IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week8/Chemical and Light

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MIT lacZ parts RE digests 08/11

8-11 gel 1 MXHTA.jpg 1% agarose
Lane Table of Contents
1 1 kb ladder
2 P109 MIT cut ES (cut out insert 3093; vector 2079)
3 P110 MIT cut XP (insert 534; vector 3953)
4 P111 MIT cut XP (insert 534; cut out vector 3755)
5 P113 MIT cut XP (insert 534; vector 3973)
6 P112 MIT cut XP (cut out insert 534; vector 3722)

P108+45 colony PCR, P1+mtrB test digest 8/11

8-11 col pcr mxh.jpg 1.2% E-gel visualized using EtBr/UV
Lane Contents (approx. expected band size)
1-4 P108+45 putative colony PCR #12-15 (2.3kb)
2 P1+mtrB not BB test digest (ES) #82 (2.2+2.75kb)
3 P1+mtrB not BB test digest (ES) #87 (2.2+2.75kb)
4 P1+mtrB not BB test digest (ES) #95 (2.2+2.75kb)
5-12 1kb ladder

Nothing looks hopeful, so we'll wait until sequences come back.

Sequence results

All of the P1+mtrB not BBs aren't correct. 82 has not hits in BLAST, which 87 and 95 BLAST to pHELLSGATE- apparently some cloning vector.

Ligations/transformations 08/11

We ligated P109+63 to get RBS+full LacZ+terminator.

Plasmid Strain Number of colonies
P63 vector control DH5-alpha 0
P63+P109 (1:2 molar ratio) DH5-alpha 17
P63+P109 (1:2 molar ratio) TOP10 112

Colony PCR

We PCRed five colonies from each plate to check if they were the right size. We also used P109 as a positive control.

8-12 gel 1 MXHTA.jpg

Lane 1: 1 kb plus ladder

Lane 2: P109 positive control (3093)

Lane 3: P109+63A (3188)

Lane 4: P109+63B (3188)

Lane 5: P109+63C (3188)

Lane 6: P109+63D (3188)

Lane 7: P109+63E (3188)

Lane 8: P109+63F (3188)

Lane 9: P109+63G (3188)

Lane 10: P109+63H (3188)

Lane 11: P109+63I (3188)

Lane 12: P109+63J (3188)

CDF

Alternative method

Using XbaI and NheI sites around the origin in pCDF, we can cut it out and ligate it into an XbaI digested P48. Depending on the orientation of the insert, the NheI-XbaI mixed site can be either just ahead of the Cm resistance (what we want) or after the E site in the BBMCS. This would destroy the prefix, so we'll use digest multiple colonies with XS for the correctly inserted origin-resistance part to put into the base pSB3K3.

pCDF, P48 digest results 8/12

The pCDF Xbal-NheI double digest (overnight, 25μL, 0.75μL of each enzyme) appeared fine, and the 800bp insert was gel purified.

P48 Xbal mysteriously yielded 2 bands, so is going to be repeated with another tube of P48.

ompR (P115) digests 8/12

Since ompR #73 and #64 aligned to ompR, we're cutting it out with XP to put the whole promoter-RBS-ompR-term part into a vector with either a CDF or SC101 origin. However, these digest products were inexplicable. One had a bright 4kb band and a faint 3kb band. The other had a bright 3kb band and a very faint kb band. Although the latter is similar to what is expected, the insert was extremely faint. Digest was repeated.

8-12 gel 2 MXHTA.jpg

Lane 1: 1 kb ladder

Lane 2: P115 uncut (~3750)

Lane 3: P115A cut XP (~1000)

Lane 4: P115B cut XP (~1000)

Lane 5: P48 uncut (3477)

Lane 6: P48 cut XbaI (3477)

PCR

8/12

Since none of the alternative strategy mtrB or P105 is working, we tried to PCR out the parts again with Phusion, as it is a more processive polymerase and our products are all >2kb.

Phusion protocol says to use Tms ≥ 60°C, so we tried both a high and low temp set for each Rx. The low temp was optimal temp from using Platinum Taq; the high temp was the highest that indicated some form of a band.

The high temperature set used as template previous iterations of the same PCR. The low temperature set used either miniprep DNA or a colony.

