Monday - 8/5/13

One of the plates with the gel purified promoter+RBS produced a few colonies, so those should be the correct construct. I overnighted one of those as well as several colonies from each of plates with unpurified insert for screening purposes. Joe had me digest and gel purify several backbones as well as the RTX construct so that we can get some things into Freiburg standard in order to make fusion proteins. These digests were ligated along with several parts that were given to us by the A-team. 2 μL from each ligation reaction were transformed after a short incubation period before putting the rest of the reaction into the thermocycler overnight in an attempt to save a day. If these transformations do not turn out tomorrow, we can go back and transform from the overnight ligations.

Make A plates, 100 mL batch of LB
Restriction digests (500 ng / 50 μL)

	FMN		FMN		pSB1C3		RTX
DNA	4.83 μL	DNA	4.83 μL	DNA	2.31 μL	DNA	30.0 μL
ddH2O	38.17 μL	ddH2O	38.17 μL	ddH2O	40.69 μL	ddH2O	13.0 μL
Cutsmart	5.0 μL	Cutsmart	5.0 μL	Cutsmart	5.0 μL	Cutsmart	5.0 μL
NgoMIV	1.0 μL	XbaI	1.0 μL	EcoRI	1.0 μL	NgoMIV	1.0 μL
SpeI	1.0 μL	AgeI	1.0 μL	PstI	1.0 μL	SpeI	1.0 μL

Gel purification

Make a 1% agar gel w/ 7.5 μL SYBR®Safe Stain
Use QIAquick Gel Extraction Kit

Elute w/ 25 μL of Buffer EB

1	2	3	4	5	6	7	8
Ladder	FMN - NS		FMN - XA		pSB1C3		RTX - NS

Ligation reactions (20 μL)

(1) - amilCP on pSB1A3 (NgoMIV & SpeI)
(2) - RTX on pSB1A3 (NgoMIV & SpeI)
(3) - XbaI on pSB1A3 (NgoMIV & SpeI)
(4) - amilCP on pSB1A3 (XbaI & AgeI)
(5) - EcoRI MTase on pSB1A3 (XbaI & AgeI)
(6) - PstI on pSB1A3 (XbaI & AgeI)
(7) - PstI MTase on pSB1A3 (XbaI & AgeI)

Add the following to a 0.2 mL PCR tube

2 μL - insert
2 μL - backbone
2 μL - T4 buffer
1 μL - T4 ligase
13 μL - ddH2O

Incubate @ R.T. for ~10 min.

Remove 2 μL for transformations

Incubate overnight @ 16° C

Transformations

Transform (1) through (7) after short incubation period

Will try again with overnight ligation if these are unsuccessful

Make overnights

1x - colony from gel purified P100+RBS plate (A)
3x - colony from unpurified P100+RBS plate (A)
3x - colony from unpurified P100+RBS plate (A)

Tuesday - 8/6/13

Today I miniprepped all of the P+RBS on pSB1A3 overnights for screening. These products were then digested with PvuI and run on a gel to verify if the plasmid size is correct. I also digested the FMN construct to gel purify the Freiburg backbone using the Qiagen gel extraction kit. I used a different buffer and also added an extra μL of AgeI in hopes of getting a complete digest.

Miniprep overnights

P100+RBS (1) - 70.1 ng/μL
P100+RBS (2) - 91.8 ng/μL
P100+RBS (3) - 96.6 ng/μL
P108+RBS (1) - 93.9 ng/μL
P108+RBS (2) - 102.5 ng/μL
P108+RBS (3) - 77.0 ng/μL
Gel purified P100+RBS - 109.8 ng/μL

Restriction digests

	P100 (1)	P100 (2)	P100 (3)	P108 (1)	P108 (2)	P108 (3)	GP P100
DNA	3.57 μL	2.72 μL	2.59 μL	2.66 μL	2.44 μL	3.25 μL	2.28 μL
ddH2O	18.43 μL	19.28 μL	19.41 μL	19.34 μL	19.56 μL	18.75 μL	19.72 μL
NEB #4	2.50 μL	2.50 μL	2.50 μL	2.50 μL	2.50 μL	2.50 μL	2.50 μL
PvuI	0.50 μL	0.50 μL	0.50 μL	0.50 μL	0.50 μL	0.50 μL	0.50 μL

