Team:Goettingen/NoteBook w4

Transformation

No colonies visible à incubation till evening, possibly clones to slow in growing because of high Cm concentration?...

 

Agarose gel to test all vectors and inserts used for ligation

1xTAE-1 % agarose gel (from yesterday)

Loaded with at least 30 – 40 ng of each DNA sample supplied with 1 μl of 5x LD

Marker: log-ladder (3 μl)

Run at 100 V in 1xTAE

EtBr staining and destaining

Gel 1:                                                            Gel2

è     For all samples, expected fragments were detected à no degradation of inserts and vectors

Test of clone growth with different chloramphenicol concentrations

-          Both negative control clones and the clones of pSB1C3 transformation grew on the 5 µg/ml Cm plates, but not in broth LB medium with 35 µg/ml Cm

-          the other clones (linearized plasmid part 7 and linearized pSB1C3) grew in broth medium and on plates

-          all other clones were able to grow in broth LB supplied with 35 µg/ml Cm

è     Cm concentration is too low to apply sufficient selective pressure for selection after transformation

è     Preparation of 10 ml Cm stock (35 mg/ml à 350 mg in 10 ml) in 70% EtOH (1000x)

è     2x 500 ml LB plates containing 35 µg/ml

è     Repetition of all ligations and transformation as before using new plates

 

Preparation of reactions for sequencing

Plasmids part 1 – part 8; part 2 C1 and part 2 C2; Plasmids DarRop C1 to C4 and C6

Sequencing: ca. 300 ng plasmid/5 µl

è     For all samples 4 µl plasmid + 1 µl primer (VR = iGEM_39 or VF2 = iGEM_38) (for some plasmids less than 300 ng plasmid used due to too low concentration); only samples for part 2 C2 (2.5 µl plasmid) and part 3 (2 µl plasmid), the plasmid concentration where so high, that they had to be diluted)

Loading:

	1	2	3	4
A	Part 1 VF2	Part 4 VF2	Part 8 VF2	DarRop C4 VF2
B	Part 1 VR	Part 4 VR	Part 8 VR	DarRop C4 VR
C	Part 2 C1 VF2	Part 5 VF2	DarRop C1 VF2	DarRopC6 VF2
D	Part 2 C1 VR	Part 5 VR	DarRop C1 VR	DarRop C6 VR
E	Part 2 C2 VF2	Part 6 VF2	DarRop C2 VF2
F	Part 2 C2 VR	Part 6 VR	DarRop C2 VR
G	Part 3 VF2	Part 7 VF2	DarRop C3 VF2
H	Part 3 VR	Part 7 VR	DarRop C3 VR

Transformation	Colonies on plate	Streak-out on LBCm (5 µg/ml Cm)	Inoculation in LBCm (35 µg/ml)
Negative Control pSB1C3	Several…	1 clone	1 clone
psB1C3	Several tiny ones	-	Clones 1 - 3
Negative Control DarR/Riboswitch biobricks and GFP-Terminator plasmid	Tiny colonies (1 was bigger à picked)	1 clone	1 clone
Without-insert control of Terminator plasmid	Colonies	3 clones	3 clones
Without-insert control of linearized pSB1C3	Colonies	3 clones	3 clones
GFP-Terminator	Many colonies	-	Clones 1 - 5
DarR biobrick	Many colonies	-	Clones 1 - 5
Riboswitch A (iGEM_42/43)	Fewer colonies than on other riboswitch plates	-	Clones 1 - 5
Riboswitch B (iGEM_41/42)	Fewer colonies than on other riboswitch plates	-	Clones 1 - 5
Riboswitch C(iGEM_40/43)	Several colonies	-	Clones 1 - 5
Riboswitch D (iGEM_40/41)	Many colonies	-	Clones 1 - 5

è     Tiny colonies on neg. control plate suggest, that E. coli without plasmid can grow, but poorly. It could be that the Cm concentration in the plates is too low à 5 µg/ml (as in our plates) are usually used for B. subtilis. But iGEM webpage recommends 35 µg/ml for selection of E. coli transformants.