Rx mix: 10μL 5x HF buffer
1μL 10mM dNTPs
1.25μL each primer
0.5μL template 0.5μL Phusion polymerase 35.5 H2O

P1/3

Rx conditions: 30s @ 98°C → 5x[10s @ 98°C → 30s @ (45 or 54)°C → 1min @ 72°C] → 10x[10s @ 98°C → 30s @ (45 or 54)°C → 1min5s @ 72°C] → 7min @ 72°C → ∞ @ 4°C

45°C worked- band purified, while 54°C did not (giant smear). AK digested and column purified.

mtrB not BB

Rx conditions: 2:30s @ 98°C → 5x[10s @ 98°C → 30s @ (46 or 52)°C → 35s @ 72°C] → 10x[10s @ 98°C → 30s @ (46 or 52)°C → 40s @ 72°C] → 7min @ 72°C → ∞ @ 4°C

46°C worked- band purified, while 52°C did not (giant smear). AK digested and column purified.

mtrB not BB ligations/transformations 08/14

Plasmid Strain Number of colonies
P1'BB vector control DH5-alpha 4
P1+mtrB not BB TOP10 24
P1+mtrB not BB DH5-alpha 4
P3 not BB vector control DH5-alpha 11
P3+mtrB not BB TOP10 104
P3+mtrB notBB DH5-alpha 24
8/15 P1/3+mtrB colony PCR

PCR conditions

  • 98 °C 2'30"
  • Repeat 35 times
    • 98 °C 10"
    • 55 °C 10"
    • 72 °C 45"
  • 72 °C 7'
  • 4 °C for ever
1-30

1-30.png

Lane 1: P1 vector control ligation

Lane 2: P3 vector control ligation

Lanes 3-30: P1+mtrB not BB (2.4kb)

  • 10, 22 picked for mL cultures.
31-96

31-96.png

Lanes 31-96: P1+mtrB not BB (2.4kb)

  • 44, 90 picked for 5mL cultures.

P105

P105C used for low temperature template.

Rx conditions: 2:30s @ 98°C → 5x[10s @ 98°C → 30s @ (57.8 or 60)°C → 35s @ 72°C] → 10x[10s @ 98°C → 30s @ (57.8 or 60)°C → 40s @ 72°C] → 7min @ 72°C → ∞ @ 4°C

57.8°C yielded a close double band a little bigger than 2kb (which is where the expected product is), while 60°C did not work (giant smear). We're not sure what the doublet is, so we are sending P105 for sequencing.

Redo 8/16

Template used is P105D. The expected band size is 2.2 kb. Bands excised, gel purified, AK digested, and column purified.

8-16 gel 1 MXHTA.jpg

RE digests: Tet QPI, HO-pcyA

08-13 mxhta.jpg 1% agarose
Lane Contents (approx expected size)
1 P116 #2 uncut (3.7 kb)
2 P116 #2 SP (3.7 kb)
3 P116 #3 uncut (3.7 kb)
4 P116 #3 SP (3.7 kb)
5 P116 #5 uncut (3.7 kb)
6 P116 #5 SP (3.7 kb)
7 P117 #1 SP (3.7 kb)
8 1 kb + ladder
9 P118 #3 uncut (4305 bp)
10 P118 #3 XP (1555+2750 bp)

The P116 and P117 digests appear to be totally wrong- there should only be 1 band. The pattern resembles an incomplete digest with BB enzymes on opposite sides of the part, but we used SP.

P118 band pattern matches ~ with an incomplete digest. The insert was cut and purified.

Making Thermoinducible cI System

Ligation of P104 and P63+P18 with cI RBS from TOPO

Digestion of cI RBS from TOPO and P18+P63 8/8

P18+ P63 not confirmed by sequencing yet.

