07/18/13

07/17/13

07/11/13

07/25/13

08/02/13

QIAPrep Spin Miniprep Kit (250)

08/02/13

#2 Aadc+ 500ng (^1&#956L⁄_85.9ng) = 6.0μL

#15 Aadc+ 500ng (^1&#956L⁄_117.5ng) = 4.5μL

#20 Aadc+ 500ng (^1&#956L⁄_92.1ng) = 5.5μL

#23 Aadc- 500ng (^1&#956L⁄_184.8ng) = 3.0μL

#6 Aadc- 500ng (^1&#956L⁄_218.1ng) = 2.5μL

#17 thl+ 500ng (^1&#956L⁄_100.0ng) = 5.0μL

#3 thl+ 500ng (^1&#956L⁄_84.4ng) = 6.0μL

#10 thl+ 500ng (^1&#956L⁄_90.4ng) = 5.5μL

#14 thl- 500ng (^1&#956L⁄_85.7ng) = 6.0μL

#21 thl- 500ng (^1&#956L⁄_89.1ng) = 5.5μL

#4 thl- 500ng (^1&#956L⁄_131.8ng) = 4.0μL

#9 Ptb+ 500ng (^1&#956L⁄_81.4ng) = 6.0μL

#1 Ptb+ 500ng (^1&#956L⁄_124.2ng) = 4.0μL

#26 Ptb+ 500ng (^1&#956L⁄_206.7ng) = 2.5μL

#18 Ptb- 500ng (^1&#956L⁄_103.7ng) = 5.0μL

#8 Ptb- 500ng (^1&#956L⁄_150.1ng) = 3.5μL

#5 Ptb- 500ng (^1&#956L⁄_216.0ng) = 2.5μL

08/02/13

QIAPrep Miniprep Kit (250)

08/01/13

08/1/13

#16-6D 500ng (^1&#956L⁄_108.6ng) = 4.5μL

#14-20E 500ng (^1&#956L⁄_235.2ng) = 2.0μL

#12-20G 500ng (^1&#956L⁄_135.1ng) = 3.5μL

#8-20G 500ng (^1&#956L⁄_192.3ng) = 2.5μL

#14-20A 500ng (^1&#956L⁄_156.5ng) = 3.0μL

#9-6D 500ng (^1&#956L⁄_`99.7ng) = 2.5μL

#10-4P 500ng (^1&#956L⁄_131.8ng) = 4.0μL

#7-20G 500ng (^1&#956L⁄_232.8ng) = 2.0μL

#3-23B 500ng (^1&#956L⁄_90.0ng) = 5.5μL

#16-23B 500ng (^1&#956L⁄_123.5ng) = 4.0μL

#9-4P 500ng (^1&#956L⁄_145.2ng) = 3.5μL

#9-20G 500ng (^1&#956L⁄_106.8ng) = 4.5μL

#15-20A 500ng (^1&#956L⁄_150.1ng) = 3.5μL

#11-20E 500ng (^1&#956L⁄_126.2ng) = 4.0μL

08/1/13

QIAPrep Spin Miniprep Kit (250)

08/01/13

To make 40mL of ampicillin 1x antibiotic

1. 2.0g ampicillin 1000x stock from 4°C refrigerator
2. Pour the amipicillin ino a 50mL falcon
3. Fill flacon wih dH₂O to 40mL mark
4. Use 10mL syringe with puradisc 25mm filter to dispense 10mL of ampicillin 1x into four 10mL falcons (leaves alternative stocks if one is contaminated)



Ampicillin 1000x = 50ng/mL

50ng/mL x 40mL = 2.0g Ampicillin 1000x


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07/31/13

Promoter and Terminator Culture Preparation




Used 2XYT liquid medium instead of LB to increase carbon amount in order to enhance growth of cultures.

-3mL of 2XYT with Chloramphenicol antibiotic

-Pellet of culture

Incubated at 37°C with shaking at 220rpm


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07/31/13

2XYT Medium Preparation



• (Agar: 10g)
• Bacto Tryptone: 16g
• Yeast Extract: 10g
• Sodium Chloride: 5g
• Distilled Water: 1000mL



Fill 1L jar with 500mL of dH₂O. Pour components and magnetic stirring rod into jar. Fill jar with another 500mL of dH₂O until the shoulder of the jar is reached.