	FMN
DNA	4.83 μL
ddH2O	37.67 μL
NEB #4	5.0 μL
XbaI	1.0 μL
AgeI	2.0 μL

Gel screening

1	2	3	4	5	6	7	8	9	10
Ladder #N3232S	P100 (1)	P100 (2)	P100 (3)	P108 (1)	P108 (2)	P108 (3)	P100 (GP)		FMN (X,A)

Wednesday - 8/7/13

I had to redo the screening process for the P+RBS constructs today because I realized last night that there I could not tell definitively if I had the correct construct with the information I had obtained yesterday. This time I choose an enzyme that will cut only at the promoter, so this will tell me for sure if I have the correct construct. The transformations of the G-block parts seemed to have worked, so we overnighted those transformants for further analysis tomorrow. However, we still need to obtain a Freiburg chloramphenicol backbone in order to proceed with these constructs. Also, Bill did a competent cell efficiency test for our new batch of competent cells so that we can document that experiment properly.

Restriction digests

	P100 (1)	P100 (2)	P100 (3)	P108 (1)	P108 (2)	P108 (3)	GP P100
DNA	3.57 μL	2.72 μL	2.59 μL	2.66 μL	2.44 μL	3.25 μL	2.28 μL
ddH2O	18.43 μL	19.28 μL	19.41 μL	19.34 μL	19.56 μL	18.75 μL	19.72 μL
NEB #4	2.50 μL	2.50 μL	2.50 μL	2.50 μL	2.50 μL	2.50 μL	2.50 μL
AvrII	0.50 μL	0.50 μL	0.50 μL	0.50 μL	0.50 μL	0.50 μL	0.50 μL

Gel screening

1	2	3	4	5	6	7	8	9
Ladder N3232S	P100 (1)	P100 (2)	P100 (3)	P108 (1)	P108 (2)	P108 (3)	P100 (GP)	FMN (X,A)

Gel purification (pSB1A3 - X,A)

Make a 0.7% agarose gel w/ 7.5 μL SYBR®Safe Stain
Use QIAquick Gel Extraction Kit

Elute w/ 25 μL of Buffer EB

Make 5 mL overnight cultures

1x - amilCP on pSB1A3 (NgoMIV & SpeI)
2x - RTX on pSB1A3 (NgoMIV & SpeI)
2x - XbaI on pSB1A3 (NgoMIV & SpeI)
2x - amilCP on pSB1A3 (XbaI & AgeI)
2x - EcoRI MTase on pSB1A3 (XbaI & AgeI)
2x - PstI on pSB1A3 (XbaI & AgeI)
2x - PstI MTase on pSB1A3 (XbaI & AgeI)

Thursday - 8/7/13

I did minipreps of all of the overnights, followed by digests and then ran a gel to verify the insert size. Unfortunately only amilCP produced the expected bands, so most of this effort was wasted. This will set back putting together the constructs by several days, but Bill and I will be working on PCR colony screening tomorrow to try and isolate several more of the parts. We still may have a chance to get the constructs put together in 2 weeks, but will not have time to do any of the experiments to purify and test the functionality of the enzymes.

Miniprep overnights

amilCP (N&S - overnight ligation) - 59.9 ng/μL
RTX (N&S - quick ligation) - 81.3 ng/μL
RTX (N&S - overnight ligation) - 78.4 ng/μL
XbaI (N&S - quick ligation) - 78.0 ng/μL
XbaI (N&S - overnight ligation) - 88.8 ng/μL
amilCP (X&A - quick ligation) - 81.7 ng/μL
amilCP (X&A - overnight ligation) - 102.0 ng/μL
EcoRI MTase (X&A - quick ligation) - 83.3 ng/μL
EcoRI MTase (X&A - overnight ligation) - 66.3 ng/μL
PstI (X&A - quick ligation) - 66.2 ng/μL
PstI (X&A - overnight ligation) - 45.7 ng/μL
PstI MTase (X&A - quick ligation) - 64.9 ng/μL
PstI MTase (X&A - overnight ligation) - 50.6 ng/μL