è     Alternative: Competent cells could contain Cm-resistant E. coli

è     Test competent cells

è     Transformation: Actually, the transformation should be repeated!!!

è     Inoculation of clones from each plate in 4 ml LB containing 35 µg/ml Cm to test which of them are able to grow with such a high selective pressure à Mini-Prep + streak-out of those that grow

è     Control of plates: streak-out of clones from control reactions on LB plates with 5 µg/ml and inoculation of the same clones in LB with 35 µg/ml, to verify if they only grow with low Cm concentrations

Plasmid-Mini Prep for DarR operator clone C6 (clone C5 discarded)

Elution of plasmid in 50 µl HPLC water (pre-warmed)

NanoDrop:

Clone	Concentration (ng/µl)	A260nm/A280nm	A260nm/A230nm
C6	73.2	1.87	2.08

Stored at -20 °C

Purification of Riboswitch PCR products from 24.6.

PCR clean-up with Qiagen kit

Elution with 30 µl HPLC water (pre-warmed)

NanoDrop:

Riboswitch PCR reaction with primers…	Concentration (ng/µl)	A260nm/A280nm	A260nm/A230nm
iGEM_42 iGEM_43	35.7	1.85	1.56
iGEM_42 iGEM_41	36.8	1.84	2.26
iGEM_40 iGEM_41	45.7	1.86	2.21
iGEM_40 iGEM_43	47.7	1.81	2.04

 

Transformation of pSB1C3 from Distribution kit (Plate 5 A3)

Whole transformation on one plate + control plate w/o DNA

Cloning of DarR biobrick and Riboswitch biobricks

-          PCR purification with Qiagen kit for test reactions of all four riboswitch biobrick PCRs

-          Agarose gel to test success of PCR

3 µl 2 log ladder, 4 µl of each water control + 1 µl 5x LD; 2 µl of each purified PCR product + 1 µl 5x LD

Gel:

è     Unspecific products/ PCR product oligomers, when primers iGEM_40 or iGEM_41 used

è     Increase annealing temperature, gel extraction of bands with expected product sizes

è     Here: too low amounts obtained after purification à used for cloning (pick more clones for restriction analysis)

 

-          Restriction of DarR biobrick PCR product and Riboswitch biobrick PCR products with EcoRI-HF and PstI

1.) Preparation of Plasmid DNA dilutions (500 ng)

	Insert	Plasmid		dH2O
	DarR biobrick	8 µl Primer-PCR reaction	12.9 µl (250 ng)	13.6 µl
		4 µl Primer-PCR reaction	16.5 µl (250 ng)
Riboswitch PCR product of primers…	iGEM_40/41	10.9 µl		32.1 µl
	iGEM_40/43	10.5 µl		32.5 µl
	iGEM_42/41	13.6 µl		29.4 µl
	iGEM_42/43	14.0 µl		29 µl
		43 µl in total

2.) 6x Master Mix

Component	Volume
10x NEBuffer 2.1	30 µl
PstI	6 µl (only 75 % activity in NEB 2.1)
EcoRI-HF	6 µl
è     Add 7 µl to 500 ng plasmid DNA in dH2O

-          Incubation for 15 min at 37 °C

-          Heat-kill for 20 min at 80 °C

 

PCR purification to purify samples after restriction digestion

Qiagen kit; elution with 50 µl HPLC-water (pre-warmed)

NanoDrop:

PCR reaction	Concentration (ng/µl)	A260nm/A280nm	A260nm/A230nm
iGEM_40 iGEM_41	7.1	2.49	2.15
iGEM_42 iGEM_43	5.9	1.84	1.73
iGEM_42 iGEM_41	15.1	1.51	0.91
iGEM_40 iGEM_43	11.7	1.48	1.01
Insert DarR	6.8	2.11	1.86

 

Digestion of pSB1C3 (for 6 reactions)

Mastermix:

5 µl NEB2

0.5 µl EcoRI-HF

0.5 µl PstI

18 dH₂0

 

