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

=MIT lacZ parts RE digests 08/11=

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

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.

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.



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.



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.

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


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


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.



=RE digests: Tet QPI, HO-pcyA=

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=

Digestion of cI RBS from TOPO and P18+P63 8/8
P18+ P63 not confirmed by sequencing yet.

Gel results 8/9
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.

Gel of Digestion 8/10
Extracted and purified 8/10.

Ligation 8/11
Ligated P18 + P63 clones 1 and 2, and P104, with RBS from TOPO.

Colony PCR of Transformations 8/12
=RE digests 08/14=

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.

Colony PCR 8/16
If mtrB is incorporated, the fragment should be ~2.2kb.

1-25 mtrB in TOPO ES + 63EX


1 picked for 5mL culture

26-60 mtrB BB PCR ES + 63EX


None look hopeful.

61-96 mtrB BB PCR XP + 97SP


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

=RE digests 08/15= We digested with the Fermantas enzymes for 25 minutes



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)

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



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



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.

Lanes 1-20: P108+45 (Master plate 1 23-42)


30, 41, 42 picked for 5mL cultures.

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


6 (lane 68) picked for 5mL culture.

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


=RE digests 08/17= We digested with Fermantas enzymes for 20 minutes.



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)