IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week4/Chemical and Light

=PCR/primers=

HO+pcyA, P3 vector w/o GFP
Primers were designed to PCR out the heme oxygenase and pcyA coding regions from the UT Austin plasmids. Due to the RE sites in these nonBB plasmids, their ends are given ApaLI and KpnI sites. The same is done for the P3 vector (the primers are such that we can PCR out the backbone, along with the RBS and terminator. The "P3-GFP" primers should also work for P1. However, I'm a little concerned, b/c the annotation of the P1 and P3 vectors disagrees with the sequence given for the RBS and terminator subparts.

mtrB
The same thing was done for mtrB (as for HO+pcyA). This gives us constitutive mtrB for testing the complement.

7/17: PCR
The primers arrived today, so PCR was performed using a colony of Shewanella Δ EnvZ (which should still have mtrB). Rx mix: 90μL PCR supermix, 2μL mtrB-ApaLI-F primer (20μM), 2μL mtrB-KpnI-R (20μM), swirl of colony Rx: 10min @ 94°C → 10x[45s @ 94°C → 45s @ 52°C → 2m30s @72°C] → 20x[45s @ 94°C → 45s @ 54°C → 4m45s @72°C]→ 5x[45s @ 94°C → 45s @ 56°C → 4m45s @72°C]→5min @ 72°C → ∞ @ 4°C

PCR failed- perhaps annealing temperature was too high or too many cells. Will retry.

7/20: Retry (Gradient)
Rx mix (split into 12 samples): 180μL PCR supermix, 4μL mtrB-ApaLI-F primer (20μM), 4μL mtrB-KpnI-R (20μM), swirl of WT Shewanella colony, 12μL water Rx: 5min @ 94°C → 35x[45s @ 94°C → 45s @ {43.0, 43.2, 43.8, 44.5, 45.5, 46.9, 48.4, 49.7, 50.6, 51.3, 51.8, 52.0}°C → 2m38s @72°C] → 5min @ 72°C → ∞ @ 4°C

P38, P39 Oligos
5' phospho oligos were made to anneal into what is effectively P38 and P39 digested with ES.

P11
7/16 P11 (a piece of the filter paper) was PCRed: 2μL EB, 45μL PCR supermix, 1μL BBpfx primer (20μM), 1μL BBsfx (20μM) Rx: 5min @ 94°C → 15x[45s @ 94°C → 45s @ 54°C → 4m45s @72°C] → 20x[45s @ 94°C → 45s @ 56°C → 4m45s @72°C]→ 5min @ 72°C → ∞ @ 4°C 7/17

PCR failed- no 4kb band

CDF
8 100μL PCR reactions set up (standard mix): DNA template is S1 P13, 40 cycles, annealing temp is 58°C, extension time is 1:15

Run on gel; bands cut and frozen.

mtrB
8 100μL PCR reactions set up (standard colony mix) w/ WT Shewanella

Rx: 5min @ 94°C → 10x[45s @ 94°C → 45s @ 52°C → 2m45s @72°C] → 25x[45s @ 94°C → 45s @ 55°C → 2m38s @72°C]→ 5min @ 72°C → ∞ @ 4°C

Run on gel; bands cut and frozen.



=Ligations=

Results from 7/12 ligations (transformed into E1), 7/16 transformations
We retransformed all of these on 07/15 using 5 μL DNA for 50 μL DH5α cells. We put the cells directly into liquid cultures after transforming them.

=RE digests 07/15= We digested P53/54 with ES and P58/59 with EX. We intend to ligate these parts together. This will give us RBS + TetR/LacI + terminator + pTet/pLac + RBS + GFP + terminator.

We also digested P90 with SP and P48/P49 with XP. This will give us the CDF origin in a p15A vector with an Amp or Cm resistance cassette.

We followed the standard digest protocol under the "General Protocols" section.