Autoclave for 20 minutes at slow exhaust (Setting #2)


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07/31/13

Gel of DNA Preps from R. eutropha




Run a gel with 3μL of dye and 5μL of DNA extract for each of our R. eutropha extracts.




Row #1
Lane Number	Sample
1	Ladder
2	Phenol Chloroform (27 July)
3	R. eutropha 1 (24 July)
4	R. eutropha 2 (24 July)
5	Power Soil (25 July)
6	Phenol Chloroform (25 July)
7	Empty
8	Empty




NOTE: Lane six shows chewed up DNA by nucleases

Disposed all samples except for Power Soil because it has the highest efficacy







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07/30/13

Plasmid Purification




Plasmid purification of top 10 cells transformed with ligation of pSB1C3 and Ca promoters (Ptb-RBS, Ptb+RBS, thl+RBS, thl-RBS, Aadc+RBS, AaDc-RBS)









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07/30/13

Plasmid Extraction of Terminators and Promoters




Used QIAPrep Spin Miniprep Kit (250)




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07/27/13

Dual Re gel of Regulatory Terminators and Promoters




Row 1
Lane Number	Sample
1	Ladder
2	#14-20A
3	#17-20G
4	#14-20E
5	#11-20E







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07/27/13

Dual Digest of pSB1C3 and mlsR Part




Row 1
Lane Number	Sample
1	Ladder
2	#10-4p
3	#14-20E
4	#14-20A
5	#7-20G
6	#11-20E
7	#16-20E
8	#9-4P
9	#15-20A







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07/11/13

Dual Digest of pSB1C3 and mlsR Part




500ng in 20μL with 2μL for each digest




#11-20E 500ng (^1&#956L⁄_58.1ng) = 8.5μL

#9-4P 500ng (^1&#956L⁄_40.2ng) = 12.5μL

#14-20A 500ng (^1&#956L⁄_43.5ng) = 12.5μL

#7-20G 500ng (^1&#956L⁄_59.4ng) = 12.5μL

#14-20E 500ng (^1&#956L⁄_37.5ng) = 12.5μL

#10-4P 500ng (^1&#956L⁄_43.6ng) = 12.5μL

#15-20A 500ng (^1&#956L⁄_51.6ng) = 12.5μL

#16-23B 500ng (^1&#956L⁄_52.5ng) = 12.5μL




Sample	EcoR1	Pst1	10x Buffer	DNA	PCR dH2O	Total
#11-20E	2μL	2μL	2μL	8.5μL	5.5μL	20μL
#9-4P	2μL	2μL	2μL	12.5μL	1.5μL	20μL
#14-20A	2μL	2μL	2μL	11.5μL	2.5μL	20μL
#7-20G	2μL	2μL	2μL	8.5μL	5.5μL	20μL
#14-20E	2μL	2μL	2μL	13.5μL	0.5μL	20μL
#10-4P	2μL	2μL	2μL	11.5μL	2.5μL	20μL
#15-20A	2μL	2μL	2μL	9.5μL	4.5μL	20μL
#16-23B	2μL	2μL	2μL	9.5μL	4.5μL	20μL


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07/26/13

Nano Drop of Isolated DNA from July 25









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07/26/13

Gel of BktB piece 3 PCR using DNA Extracts




Row #1
Lane Number	Sample
1	Ladder
2	R. eutropha extract (27 July)
3	R. eutropha 1 (24 July)
4	R. eutropha 2 (24 July)
5	Power Soil (25 July)
6	Phenol Chloroform (25 July)
7	Empty
8	Empty







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07/26/13

Gel Repeat of BktB PCR Gradient (25 July)




Repeated gel with larger loading of DNA. Removing only loaded 5μL of dye pls products. Wanted to see if bands appear brighter.