' cI857 RBS TOPO ES 1 cI857 RBS TOPO ES 2 cI857 RBS TOPO ES 3 cI857 RBS TOPO ES 5 P18+ P63 ES 1 P18+ P63 ES 2 P18+ P63 ES 3 P18+ P63 ES 4 E1 P63 + RBS PP B E1 P104 + RBS PP A E1 P63c + RBS PP G
DNA Fill in Fill in Fill in Fill in Fill in Fill in Fill in Fill in Fill in Fill in Fill in
100X BSA 0.25 uL 0.25 uL 0.25 uL 0.25 uL 0.25 uL 0.25 uL 0.25 uL 0.25 uL 0.25 uL 0.25 uL 0.25 uL
10X Buffer 2 2.5 uL 2.5 uL 2.5 uL 2.5 uL 2.5 uL 2.5 uL 2.5 uL 2.5 uL 2.5 uL of Buffer 3 2.5 uL of Buffer 3 2.5 uL of Buffer 3
EcoRI 1 uL 1 uL 1 uL 1 uL 1 uL 1 uL 1 uL 1 uL 1 uL XbaI 1 uL XbaI 1 uL XbaI
Enzyme 2 1 uL SpeI 1 uL SpeI 1 uL SpeI 1 uL SpeI 1 uL XbaI 1 uL XbaI 1 uL XbaI 1 uL XbaI 1 uL PstI 1 uL PstI 1 uL PstI
Water Fill in Fill in Fill in Fill in Fill in Fill in Fill in Fill in Fill in Fill in Fill in
Volume 25 uL 25 uL 25 uL 25 uL 25 uL 25 uL 25 uL 25 uL 25 uL 25 uL 25 uL

Gel results 8/9

8-9-08 digests al.jpg 1% agarose gel visualized using EtBr/UV
Lane Sample
1 1 kB Ladder
2 cI857 RBS TOPO ES 1
3 cI857 RBS TOPO ES 2
4 cI857 RBS TOPO ES 3
5 cI857 RBS TOPO ES 5
6
7 1 kB Ladder
8 P18+ P63 EX 1
9 P18+ P63 EX 2
10 P18+ P63 EX 3
11 P18+ P63 EX 4
12 1 kB Ladder
13 E1 P63 + RBS PP B
14 E1 P104 + RBS PP A
15 E1 P63c + RBS PP G

RBS TOPO 1,3, and 5, and P18+ P63 EX 1-4 were extracted and purified.

Update: At least P18+ P63 1-2 have been sequence confirmed.

Digest of P104 and P63 8/9

' P63 EX P104 EX
DNA 5 uL 15 uL
100X BSA 0.25 uL 0.25 uL
10X Buffer 2.5 uL of Buffer 2 2.5 uL of Buffer 2
EcoRI 1 uL 1 uL
Enzyme 2 1 uL XbaI 1 uL XbaI
Water 15.25 uL 5.25 uL
Volume 25 uL 25 uL

Gel of Digestion 8/10

8-10-08 p63and104 ls.jpg 1% agarose gel visualized using EtBr/UV
Lane Sample
1 1 kB Ladder
2 P104 EX
3 P63 EX

Extracted and purified 8/10.

Ligation 8/11

Ligated P18 + P63 clones 1 and 2, and P104, with RBS from TOPO.

Plate Results 8/12

Plate Marker # Colonies Description
E1 P104 Dephos only Kan 0 colonies
E1 P104 + RBS TOPO 1 Kan 2 0.25 mm colonies?
E1 P104 + RBS TOPO 3 Kan 2-3 0.25mm colonies?
E1 P104 + RBS TOPO 5 Kan 1-2 0.25mm colonies?
E1 P104 + RBS TOPO 1 6:2 Kan 1-2 1mm colonies
E1 P63 + P18 Dephos only Amp 0 colonies
E1 P63 + P18 + RBS TOPO 1 Amp 1 1mm colony, 1 2mm colony
E1 P63 + P18 + RBS TOPO 3 Amp 11 1mm colonies not evenly distributed
E1 P63 + P18 + RBS TOPO 5 Amp 5-6 0.25mm colonies, 1 0.5mm colony
E1 P63 + P18 + RBS TOPO 3 6:2 Amp 0 colonies
E1 P63 + P18 2 Dephos Only Amp 2 0.25mm colonies
E1 P63 + P18 2 + RBS TOPO 1 Amp 22 1mm colonies Not evenly distributed.
E1 P63 + P18 2 + RBS TOPO 3 Amp ~23 1mm colonies Not evenly distributed.
E1 P63 + P18 2 + RBS TOPO 5 Amp 16 1mm colonies
Kan (-) ctrl Kan ~50 colonies
Amp (-) ctrl Amp 0 colonies
puc 19 (+) ctrl Amp 0 colonies