Gel 1



Lane 1: 1 kb ladder

Lane 2: P48 cut XP (769 bp)

Lane 3: P48 uncut (3477 bp)

Lane 4: P49A cut XP (943 bp)

Lane 5: P49A uncut (3651 bp)

Lane 6: P49B cut XP (943 bp)

Lane 7: P49B uncut (3651 bp)

Lane 8: P49C cut XP (943 bp)

Lane 9: P49C uncut (3651 bp)

Lane 10: P53 cut ES (840 bp)

Gel 2



Lane 1: P53 uncut (2919 bp)

Lane 2: P54 cut ES (1308 bp)

Lane 3: P54 uncut (3387 bp)

Lane 4: P58 cut EX (~3016 bp)

Lane 5: P58 uncut (3016 bp)

Lane 6: P59A cut EX (~3201 bp)

Lane 7: P59A uncut (3201 bp)

Lane 8: P59B cut EX (~3201 bp)

Lane 9: 1 kb ladder

Gel 3



Lane 1: P59B uncut (3201 bp)

Lane 2: P59C cut EX (~3201 bp)

Lane 3: P59C uncut (3201 bp)

Lane 4: P90A cut SP (~3650 bp)

Lane 5: P90A uncut (~3650 bp)

Lane 6: P90A cut SP (~3650 bp)

Lane 7: P90A uncut (~3650 bp)

Lane 8: P90A cut SP (~3650 bp)

Lane 9: P90A uncut (~3650 bp)

Lane 10: blank

Lane 11: 1 kb ladder

=RE digests 07/16= We digested
 * P63, P76 (A, B, C, D), P91 (A, B, C) with EX
 * P90 (α, β) with SP
 * P48, P49 (A, B, C) with XP

We used the standard digestion protocol.

We plan to ligate
 * P90 with P48/P49. This will give us the CDF origin in the P1 vector with Amp and Cm resistance cassettes. We will also add P63, a terminator, and the high and low constitutive promoters (P38 and P39) later.
 * P91/P76 with the high and low constitutive promoters. This should give us the entire Lac and Tet plasmids.

Gel 1


 * 1 kb ladder
 * P48 cut XP
 * P48 uncut
 * P49A cut XP
 * P49A uncut
 * P49B cut XP
 * P49B uncut
 * P49C cut XP
 * P49C uncut
 * P63 cut EX
 * P63 uncut
 * P76A cut EX
 * P76A uncut
 * P76B cut EX
 * P76B uncut

Gel 2
 * 1 kb ladder
 * P90α cut SP
 * P90α uncut
 * P90β cut SP
 * P90β uncut
 * P91A cut EX
 * P91A uncut
 * P91B cut EX
 * P19B uncut

We extracted and purified P48, P49A, P49B, P63

=RE digests 07/17= We digested
 * P11 with XP
 * P90α/β with SP (we plan to add the Amp/Cm resistance cassettes, P48 and P49)
 * P91D/E with EX (we plan to add the high and low constitutive promoters, P38 and P39)
 * P80 with ES (we think this is P90 with the Amp resistance marker; we plan to add terminators, P63)
 * P76E/F with EX (we plan to add the high and low constitutive promoters, P38 and P39)

We digested more DNA (scaled everything up) to improve our yields from the gel extraction.

Gel 1




 * Lane 1: 1 kb ladder
 * Lane 2: P76C cut EX (3857 bp)
 * Lane 3: P76C uncut (~3857 bp)
 * Lane 4: P76D cut EX (3857 bp)
 * Lane 5: P76D uncut (~3857 bp)
 * Lane 6: P76E cut EX (3857 bp)
 * Lane 7: P76E uncut (~3857 bp)
 * Lane 8: P76F cut EX (3857 bp)
 * Lane 9: P76F uncut (~3857 bp)
 * Lane 10: P80 cut ES (~1800 bp)