Restriction digests (500 ng / 25 μL)

All volumes in μL

	(1)	(2)	(3)	(4)	(5)	(6)	(7)	(8)	(9)
	PstI (1)	PstI (2)	M.P (1)	M.P (2)	aml (1)	aml (2)	aml (3)	Rtx (1)	Rtx (2)
DNA	7.55	10.94	7.70	9.88	6.12	4.90	8.35	6.15	6.38
ddH2O	13.95	10.56	13.80	11.62	15.38	16.60	13.15	15.35	15.12
NEB#4	2.50	2.50	2.50	2.50	2.50	2.50	2.50	2.50	2.50
EcoRI	0.50	0.50	0.50	0.50	0.50	0.50	0.50	0.50	0.50
AgeI	0.50	0.50	0.50	0.50	0.50	0.50	0.50	0.50	0.50

	(10)	(11)	(12)	(13)	(14)	(15)	(16)	(17)	(18)
	PstI (1)	PstI (2)	M.P (1)	M.P (2)	aml (1)	aml (2)	aml (3)	Rtx (1)	Rtx (2)
DNA	7.55	10.94	7.70	9.88	6.12	4.90	8.35	6.15	6.38
ddH2O	13.95	10.56	13.80	11.62	15.38	16.60	13.15	15.35	15.12
NEB#4	2.50	2.50	2.50	2.50	2.50	2.50	2.50	2.50	2.50
NgoMIV	0.50	0.50	0.50	0.50	0.50	0.50	0.50	0.50	0.50
PstI	0.50	0.50	0.50	0.50	0.50	0.50	0.50	0.50	0.50

	(19)	(20)		(21)
	M.PstI (1)	M.PstI (2)		P100+RBS
DNA	7.70	9.88	DNA	4.55
ddH2O	13.80	11.62	ddH2O	16.95
NEB#4	2.50	2.50	NEB#4	2.50
XbaI	0.50	0.50	EcoRI	0.50
PstI	0.50	0.50	SpeI	0.50

	(22)	(23)		(24)	(25)
	M.Eco (1)	M.Eco (2)		M.Eco (1)	M.Eco (2)
DNA	6.00	7.54	DNA	6.00	7.54
ddH2O	15.50	13.96	ddH2O	15.50	13.96
NEB#4	2.50	2.50	NEB#4	2.50	2.50
XbaI	0.50	0.50	XbaI	0.50	0.50
PstI	0.50	0.50	PstI	0.50	0.50

Gel verification

1	2	3	4	5	6	7	8	9	10	11	12
Lad.	10	11	12	13	14	15	16	17	18	24	25

Friday - 8/9/13

Today I took a step back and began making preparations to obtain a good working stock of Freiburg pSB1A3 backbone. It seemed that the DNA concentrations in our previous stocks were unacceptably low. First, I re-transformed FMN from our original stock that had been verified through sequencing so that we can miniprep a large amount of it on Monday to digest and gel purify. Next I digested the remaining FMN stock to run a gel purification experiment with the Qiagen gel extraction kit. The gel showed that the digest did not go to completion, which was odd because the only difference from the previous digest was that I used a larger PCR tube to run the reaction. A rough estimate of the concentration predicts a concentration of about 25 ng/μL, but the nanodrop reading was 7.9 ng/μL. Obviously we are not getting a very good recovery with this method, so it would most likely be preferable to use Alex’s gel purification method moving forward.

Transform FMN (on ampicillin)

Original sequence stock
1 μL DNA / 40 μL cells
Recover in 200 μL SOC
Plate 25 μL of transformed cells

2x - Restriction Digests (500 ng / 50 μL)

Ran in 0.5 mL PCR tube

DNA (FMN) - 4.83 μL
ddH2O - 38.17 μL
NEBuffer #4 - 5.0 μL
NgoMIV - 1.0 μL
SpeI - 1.0 μL

Gel purify pSB1A3 Freiburg backbone

Evenly distribute digest over 6-8 lanes
Cut out and use Qiagen Gel Extraction Kit

Nanodrop reading - 7.9 ng/μL

PCR colony screening

Bill spent all day on this and I tried to help where I could