IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week7/Chemical and Light

=PCR=

8/4: Cph/EnvZ, P1, P3, mtrB not BB
HO-pcyA and ompR bands excised, purified, and digested (ApaLI & Kpn overnight).

8/4 Colony PCR of putative mtrB samples
We picked 24 colonies each of old putative P98 and newly transformed putative P98+63 for colony PCR using the BioBrick primers and 55°C annealing for 30 cycles. If the colonies contained mtrB, a band at ~2.1 kb should appear. No colonies appeared to contain mtrB.

All gels with 1kb plus ladder

8/4 CphEnvZ, P1, P3, mtrB
Pl05 (CphEnvZ w/ PstI site mutated out) conditions: 5min @ 94°C → 40x [30s @ 94°C → 30s @ (50.8, 51.5, 52.6, 53.9, 55.4, 56.7, 57.7, 58.3)°C → 2:30 @ 72°C] → 7min @ 72°C → ∞ @ 4°C

8/5: gels
Bands excised and digested: Cph/P105 (AK), P3 (AK), mtrB (XP, ES)

Bands excised and saved: ompR, HO-pcyA, CDF

Ligations and transformations 08/06
We ligated P105 and P1/P3 not BB, and mtrB not BB and P1/P3 not BB. We tried letting the ligation reactions run for 5 and 10 minutes. We used P1/P3 not BB from 08/05 and from another date (older). We transformed using TOP10 and DH5α. We also retransformed P108, the Lac QPI with a high constitutive promoter in a p15A vector.

Results below

=TOPO cloning=

P1, mtrB
These had a few white colonies. Colony PCR, however, indicated that the correct PCR product was not incorporated.

ompR
All colonies were blue.

HO-pcyA
No colonies.

=Transforming in XL10-Gold cells=

=Ligations=

HO-pcyA, ompR, CDF
All three ligations yielded >50 colonies on the TOP10 cell plate with a 5 minute ligation in all combinations (HO-pcyA and ompR with P1 and old P3; CDF with P3). The P90 (CDF+P3) did not fluoresce. The DH5α cells did not have nearly as many (<5) and ligation times (10min, 30 min) did not appear to be significant.

Colony PCR 8-6
We picked at least 18 colonies from each ligation set, patched a master plate, inoculated liquid cultures, and performed 12.5 μL colony PCR reactions. The BBp/sfx primer pair was used, annealing temperature was 50°C for first 10 cycles and 54°C for rest of the cycles. 2 controls were performed: one using a picked P20 colony, and another with miniprepped P20 plasmid.

Digests, 1-8
We ran the digests to see if PCR mutations could have introduced an internal cut site. However, even with the small amount of purified digest we had left, we could see a properly sized band for each of P1, ompR, and HO-pcyA PCR products.

86-94, P20 controls
=Making Thermoinducible cI System=

Ligation from 8/1
Plate Results (also listed in Week 6):

Fluorescent colony was picked for liq culture and restreaked. Miniprep sent for sequencing-- ligation confirmed by sequencing (8/5).

Analytical Digest Gels from TOPO Cloning 8/5
Strange bands, but sent several clones for sequencing anyway.

Digestion and Ligation of P18+P63 and P75+P63
Done in tandem and side by side with Christina (except for digestion).

Ligation of P18+P63 and P75+P63 8/5
Used dephosphorylated and undephosphorylated vector.

Digestion of cI857 PCR product and P63 8/5
Christina digested the same samples of P63, cI857 RBS GP and PP, and cI857 BIG GP and PP with EcoRI buffer. P104 was cut 8/6 in the morning.

Ligation of P63, P63c, and P104 with cI857 8/6
Accidentally mixed P63c and P104 so plated each of those ligations in Amp and Kan selective plates.

Colony PCRs of 8/6 Ligations 8/7
E1 P63 + RBS PP B to be sequenced Monday. All of the bands in the third gel indicated re-ligated vector.

Ligation of P63, P63c, and P104 with cI (repeat) 8/7
Ligations were re-done with newly digested vector because of mishap in 8/6 ligations.

Gel of Digestion 8/7
Bands were extracted, purified, and dephosphorylated.

Ligation of P63, P63c, and P104 with cI 857 8/7
Vector backbones were ligated with cI857 digested 8/5.

Plate Results 8/8
Restreaked some non-fluor colonies from RBS ligations/ transformations to see if repressor is being repressed-- grew in 37 and 42 degrees. No perceived difference in fluor/ no induction.