Gel 2




 * Lane 1: 1 kb ladder
 * Lane 2: 80 uncut (~4500 bp)
 * Lane 3: P90α cut SP (~3600 bp)
 * Lane 4: P90α uncut (~3600 bp)
 * Lane 5: P90β cut SP (~3600 bp)
 * Lane 6: P90β uncut (~3600 bp)
 * Lane 7: P91C cut EX (4327 bp)
 * Lane 8: P91C uncut
 * Lane 9: P91D cut EX (4327 bp)
 * Lane 10: P91D uncut

Gel 3




 * Lane 1: 1 kb ladder
 * Lane 2: P91E cut EX (4327 bp)
 * Lane 3: P91E uncut
 * Lane 4: P11 cut XP (4333 bp)

=RE digests 07/18= Gel 1




 * Lane 1: 1 kb ladder
 * Lane 2: P90α cut SP (~3600)
 * Lane 3: P90α uncut
 * Lane 4: P90β cut SP (~3600)
 * Lane 5: P90β uncut
 * Lane 6: P90γ cut SP (~3600)
 * Lane 7: P90γ uncut
 * Lane 8: P90δ cut SP (~3600)
 * Lane 9: P90δ uncut
 * Lane 10: ligated P76 cut EX (3857)
 * Lane 11: ligated P76 uncut (3857)

Gel 2




 * Lane 1: 1 kb ladder
 * Lane 2: P90ε cut SP (~3600)
 * Lane 3: P90ε uncut
 * Lane 4: P90ζ cut SP (~3600)
 * Lane 5: P90ζ uncut
 * Lane 6: Christina's plasmid uncut (?)
 * Lane 7: Christina's plasmid cut EX (?)
 * Lane 8: Christina's plasmid cut ES (~1300)
 * Lane 9: Christina's plasmid cut XP (~1300)
 * Lane 10: ligated P91 cut EX (4327)
 * Lane 11: ligated P91 uncut (4327)
 * Lane 12: mtrB PCR (2200)



=RE digests 07/19= We digested
 * P17, P51, P52 (QPIs) with EX so we can add high/low constitutive promoters
 * P63 (terminator) with EX
 * P48, P49a, P49B (Amp, Cm resistance cassettes) with XP so we can add them to P90 (P1 + CDF)



=Ligations 07/15= We ligated
 * P90 SP with P48 XP
 * P90 SP with P49 XP
 * P58 EX with P53 ES
 * P59 EX with P53 ES

These ligations were transformed into TOP10 cells that we made chemically competent.

Results 7/16
Transformed in E2:

=Cross Transformations in S1=

We transformed cells with repressor plasmid (P27), and duet vector (P12 and P13) with LacI with p59 (pLac + GFP) and vice versa in order to test the inducible system in S1.

Testing Cultures with IPTG 7/15
=Thermoinducible Lac System=

Grew up cultures of P84 (Lac mut265) and P85 (Lac mut 241) according to protocol derived from [Chao et al. 2002] and [Yabuta et al. 1995]

Testing Thermoinducible Cultures 7/14

 * 1) Grow up 10mL culture overnight with antibiotic at 200 RPM, 37 degrees.
 * 2) Dilute 2.5mL of overnight culture into 50mL of fresh LB.
 * 3) Grow at 200 RPM, 30 degrees until OD660 = 0.2.
 * 4) Split culture into two 25mL cultures, and put one in 30 degrees and the other in 40 degrees.
 * 5) Grow for 4 hours at these separate temperatures at 200 RPM.
 * 6) OD and measure YFP fluorescence (Ex: 514, Em: 527).

Results from First Thermoinducible Test 7/14
Controls:

Results:

=Putting Thermoinducible Lac and pLambda + GFP System on p15a Vector=

Gel 7/14
P1 and P74 cut and uncut bands are in the right place, but P84 and P85 both have 2079 bp backbones, which does not account for the ~900 bp bands. We searched the sequence of the backbone in the registry for internal cut sites that could have produced the band, but found none.

Only P1 and P74 were extracted and gel purified.

