IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week6/Chemical and Light

=Sequencing Parts= =Making Thermoinducible cI System =

Gel of Digestion 7/29
Gel here-- P104 was cut correctly but I made a mistake and thought it hadn't been cut properly, so discarded gel and repeated digest.

Gel for Digestion 7/30
P104 was extracted and gel purified. It was dephosphorylated with the rAPid Alkaline Phosphatase.

Ligation of P104 and cI 7/30
Transformed E1 with 5 uL of each ligation reaction.

Re-Ligation of P104a + cI 7/31
Used the same purified fragments as before and ligated again in TOP10 cells.

Ligation of P75 and P63 8/1
Transformed in E1.

TOPO TA cloning with gel extracted cI 8/1
Transformed in E1.

Plate Results 8/2
Includes results for ligation of P75a and P75b with P63 (terminator). RBS indicates primer set RBS, and BIG indicated primer set BIG. These TOPO cloning reactions were done with gel purified cI857. Volumes of DNA in parentheses indicate the amount of DNA added to the cloning reaction (0.5, 2, or 4 uL).

Gel of PCR 8/1
Used in TOPO TA Cloning Reaction and transformation with E1.

TOPO TA with Fresh cI PCR Product 8/1
Transformed in E1-- added either 2 or 4 uL of cloning reaction. Had limited and suspect X-gal so only applied to some plates, and included X-gal control for contamination.

Plate Results 8/2
Includes results for control reactions for TOPO TA cloning system. RBS indicates primer set RBS, and BIG indicated primer set BIG. These TOPO cloning reactions were done with fresh PCR product of cI857 (confirmed with E-gel). Volumes of DNA in parentheses indicate the amount of DNA added to the cloning reaction (0.5, 2, or 4 uL). Volumes of DNA rxn indicates how much of the cloning reaction was added to the competent cells (either 2 or 4 uL). Presence or absence of X-gal indicated in Description.

=Western Blot to Test IPTG and Heat Induction of Lac Systems=

Induction and Preparing Lysate 7/29
=Transformations in S1=

Transformed mtrB constitutive system into S1 7/28
Electroporated and transformed S1.

Restreaked plates and picked two colonies per plate for liquid cultures and glycerol stocks.

=Housekeeping=

Made S1 Electrocompetent Cells 7/29
Made ~250 aliquots of wildtype and mtrB- Shewie-- 80 uL in each aliquot.

=RE digests 07/29= We digested several samples of P17 in the P1/P3 vector with EX. We will add the high/low constitutive promoters.



Lane 1: 1 kb ladder

Lane 2: uncut E2 P3+17 B (3652)

Lane 3: E1 P1+17 A cut EX (3652)

Lane 4: E1 P1+17 B cut EX (3652)

Lane 5: E1 P1+17 C cut EX (3652)

Lane 6: E1 P1+17 D cut EX (3652)

Lane 7: E2 P1+17 A cut EX (3652)

Lane 8: E2 P1+17 B cut EX (3652)

Lane 9: E2 P3+17 A cut EX (3652)

Lane 10: E2 P3+17 B cut EX (3652)

Ligation with promoters
We attempted ligations of P38 and P39 with the proper sized P17s excised from the gels in a 2:6 ratio with TOP10 cells. No plates (Kan) had colonies.

=Mutagenesis of Cph1 07/29= We set up a reaction to remove the PstI site from cph1 (P87A and B) according to the QuikChange kit protocol. We ran a PCR positive control and a transformation positive control. We transformed all of the samples in XL 1-Blue cells and P87B in TOP10 as well.

Transformation results 07/30
We used the TOP10 transformation protocol for both cell types. We plated 125 μL of each sample on two plates.

=Ligations of digested 7/26 minipreps= See last 2 gels of last week's notebook.

All cells are of E1 and were on Kan plates.

1-2 non-fluorescent colonies were picked for each sample with colonies.

P3A+(E1 P39+51 C) didn't grow in liquid culture.

=RE digests 07/30=

Lane 1: 1 kb ladder

Lane 2: P3A + (E1 P38 +51 D) uncut (3155)

Lane 3: P3A + (E1 P38 +51 D) cut SP (3155)

Lane 4: P3A + (E1 P38 +51 C) cut SP (3155)

Lane 5: P3A + (E1 P38 +51 B) cut SP (3155)

Lane 6: P3A + (E1 P39 +51 A) uncut (3155)

Lane 7: P3A + (E1 P39 +51 A) cut SP (3155)

Lane 8: P3A + (E1 P39 +51 B) cut SP (3155)

Lane 9: P3A + (E1 P39 +51 C) cut SP (3155)

Lane 10: P3A + (E1 P39 +51 D) cut SP (3155)

Lane 11: E1 P63+98 B uncut

Lane 12: E1 P63+98 B cut XP (~2200)

Lane 13: E1 P63+98 A cut XP (~2200)

Lane 14: E2 P63+98 B cut XP (~2200)

Lane 15: E2 P63+98 A cut XP (~2200)

.