Colony PCR of Ligations 8/8
3 bands in second gel indicate re-ligated vector.

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.

=mtrB, P97, P63 digests 08/06= We digested mtrB with ES and XP so that we can put it into the P63 (terminator) and P97 (RBS) vectors. We also digested P97 with SP (and dephosphorylated it).

Colony PCR
Ladders (end of each block) alternate as low range (100, 200, 400, 800, 2000bp) and 1kb plus. Expected band sizes indicated. (Lanes rearranged from 2% agarose 96 well E-gel)

1-16


Lane 1: Plasmid control P85 (2.3kb) Lane 2: Colony control P88 (2.2kb) Lanes 3-16: P105+P3 ligation colony PCR (2.5kb)

4, 6, 14 look like they have potentially correct products, so liquid cultures were grown up.

14 is incorrect- it's just P1 in pSB3K3.

17-24: P105+P1 ligation colony PCR (2.5kb)


None appear to be correct.

25-48: mtrB+P63 ligation colony PCR (2.2kb)


27, 28 picked.

RE digests 08/09


Lane 1: 1 kb ladder

Lane 2: mtrB+63 #28 cut XP (~2200+3.1kb)

Lane 3: E1P3A-P38+51C cut SP (4155)

Lane 4: E1P3A-P38+51D cut SP (4155)

Lane 5: E1P3A-P38+51B cut SP (4155)

49-64: mtrB+P97 ligation colony PCR (2.1kb)


51 picked.

65-80: mtrB'+P3 ligation colony PCR (2.3kb)


65, 69, 70, 78, 79, 80 picked.

65 sequence did not read properly.

81-96: mtrB'+P1 ligation colony PCR (2.3kb)


82, 87, 95 picked.

82 is wrong- mystery sequence. 87 and 95 are BLAST to pHELLSGATE ?!

=RE digests 08/07=

Lane 1: 1 kb ladder

Lane 2: E1P1+P17A cut EX (3652)

Lane 3: E1P1+P17C cut EX (3652)

Lane 4: E1P1+P17D cut EX (3652)

Lane 5: E2P1+P17A cut EX (3652)

Lane 6: E2P1+P17B cut EX (3652)

Lane 7: P108 cut SP (4155)

Lane 8: P45 cut XP (876; 2079)

Ligations 08/08

 * We ligated P17 in a p15A vector (P1 or P3) to P38 and P39. We used a vector to insert ratio of 1:7. We also prepared a ligation reaction with 1 μL vector (P17) and 7 μL water as a transformation control. We transformed 50 μL TOP10 cells with 7 μL DNA and 100 μL DH5α cells with 7 μL DNA. We also used 7 μL of the vector-only ligation to transform 7 μL DH5α cells.


 * To test the Lac QPI system, we ligated P45 (RBS+GFP+terminator) to P108. We used a vector to insert ratio of 2:6. We also prepared a ligation reaction with 1 μL vector (P108) and 7 μL water as a transformation control. We transformed 100 μL DH5α cells with 7 μL DNA and we also used 7 μL of the vector-only ligation to transform 7 μL DH5α cells.

Colony PCR 8/9
We picked all of the noncontrol colonies and one control colony for colony PCR using the BBp/sfx primers.

Since the bands are at least plausible, the P38/39 in P1/3 samples were grown up and miniprepped.

Retransformation 8/9
We retransformed 5μL of the P39+17 in P1/3 ligation into 50μL DH5α and obtained two colonies.

Colony PCR shows that one of these does not contain the right construct. The other was set up in 5ml culture.



1kb ladder, colony A, colony B (both should have ~950 products if correct)

New ligations and transformations 08/10
We retransformed our old P108+45 (2:6 vector to insert) ligation and the old vector control. We also did this ligation over with a 2:1 molecular ratio of insert to vector. Both P108 (4155 bp) and P45 (876 bp) were about 17 ng/μL. We used a ratio of 5.4:2.3 vector to insert by volume.

Transformation results
=Transforming LacZ parts= We attempted to transform the following parts: I732017, E0435, E0435, I732005 into our competent DH5α. E0435 (on CM) had 0 colonies). The others (on Carb) had 10-20 extremely small colonies (after 16+hrs of incubation) each. We picked 2 of each for liquid culture, but none grew.

We requested the parts directly from MIT.