Gel from PCR 7/15
P84 and P85 bands were extracted and gel purified so they could be digested with XP.

Digestion of PCR Products with XP 7/15
After digestion, samples were PCR purified (7/16).

Single and Triple Digests of P84-5 7/14
P84 and P85 were found to have an internal XmnI cut site, so single cut digests were performed for both samples with XbaI, PstI, and XmnI. Triple digests were also done with all three enzymes. Digestions were incubated overnight at 37°C.

Gel of Single and Triple Digests 7/15
The 2314 bp bands for the triple cuts of P84-5 were extracted and gel purified.

Ligation 7/15
The P84-5 inserts are roughly the same size as the vector (P1) so we tried different ratios of insert to vector.

Ligations were then transformed in DH5α cells.

Transformation of P1/P74 and P1/P84-5 Triple Cuts 7/15
5 uL Ligation in 50 uL chemically competent E1 cells (old batch). Plates were incubated overnight at 37°C.

For positive control plates, see the Competent Cell Test Plates under Housekeeping.

Picked Colonies for Liquid Culture 7/16
All cultures grew, except for (-) ctrl.

Ligation of P84-5 PCR with Dephos P1, 7/16
P1 was dephosphorylated the day before, and used for this ligation reaction as well.

Transformation of P92-3 PCR into E1, 7/16
5 uL Ligation in 50 uL chemically competent E1 cells (old batch). Plates were incubated overnight at 37°C.

Transformation of P75 and P92-3 (from 3x Cut) into S1 7/17
Used S1 cells frozen for electroporation.

1mm white/ cream colonies present on all plates after the 2 day incubation resemble the contamination we have observed in other E. coli plates. This is of interest because Kan is not as vulnerable to degradation as Carb/ Amp are.

P75a and P75b were both very bright in S1. Thermoinducible cI857 was ordered from [ATCC].

The plates of P84-5 originally transformed in E. coli from the registry were used as a standard for fluorescence for the thermoinducible Lac colonies. They were left overnight at 37°C to stimulate thermosensitivity, which should lead to YFP expression. Although they were not very bright, they glowed. Compared to these plates, the P93 1:3 transformed in S1 was noticeably dimmer, though it had been incubated at 30°C O/N. Plate will be restreaked and grown at different temperatures after the weekend.

Transformation of P92-3 (from PCR) into S1 7/18
=Test of IPTG Inducible System=

7/15/08 S1 containing 2 vectors one coding for LacI and one w/ a lac promoter controlling GFP expresssion were tested with IPTG

7/17/08:

=Overnight cultures 07/18= We made overnight cultures of the parts we got from MIT
 * P96
 * P95
 * P51
 * P52
 * P17

We also made cultures for plasmids we will need later
 * P1 (A and B)
 * P3 (A and B)
 * P46 (A and B)
 * P63 (A and B)
 * P48
 * P49 (a and B)
 * P63 (A and B)

We are not completely sure if the LB Kan we used contains Kan or if it is just plain LB. So we also made a culture of P95, which is Amp resistant, in the this medium. So if it doesn't grow, then Kan was added to that bottle of LB.

07/19: All of these cultures grew except for P46A. The LB Kan really does have Kan.

=Housekeeping!=

Daily

 * Pour LB-Amp plates.
 * Ensure we have petri dishes, LB, LB agar, and agarose.
 * Make LB-antibiotic medias as necessary.

Making New Competent Cells 7/22
As per Jason's lab's protocol.

Transformation of P86, P87, P97
We transformed each of the UTAustin plasmids (individually) and a new strong RBS into TOP 10 cells. The RBS has been miniprepped. The UTA plasmid transformants formed a lawn, so were restreaked first. Liquid cultures have been grown up. P87 requires mutagenesis to remove a PstI site ~100 bp into the coding region of the Cph/EnvZ fusion. We miniprepped P86A, P86B, P87A, P87B (all in E2) from liquid cultures. The amp and cm negative controls did not have any growth.