Row #1
Lane Number	Sample	Temperature (°c)
1	Kb Plus Ladder
2	Piece 1	50
3	Piece 1	52.7
4	Piece 1	54.5
5	Piece 1	56.4
6	Piece 1	60
7	Piece 2	50
8	Piece 2	52.7
9	Piece 2	54.5
10	Piece 2	56.4
11	Piece 2	60
12	Ladder
13	Empty
14	Empty
15	EMpty
16	Empty









Row #1
Lane Number	Sample	Temperature (°c)
1	Kb Plus Ladder
2	Piece 3	50
3	Piece 3	52.7
4	Piece 3	54.5
5	Piece 3	56.4
6	Piece 3	60
7	Whole gene	50
8	Whole gene	52.7
9	Whole gene	54.5
10	Whole gene	56.4
11	Whole gene	60
12	Ladder
13	Empty
14	Empty
15	Empty
16	Empty







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07/11/13

PCR of BktB piece 3 using Different DNA Extraction




Wanted to identify which extracts may have DNA present

Previous gradient seems to have produced a PCR product for 3 piece. Used temperature 55°C, since this is the predicted optimal temperature for the SDM2 forward and the BktB_Rev primers




Templates used:

• R. eutropha = 27 July_ 16.9
• R. eutropha = 24 July_ 25
• R. eutropha = 24 July_ 38.9
• Power Soil Extraction = 25 July_ 28.3
• Phenol Chloroform Extraction = 25 July_ 637





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07/25/13

Gel of Gradient PCR for BktB Site-Directed Mutagenesis




Row #1
Lane Number	Sample	Temperature (°c)
1	Kb Plus Ladder
2	Forward + SDM1 Reverse	50
3	Forward + SDM1 Reverse	52.7
4	Forward + SDM1 Reverse	54.5
5	Forward + SDM1 Reverse	56.4
6	Forward + SDM1 Reverse	60
7	SDM1 Forward + SDM2 Reverse	50
8	SDM1 Forward + SDM2 Forward	52.7
9	SDM1 Forward + SDM2 Reverse	54.5
10	SDM1 Forward + SDM2 Reverse	56.4
11	SDM2 Forward + Reverse	60
12	Empty
13	Empty
14	Empty
15	EMpty
16	Empty
Row #2
Lane Number	Sample	Temperature (°C)
1	Ladder
2	SDM2 Forward + Reverse	50
3	SDM2 Forward + Reverse	52.7
4	SDM2 Forward + Reverse	54.5
5	SDM2 Forward + Reverse	56.4
6	SDM2 Forward + Reverse	60
7	Forward + Reverse	50
8	Forward + Forward	52.7
9	Forward + Reverse	54.5
10	Forward + Reverse	56.4
11	Forward + Reverse	60
12	Empty
13	Empty
14	Empty
15	Empty
16	Empty







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07/25/13

DNA Extraction from R. eutropha




If DNA template is the problem with the PCR, then one of the following procedures may correct it.




1) Phenol Chloroform extraction using the protocol from July 24, 2013 with the following modification: 2μL of lysozyme added in the 3rd step with the 10% SDS, to encourage membrane disolution




2)Using the MoBio Power Soil DNA extraction kit and included protocol.







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07/25/13

Transformation of Ca Promoters in pSB1C3




Most plates had growth. Plates are going to be incubated at 37°c for another night. Tomorrow a library will be made.


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07/26/13

Transformation of Ca Promoters in pSB1C3




Colonies were larger. Transformant libraries were made of Ca promoters in addition to ter, Adh6, and C. cell Adh. Plates were parafilmed and placed in 37°C incubator to prevent drying out over the weekend. Monday, plates will be streaked, Tuesday, plasmids will be extracted, and Wednesday, plasmids will be sent for sequencing.


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07/25/13

PCR of BktB for Site-Directed Mutagenesis




Repeated PCR of BktB using primers for sie-directed mutagenesis

Also included one PCR series using the Forward and Revers primers for the whole gene




Used only undiluted templates




Temperature gradient was selected to identify correct temperature for each pair of primers (assuming template is present)




Used wells:

1: T = 50&#176:c

4: T = 52.7&#176:c

6: T = 54.5&#176:c

8: T = 56.4&#176:c

12: T = 60&#176:c







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07/25/13

Gel of BktB Site-Directed Mutagenesis PCR




Row #1
Lane Number	Sample	Dilution
1	Empty
2	Kb plus ladder
3	Forward + SDM1 Reverse	1:10
4	Forward + SDM1 Reverse	1:100
5	Forward + SDM1 Reverse	1:1000
6	Forward + SDM1 Reverse	1:10000
7	SDM1 Forward + SDM2 Reverse	1:10
8	SDM1 Forward + SDM2 Forward	1:100
9	SDM1 Forward + SDM2 Reverse	1:1000
10	SDM1 Forward + SDM2 Reverse	1:10000
11	SDM2 Forward + Reverse	1:10
12	SDM2 Forward + Reverse	1:100
13	SDM2 Forward + Reverse	1:1000
14	SDM@ Forward + Reverse	1:10000
15	Kb plus Ladder
16	Empty









No PCR products.