4 µl pSB1C3

4 µl Mastermix

37°C/20min  heat kill: 80°C/20min

 

Ligation reaction (pSB1C3 + riboswitch):

2 µl digested pSB1C3 (25 ng)

equimolar amount of insert (here: 1 µl of dilutions corresponding approximately the equimolar amount) (calculated with online ligation calculator:

http://www.insilico.uni-duesseldorf.de/Lig_Input.html )

1 µl T4 DNA Ligase buffer

0.5 µl T4 Ligase

Add dH₂O up to 10 µl

(use of further diluted inserts for better pipetting)

room temperature 20 min  heat kill:80°C/20min

(remaining digested insert and vector stored at – 20°C)

 

Transformation

(according to Method collection)

put into 37°C incubator

Digestion of part 6 and 7

 

Part6:                                 

5.6 µl plasmid

1 µlEcoRI-HF                            

1 µl SpeI                                 

5 µlNEB2.1                              

37.4 µl dH₂O  

 10 – 15 min/37°C   Heat kill: 20min/80°C                    

 

Gel extraction                                    

-          pour thick garose gel 1% in 1xTAE

-          load marker, uncut plasmid and sample (leave 1 well empty between sample and marker/uncut plasmid!!!)

(here: 3 µl log ladder, 2.5 µl uncut plasmid part 6 + 1 µl 5x LD, 50 µl restriction digest of part 6 + 10 µl 5x LD)

-          run at 80 V in 1xTAE buffer

-          in mean time weigh 2 ml Epi tubes at exact scale (usually ca. 1 g)

-          EtBr staining for 5 min, then destaining briefly in water + quick glance at Gel doc at normal UV wavelength

-          Cut out bands of expected size (here: about 900 bp) at UV desk (other room next to 37 °C room…) at UV light with longer wavelength than normal (less DNA damage) fast

-          Switch off UV desk, cut gel stripe into smaller fragments and transfer them to the 2 ml tubes

-          Clean outer side of tubes with EtOH

-          Detect gel at Gel doc again and take a picture (to make sure that nothing of the bands of interest is left in gel)

 

Gel:

 

Purification of DNA from gel using Qiagen PCR purification kit:

-          Weigh Epi tubes with gel and calculate weight of gel fragments

-          Add 3 x Volume of QG buffer (in PCR purification kit in Bacillus-Lab) (100 mg gel = 300 µl of QG buffer)

-          Dissolve gel pieces by incubating at 50 °C for 10 min in a heating block and by vortexing every 2 – 3 min (if not dissolved after this time, prolong incubation time until gel is dissolved properly)

-          Transfer 750 µl of the solution onto the column and centrifuge (13000 rpm, 30 – 60 s, RT), discard flowthrough; repeat this step until all of the solution is used up

-          Proceed as for normal PCR purification (wash buffer PE, blank spin, elution with pre-warmed water à my personal recommendation ;P: for elution, use smaller volume than 50 µl; here: 30 µl were used yielding a concentration of 4.6 ng/µl. In total, this corresponds to >150 ng insert compared with 500 ng vector that were initially digested… it seems that one loses much of DNA during gel extraction!)

-          Measure concentration with NanoDrop (see below)

 

Part7:

7.5 µl plasmid

1 µl EcoRi-HF

1 µl XbaI

5 µl NEB2.1

35.5 µl dH₂O

 

10 – 15 min/37°C   Heat kill: 20min/80°C                    

 

PCR cleanup (see kit)

Elution with 50 µl HPLC water (pre-warmed)

Restricted and purified sample	Concentration (ng/µl)	A260nm/A280nm	A260nm/A230nm
Part 7	9.0	1.59	1.63
GFP	4.6	2.53	0.01

 

Ligation reactions (Part 6 +7):

2 µl vector (part7) (ca. 20 ng)

4 µl insert (part6) (ca. 20 ng)

2 µl 10x T4 DNA Ligase buffer

1 µl T4 DNA Ligase

11 µl dH₂O

20 µl in total

 

room temperature 20 min  heat kill:80°C/20min

(remaining digested insert and vector stored at – 20°C)

