL pGEM 4) 1.31 μL Taz 5) 0.5 μL DNA ligase

Transformations into RU 1012 1) Taz pNoTat (4)+C 2) TazBBpGEM (4)+C

Aug 20, 09 Liquid culture: Testing RU1012 (Kanr) with Taz 1 (Ampr) Negative: no growth Amp: no growth Amp+Kan: growth Kan: growth Chloremphenicol: no growth A+K+C: no growth

Week 10

1. 8/24/09: Received successful sequencing for Taz1 (p-GEM). 2. Received GINKGO ompC-RFP (Tet): • Incubated plates, inoculated cultures, mini-prepped DNA

3. Constructed Taz1 BB: • Digest (EcoR1, Pst1) and gel extraction of mini-prepped Taz1 BB (p-GEM)

Taz1 BB (p-GEM) Mini-Preps (Nanodrop Concentrations):

      Sample	                 Concentration		         260/280

1 286.0 1.93 2 127.3 1.98 3 330.1 1.92 4 292.3 1.95 5 99.6 1.98 6 415.1 1.92 7 110.5 1.94

Photograph: Gel: Taz1 BB (p-GEM):

<PHOTO TO GO HERE>

Gel Extraction Concentration: Taz1 BB (p-GEM) Sample ng/uL 260/280 1 9.8 1.48 2 13.7 1.78

• Digest (EcoR1, Pst1) and gel extraction of BB vector.

Photograph: Gel: BB Vector:

<PHOTO TO GO HERE>

Gel Extraction Concentration: BB Vector 18.1 ng/uL 260/280 = 1.83 • Overnight ligation of Taz1 BB and BB vector.

For 50 ng BB vector (2.78 uL of 18.1 ng/uL BB vector Gel Extract) (1458/2079) (3 or 6) = 105.19ng (10.73 uL of 9.8ng/uL Taz1 BB (p-GEM) Sample 1 Gel Extract) or 210.39 ng (21.47uL) Taz1 BB. • Transformation of ligation into DH5α. Incubated plates, inoculated liquid cultures, mini-prepped DNA, received successful sequencing.

4. Stratagene Mutagenesis: (i) Thaw dNTP mix once; prepare single-use aliquots (-20°C) (ii) Control Reaction 5 μL 10 x rxn buffer; 2 μL pwhitescript control plasmid (4.5 kb); 1.25 μL control primers (1), (2); 1 μL dNTP mix; 38.5 μL ddH2O After: 1 μL pFuUltra DNA polymerase

Reaction: 5 μL 20x buffer; DNA template (Taz pNoTat); primer (1 mut), primer (2 mut); 1 μL dNTP; X μL ddH2O After: 1 μL PfuUltra DNA polymerase

Transformation→ plate: no colonies for both control snad samples

5. Tested Sequencing of Taz1 (p-NOTat ): • Digest (BamH1, EcoR1) and received successful sequencing.

Taz1 (p-NOTat) Mini-Preps (Nanodrop Concentrations): Sample Concentration 260/280 1 178.4 1.97 2 188.6 1.96 3 168.7 1.96 4 371.2 1.91 5 197.2 1.95 6 137.5 2.00 7 198.4 1.97

Week 12

Sept 10, 09 (i) control: 5 μL 10x reaction buffer 2 μL pwhitescript 1.25 μL control primer 1 1.25 μL control primer 2 1 μL dNTP 38.5 μL ddH2O → then 1 μL PfuUltra HF DNA polymerase (ii) sample (same except 2 μL pNoTat template 6 (137.5 ng/μL)

Result: no colonies

Sept 13, 09 Transformation of RU1012 T1: RU1012+ OmpC-RFP→ Tet plate (3:20 pm) T2: RU1012+ OmpC-RFP→ Tet plate (3:20 pm) T3: RU1012+ OmpC-RFP + Taz→ Tet-Amp plate (3:50 pm) T4: RU1012+ OmpC-RFP+ Taz→ Tet=Amp Plate (3:50 pm) T5: RU1012→ Tet plate (3:20 pm)

Week 13

Sept 14, 09 7 am Check plates: RU1012 negative control→ lots of growth!!! Batch of plates is bad. (200 μL) RU1012+OmpC-RFP→ lots of growth (50 μL) RU1012+ OmpC-RFP→ lots of growth MC IPTG+ Xgal→ no growth Sample 1 250 μL → no growth Sample 2 250 μL→ 3 colonies TC IPTG+ X-gal→ 6-7 colonies RU1012+Taz+Ompc-RFP 1→ no growth RU1012+ Taz+ OmpC-RFP 2→ growth

Sept 15+ 16, 09 - Testing Aspartate binding of Double transformants 8:15 pm: start 10 liquid cultures in minimal media with Amp, Tet, Kan 10:15 am: move 0.5 mL to new 5 mL culture tube (minimal media+ A, T,K)

- Liquid cultures didn’t grow Redid cultures→ 16 of them (i) LB→ growth (ii) LB+A→ growth (iii) LB+T→growth (iv) LB+A+T→ no growth (v) MM→ no growth (vi) MM+A→ no growth (vii) MM+T→ no growth (viii) MM+A+T→ no growth

Plates (LB amp, tet-new plates) IPTG induced: re-streak

- IPTG induced RU1012 Double transformation o OmpC-RFP (Tetr) o Taz pNotat miniprep1 from 8/21/09(Ampr)

Redid double transformation of RU1012 with Taz-pNoTat+OmpC-RFP 2:30 pm: visualized under fluroscope→ WE SEE RED!! DOUBLE TRANSFORMATION SUCCESS.

Liquid cultures (picked only red colonies) 5 μL LB (negative)→ growth LB→ growth LB+A→ growth LB+T→ no growth LB+A+T→ no growth MM (neg)→ no growth MM→ no growth MM+A→ no growth MM+T→ no growth MM+A+T→ no growth

Sept 18, 09 To do: try liquid cultures with less Tet Single transformation of RFP (TetR) Construct→RU1012+ DH5α Plate on only Tet plate (make sure to use the TetR construct is usd and not the first KanR

            construct we received)

Week 14

Sept 21, 09 Retry liquid cultures from LB-A-T plate

Amp Tet Growth - - Yes - 0.1 μL Yes - 0.5 μL Yes - 1 μL Yes - 2 μL Yes - 4 μL Yes 5 μL 0.1 μL Yes 5 μL 0.5 μL Yes 5 μL 1 μL Yes 5 μL 2 μL Yes 5 μL 4 μL Yes

IPTG induction (5 μL) at 10:25 am 0.5 mL liquid culture 5 mL Minimal media 5 μL IPTG

Result: All fluoresced red…there is lactose in LB.

Sept 23, 09 Minimal media liquid cultures (1pm→ 10 am) MM (neg)→ no growth MM (pos)+ RU1012 with Taz 1 from 8/20→ growth MM with double transformants→ growth MM+A with double transformants→ no growth MM+T with double transformants→ no growth MM+A+T with double transformants→ no growth

Testing for RFP (5mL cultures) 1) no IPTG no aspartate 2) no IPTG 100 μL 0.1M Asp (2mM) 3) 5 μL IPTG 50 μL Asp (1mM) 4) 5 μL IPTG 100 μL Asp (2mM) 5) 5 μL IPTG 10 μL Asp (0.2 mM) 6) 5 μL IPTG (3 hours later)→ 10 μL Asp 7) 5 μL IPTG (3 hours later)→ 50 μL Asp 8) 5 μL IPTG (3 hours later)→ 100 μL Asp