Colony PCR of Transformations 8/12

8-13-08 colonypcr1 al.jpg 1% agarose gel visualized using EtBr/UV
Lane Sample
1 1 kb ladder
2 E1 P104 + RBS TOPO 1 6:2
3 E1 P63 + P18 (1) + RBS TOPO 1 A
4 E1 P63 + P18 (1) + RBS TOPO 1 B
5 E1 P63 + P18 (1) + RBS TOPO 3 A
6 E1 P63 + P18 (1) + RBS TOPO 3 B
7 E1 P63 + P18 (1) + RBS TOPO 5 A
8 E1 P63 + P18 (2) + RBS TOPO 1 A
9 E1 P63 + P18 (2) + RBS TOPO 1 B
10 E1 P63 + P18 (2) + RBS TOPO 1 C
11 E1 P63 + P18 (2) + RBS TOPO 3 A
12 E1 P63 + P18 (2) + RBS TOPO 3 B
8-13-08 colonypcr2 al.jpg 1% agarose gel visualized using EtBr/UV
Lane Sample
1 E1 P63 + P18 (2) + RBS TOPO 3 C
2 E1 P63 + P18 (2) + RBS TOPO 3 D
3 E1 P63 + P18 (2) + RBS TOPO 5 A
4 E1 P63 + P18 (2) + RBS TOPO 5 B
5 E1 P63 + P18 (2) + RBS TOPO 5 C
6 1 kb Ladder
7
8
9
10
11
12

RE digests 08/14

8-14 gel 1 MXHTA.jpg

Lane 1: P45 cut XP (876; 2079)

Lane 2: blank

Lane 3: P48 cut X (3477)

Lane 4: P63 cut EX (3284)

Lane 5: P97 cut SP (2091)

Lane 6: mtrB cut ES (~2100; 1100, 2700)

Lane 7: 1 kb plus ladder

Lane 8: mtrB cut XP (~2100; 1100, 2700)

Lane 9: P115 cut ES (~960; 2750)

Lane 10: P116 cut SP (3687)

Lane 11: P117 cut SP (3687)

Lane 12: P118 cut XP (1555; 2750)

We used the new Fermantas enzymes and digested for 6 minutes.

mtrB Ligations/transformations 08/15

Volumetric ratios not 7:1 are 2:1 molar ratios. Vector controls used plain EB to fill volume to 8μL.

Ligation Insert:Vector/Amount of vector (μL) Strain Number of colonies Selection
P97 SP vector control 0.66 E1 ~35 small colonies Carb
mtrB(BB PCR) XP + P97 SP 7.34:0.66 E2 78 Carb
mtrB(BB PCR) XP +P97 SP 7.34:0.66 E1 ~20 small colonies Carb
P97 SP vector control 1 E1 ~25 small colonies Carb
mtrB(BB PCR) XP +P97 SP 7:1 E1 8 and small colonies Carb
P63 EX vector control 3.26 E1 5 Kan
mtrB(TOPO) ES +63 EX 4.74:3.26 E2 36 Kan
mtrB(TOPO) ES +63 EX 4.74:3.26 E1 0 Kan
P63 EX vector control 1 E1 0 Kan
mtrB(TOPO) ES +63 EX 7:1 E1 1 Kan
mtrB(BB PCR) ES+P63 EX 7:1 E1 0 Kan
P63 EX vector control 2.37 E1 0 Kan
mtrB(BB PCR) ES+P63 EX 5.63:2.37 E1 1 Kan
mtrB(BB PCR) ES+P63 EX 5.63:2.37 E2 128 Kan

Colony PCR 8/16

If mtrB is incorporated, the fragment should be ~2.2kb.

1-25 mtrB in TOPO ES + 63EX

MtrB TOPO+63.png

1 picked for 5mL culture

26-60 mtrB BB PCR ES + 63EX

MtrB BB PCR+63.png

None look hopeful.

61-96 mtrB BB PCR XP + 97SP

MtrB BB PCR+97.png

62, 63, 64, 67, 85 picked for 5mL culture.