=RE digests 07/31= gel 1



Lane 1: 1 kb ladder

Lane 2: E5P105D cut ApaLI and PstI

Lane 3: E2P105A cut ApaLI and PstI

Lane 4: E5P105F cut ApaLI and PstI

Lane 5: E5P105G cut ApaLI and PstI

Lane 6: E2P105E cut ApaLI and PstI

Lane 7: E5P105B cut ApaLI and PstI

Lane 8: E5P105H cut ApaLI and PstI

Lane 9: E2P105E cut ApaLI and PstI

Lane 10: E5P105A cut ApaLI and PstI

Lane 11: E2P105B cut ApaLI and PstI

Lane 12: E2P105B uncut

gel 2



Lane 2: 1 kb ladder

Lane 3: E2P105D uncut

Lane 4: E5P105C cut ApaLI and PstI

Lane 5: E2P105C cut ApaLI and PstI

Lane 6: E2P105E cut ApaLI and PstI

Lane 7: P87A uncut

Lane 8: P87A cut ApaLI and PstI

Lane 9: P87B cut ApaLI and PstI

Lane 10: E1P63+98 E uncut

Lane 11: E1P63+98 E cut XP

Lane 12: E2P63+98 E cut XP

Lane 13: E2P63+98 F cut XP

Lane 14: E2P63+98 F cut XP

=Western Blot= Testing ts-lac inducible system



Only the constitutive plasmids (p30 and p59b) and Natalie's plasmid when induced expressed the desired protein, indicating that neither the heat-inducible lac system nor the dual plasmid lac operon work.

Upper Gel: 1- S1 p13 and p59b 2 IPTG 2- S1 p13 and p59b 2 30°C 3- E1 p30 40°C 4- E1 p30 IPTG 5- E1 p30 30°C 6- E1 p85 40°C 7- E1 p85 IPTG 8- E1 p85 30°C 9- E1 p84 40°C 10- E1 p84 IPTG 11- E1 p84 30°C 12- Ladder 13- E1 p41 40°C 14- E1 p41 IPTG 15- E1 p41 30°C

Lower Gel: 1- pGEX (Natalie's cells) 30°C 2- Ladder 3- pGEX our IPTG 4- pGEX Silver Lab's IPTG 5- S1 p59b and p13 6- S1 p59b and p13 IPTG 7- S1 p13 and p59b 1 8- S1 p13 and p59b 1 IPTG 9- S1 p27 and p59b 10- S1 p27 and p59b IPTG 11- S1 p59b 12- S1 p59b IPTG 13- S1 p13 14- S1 p13 IPTG 15- Ladder

=RE digests and ligations 08/02= Lane 1: 1 kb ladder

Lane 2: P3 cut XP, dephosphorylated (2750)

Lane 3: P39+51 cut XP (1405)





Gel extraction:

These bands were cut from the gel and melted at 65 °C. This was used in one set of ligations. Another set was done using our normal gel extraction protocol. We used ligation ratios of 2:6 and 3:5 of vector to insert. The ligations were transformed into DH5α cells.

=Results= Both transformations involving the DNA extracted using the new gel extraction protocol failed (no colonies). The the ligation with a ratio of 2:6 of P3 to P39+51 did not give any colonies, but the 3:5 ligation plate had 2 colonies. Both of these colonies were picked and grown up in liquid cultures.

=Cutting GFP out of P1=

=ON RE digests and ligations 08/03=
 * We cut P98 with ES to ligate it with a terminator, P63 cut with EX. This will give us RBS+mtrB+terminator. We also cut P98 with XmnI because the backbone and insert of P98 are around the same size, and digesting with XmnI cuts the backbone so it becomes smaller.
 * We also cut the QPIs (P17: Tet and P51: Lac) in a p15A vector with EX. We will add high and low constitutive promoters.



Lane 1: 1 kb ladder

Lane 2: P98A uncut (~4200)

Lane 3: P98A cut ESXmnI (~2102; 1623, 433)

Lane 4: P98B cut ESXmnI (~2102; 1623, 433)

Lane 5: P98C cut ESXmnI (~2102; 1623, 433)

Lane 6: P98D cut ESXmnI (~2102; 1623, 433)

Lane 7: P98E cut ESXmnI (~2102; 1623, 433)

Lane 8: P98F cut ESXmnI (~2102; 1623, 433)

Lane 9: P63 cut EX (3284)

Lane 10: P3+51# cut EX (4120)

Lane 11: P1/3+51 cut EX (4120)

Lane 12: P3+51 cut EX (4120)

Lane 13: P3+17B uncut (3652)

Lane 14: P3+17 cut EX (~3652)

Lane 15: P3+17 cut EX (~3652)

Lane 16: 1 kb plus ladder


 * Since the P63 band in lane 9 looked like it could have also had uncut DNA, we used a sample of P63 from 07/16 in the ligations.
 * We used the standard QIAGEN gel extraction protocol.
 * We ligated using 2:6 of vector to insert. We let the P98+63 ligations run for 10, 20 and 30 minutes (the other ligations only ran for 10 minutes).
 * We transformed the P39+51, P39+17, P98+63 10' ligations into TOP10 cells. All of the other ligations were transformed into DH5α. We also transformed a positive control with pUC19.
 * Since there was no SOC medium left, we incubated in LB for 2 hours after heat shock.