Will run a temperature gradient for each sample to see if temperature is affecting enzyme activity

Will preform Power Soil DNA Extraction and bead beaten with phenol: chloroform extraction and isopropanol precipitation to see if template is the problem.

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07/24/13

Site-Directed Mutagenesis PCR for BktB




Into each tube:

• Mix: 98μL
• Template: 2μL
• Total: 100μL



Dilutions used: 1:10, 1:100, 1:1000, 1:10000


Mix:

• 2x Master Mix: 250μL
• Forward Primer: 3μL
• Reverse Primer: 3μL
• PCR dH₂O: 244μL
• Total: 500μL



PCR Primers Used:

• Forward + SDM1 Reverse
• 340 base pairs
• SDM1 Forward + SDM2 Revers
• 660 base pairs
• SDM2 Forward + Reverse
• 300 base pairs






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07/24/13

DNA Extraction of R. eutropha



1. Re suspend cells in 500μL 1x STE buffer
2. Vortex until mixture is homogenous
3. Add 50μL of 10% Sodium-dodecyl sulfate (SDS)
4. IF cells insufficiently lysed, 2 minutes in bead beater
5. Add 500μL of Chloroform:Phenol pH 8
• Extract bottom layer (2 phases present)
• Minimize time the chloroform is out of fridge
6. Vortex
7. Centrifuge for 30 seconds
• 14000 rpm
8. Transfer top phase to 1.5mL microcentrifuge tube
• Don't take up white precipitant from interphase of solution
9. Add 500μL of isoamyl acetate (CIAA 24:1) to top phase
10. Vortex
11. Centrifuge for 30 seconds
• 14000 rpm
12. Transfer top phase to 1.5mL microcentrifuge tube
• Don't take up white precipitant from interphase of solution
13. REPEAT STEPS 9-12
• Note: Keep track of amount finally transfered in step 12
14. Add (2/10) volume (amount of top phase) of 3M Sodium Acetate
15. Add (7/10) volume (step 14 amount) of Isopropanol
• Note: Mix solutions with pipette
16. Centrifuge for 25 minutes
• 14000 rpm
17. Dispose of liquid portion
• Don't disturb invisible pellet
18. Dry pellet on heat block at 60°C for 10 minutes
• Leave caps open for evaporation of isopropanol
19. Re suspend pellet in 50μL of distilled water






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07/24/13

Repeat of Transformation of Ca Promoters into Top 10 Cells




Preformed as outlined on February 11, 2013

-Last time we used 2μL for transformation this time since our ligations will contain a maximum of 100ng of insert (recommended amount for transformation of top 10 cells), we are going to use the entire ligation volume (20μL)


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07/23/13

Making Beef Extract Medium




Component	Amount
Beef Extract	3g
Peptone	5g
Agar	15g
Distilled Water	1000mL




Pour components and one magnetic mixer into empty jar. Fill jar with distilled water to 1000mL mark.

Autoclave at setting number "2" for 20 minutes at slow exhaust


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07/23/13

PCR of Ca AdhE




Component	Amount
2X Master Mix	125μL
Forward Primer	4μL
Reverse Primer	4μL
PCR Water	117μL
TOTAL	250μL

1 Reaction 50μL = 49μL Mix + 1μL Template








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07/23/13

Digest of Cc Adh and Cs Adh




500ng DNA for each reaction




500ng (^1&#956L⁄_39.7ng) = 12.5 μL Cc DNA

500ng (^1&#956L⁄_36.0ng) = 14 μL Cs DNA




Component	Cc	Cs	pSB1C3
10X Buffer	2μL	2μL	2μL
EcoR1	2μL	2μL	2μL
Pst1	2μL	2μL	2μL
DNA	12.5μL	14μL	2.5μL
PCR Water	1.5μL	0μL	11.5μL
Total	20μL	20μL	20μL




500ng (^1&#956L⁄_202ng) = 2.5μL pSB1C3 backbone




Incubate at 37°C water bath for 45 minutes


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07/11/13

Repeat of Cohesive End CLoning of gBloeu Ca Promoters Ptb, AaDc, and thl into pSB1C3 Vector