RE digests 08/15

We digested with the Fermantas enzymes for 25 minutes

8-15 gel 1 MXHTA.jpg

Lane 1: 1 kb ladder

Lane 2: P108 cut SP (4155)

Lane 3: P115 #64 cut XP (~960; 2750)

Lane 4: P115 #73 cut XP (~960; 2750)

Lane 5: P116 #2 cut SP (3687)

Lane 6: P116 #3 cut SP (3687)

Lane 7: P116 #5 cut SP (3687)

Lane 8: P117 cut SP (3687)

Lane 9: P118 cut XP (1555; 2750)

Lane 10: mtrB TOPO cut ES (~2100; 3800)

Lane 11: mtrB TOPO cut XP (~2100; 1100, 2700)

Lane 12: P109 cut ES (3093; 2079)

Lane 13: P110 cut XP (534, 3419)

Lane 14: P111 cut XP (534, 3221)

Lane 15: P112 cut XP (534; 3189)


Ligations 8/16

Transformation results from 8/17:

(all ratios are 2:1 molar)

Ligation Insert Vector Selection # E2 colonies # E1 colonies
P97SP+mtrB topoXP 7.26 0.74 Carb 12 small
P63EX+mtrB topoES 5.38 2.62 Kan 10 0
P63EX+P109ES 6.51 1.49 Kan n/a 0
P116SP+P45XP 4.63 3.37 Kan 28 0
P117SP+P45XP 3.57 4.43 Kan 25 3
P108SP+P45XP 3.43 4.57 Kan 20 0
P116SP+old P112XP 2.09 5.91 Kan 70 2
P116SP+P112XP 3.67 4.33 Kan n/a 0
P117SP+old P112XP 1.37 6.63 Kan n/a 5
P117SP+P112XP 2.65 5.35 Kan n/a 0

Colony PCR 08/17

Phusion polymerase used.

Master plate 1

  • 1-17 P112+116
  • 18-22 P112+117
  • 23-42 P108+45

Master plate 2

  • 1-26 P117+45
  • 31-56 P116+45

Master plate 3

  • 1-10 mtrB(TOPO)+P63
  • 11-24 mtrB(TOPO)+P97
Strip tube PCRs (P112+116, P112+117)

Gel 1

8-17 PCR gel 1 MXHTA.jpg

Lane 1: 1 kb ladder

Lanes 2-12: P112+116 (Master plate 1, 1-11; 1471 kb)

9, 11 picked for 5mL cultures.

Gel 2

8-17 PCR gel 2 MXHTA.jpg

Lane 1: 1 kb ladder

Lanes 2-7: P112+116 (Master plate 1, 12-17; 1471 kb)

Lanes 8-12: 112+117 (Master plate 1, 18-22; 1471 kb)

21, 22 picked for 5mL cultures.

96-well plate PCRs (all the rest)
Lanes 1-20: P108+45 (Master plate 1 23-42)

108+45.png

30, 41, 42 picked for 5mL cultures.

Lanes 21-46: P117+45 (Master plate 2 1-26)

21-46.png

Lanes 47-72: P116+45 (Master plate 2 31-56)

47-72.png

Lanes 73-82: mtrB(TOPO)+63 (Master plate 3 1-10)

73-82.png

6 (lane 68) picked for 5mL culture.

Lanes 83-96: mtrB(TOPO)+97 (Master plate 3 11-24)

83-96.png

RE digests 08/17

We digested with Fermantas enzymes for 20 minutes.

8-17 digest gel 1 MXHTA.jpg

Lane 1: 1 kb ladder

Lane 2: P48 MIT cut X (3477)

Lane 3: mtrB(BB)+P97 #63 cut XP (2100)

Lane 4: mtrB(BB)+P97 #62 cut XP (2100)

Lane 5: mtrB(BB)+P97 #64 cut XP (2100)

Lane 6: mtrB(BB)+P97 #67 cut XP (2100)

Lane 7: mtrB(BB)+P97 #85 cut XP (2100)

Lane 8: mtrB(TOPO)+P63 #1 cut XP (2200)

Lane 9: P101 cut ES (3221)

Lane 10: P101 cut XP (3221)