 

Transformation

(according to Method collection)

put into 37°C incubator

Overday culture of clones from 21.6.13

(4 ml in LB^Amp)

Added Biobricks BBa_K1045000 – Bba_1045002 to the parts-registry

Preparation of new media without antibiotics and culture plates (ampicillin and chloramphenicol)

500 ml each

Dissolving of primers iGEM_44, iGEM_37 to iGEM_43 and preparation of 1:20 dilutions

DarR biobrick PCR

-          PCR reaction protocol as usual (see 10.6.13)

-          Primers iGEM_33 and iGEM_44

Testing of different Primer concenctrations for both primers since iGEM_44 forms a hairpin – 2 μl, 4 μl or 8 μl of each primer

-          5x Master Mix consisting of buffer, PhuS, dNTPs

Component	For 1x Primer amount and water control	For 2x Primer amount	For 4x Primer amount
HF buffer 5x	10 μl	10 μl	10 μl
PhuS	1 μl	1 μl	1 μl
M. smegmatis chrom. DNA/dH2O	2 μl	2 μl	2 μl
Primer fwd iGEM_44	2 μl	4 μl	8 μl
Primer rev iGEM_33	2 μl	4 μl	8 μl
dNTPs	2 μl	2 μl	2 μl
dH2O	31 μl	27 μl	19 μl
Total	50 μl	50 μl	50 μl

 

Protocol (30 cycles)

Step	Temperature	Time
Initial Denaturation	98.5 °C	5 min
Denaturation	98.5 °C	30 s
Annealing	67.5 °C	35 s
Elongation	72 °C	2 min
Final Elongation	72 °C	10 min
Hold	15 °C	∞

 

(T_m (iGEM_44) = 69.7 °C; T_m (iGEM_33) = 77.3 °C)

 

Agarose gel (1 %; 1x TAE; EtBr staining)

è     PCR worked, 2 μl of each Primer are enough (though I’m not sure, if I pipetted 4 μl reaction correctly… volume appeared to be larger than that of the other samples…)

 

PCR clean-up with Qiagen kit

Elution with 30 μl HPLC water (pre-warmed)

NanoDrop:

PCR reaction	Concentration (ng/μl)	A260nm/A280nm	A260nm/A230nm
1x Primer amount	12.2	2.29	2.19
2x Primer amount	15.2	2.37	2.11
4x Primer amount	19.4	2.22	2.05

 

 

YdaO riboswitch biobricks PCRs

	Primers	TA = 0.5 * [Tm(primer1) + Tm(Primer2)] – 6 °C	PCR product size	Name of Protocol in cycler
Riboswitch with native Promoter and RBS	iGEM_42 iGEM_43	51 °C	466 bp	Ribo1
Riboswitch with native Promoter	iGEM_42 iGEM_41	55.2°C	370 bp	Ribo2
Riboswitch only	iGEM_40 iGEM_41	59.7°C	213 bp	Ribo3
Riboswitch with native RBS	iGEM_40 iGEM_43	55.5 °C	403 bp	Ribo4

T_m (iGEM_40) = 65.7 °C

T_m (iGEM_41) = 65. 7 °C

T_m (iGEM_42) = 56.7 °C

T_m (iGEM_43) = 57.3 °C

PCR reaction and protocol as usual (50μl) (template: B. subtilis chrom.DNA)

Concentration of primer iGEM_43 doubled (4μl per reaction)

Reactions stored at -20°C in Falcon Tube

Backup-plates for all DarR operator clones and Plasmid-Mini Prep for clones 1 – 4; further incubation of clones 5 and 6 o.n. (grew only poorly over day…)

 

Elution of plasmids in 50 μl HPLC water

Clone	Concentration (ng/μl)	A260nm/A280nm	A260nm/A230nm
C1	71.1	1.89	2.11
C2	80.6	1.80	1.97
C3	54.6	1.86	2.17
C4	14.5	1.62	1.99

Stored at – 20 °C