1. Digest

A. In 1 tube contain

• Backbone: 2μL
• EcoR1: 1μL
• Pst1: 1μL
• 10x Buffer: 2μL
• Water: 14μL
• Total: 20μL



B. In each 6 tubes combine

• gBloeu Resuspension: 10μL
• EcoR1: 1μL
• Pst1: 1μL
• 10x Buffer: 2μL
• Water: 6μL
• Total: 20μL




Incubate in 37°C water bath for 1 hour

Clean with QIAquick PCR Purification Kit

Nano drop




Aadc quantification appears to be good with a reading of 1.9ng/μL, but is the lowest limit of detection for the nano drop 1000, so we can't be sure




Ligation

Since the vector is 31.7 ng/μL and the protocol calls for 50ng of DNA and the concentration of insert might be 1.5 to 2.0ng/μL due to the need of 20ng.



• pSB1C3: 2μL
• Promoter: 13μL
• Buffer: 2μL
• Ligase: 1μL
• Water: 2μL
• Total: 20μL



Incubate at room temperature overnight


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07/23/13

Gel of AdhE and AdhE1



Row #1
Lane Number	Sample	Dilution	Temperature(μ)
1	Ladder
2	Ca AdhE	1:100	53
3	Ca AdhE	1:100	50
4	Ca AdhE	1:100	56
5	Ca AdhE	1:100	60
6	Ca AdhE	1:10	50
7	Ca AdhE	1:10	53
8	Ca AdhE	1:10	56
9	Ca AdhE	1:10	60
10	Ca AdhE	1:1000	50
11	Ca AdhE	1:1000	53
12	Ca AdhE	1:1000	56
13	Ca AdhE	1:1000	60
14	Empty
15	Empty
16	Empty
Row #2
1	Ladder
2	Ca AdhE1	1:100	50
3	Ca AdhE1	1:10	50
4	Ca AdhE1	1:100	53
5	Ca AdhE1	1:100	50
6	Ca AdhE1	1:100	56
7	Ca AdhE1	1:10	53
8	Ca AdhE1	1:10	56
9	Ca AdhE1	1:10	60
10	Ca AdhE1	1:1000	50
11	Ca AdhE1	1:1000	53
12	Ca AdhE1	1:1000	56
13	Ca AdhE1	1:1000	60
14	Empty
15	Empty
16	Empty






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07/22/13

PCR of Ca AdhE and AdhE1





Sample	Dilution	50°	53°	56°	60°
Empty
Ca AdhE	1:10	✓	✓	✓	✓
Ca AdhE	1:100	✓	✓	✓	✓
Ca AdhE	1:1000	✓	✓	✓	✓
Empty
Ca AdhE1	1:10	✓	✓	✓	✓
Ca AdhE1	1:100	✓	✓	✓	✓
Ca AdhE1	1:1000	✓	✓	✓	✓



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07/22/13

PCR of Ca AdhE/AdhE1 using pSOL




Used Qia Quick Prep Plasmid Extraction Kit




AdhE and AdhE1 are supposed to be located on pSOL plasmid using pSOL as template (1:10, 1:100, 1:1000)




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07/22/13

Plasmid Extraction of Ca pSOL




Used Qia Quick Prep Plasmid Extraction Kit






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07/11/13

Dual Gel of mlsR Xformants




Row #1
Well #	Sample
1	Ladder
2	mlsR 5
3	mlsR 9
4	mlsR 10
5	mlsR 12
6	mlsR 22
7	mlsR 24






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07/22/13

Dual Digest of mlsR Xformants




EcoR1 and Pst1 Dual Digest of mlsR colonies

5, 9, 10, 12, 22, 24




500ng (^1&#956L⁄_112.2ng) = 4.45 μL → 4.5 μL

500ng (^1&#956L⁄_124.5ng) = 4.02 μL → 4.0 μL

500ng (^1&#956L⁄_117.7ng) = 4.25 μL → 4.5 μL

500ng (^1&#956L⁄_162.0ng) = 3.09 μL → 3.0 μL

500ng (^1&#956L⁄_188.7ng) = 2.65 μL → 2.5 μL

500ng (^1&#956L⁄_206.4ng) = 2.42 μL → 2.5 μL





Colony Number	EcoR1	Pst1	10x Buffer	DNA	PCR dH2O	Total
5	2μL	2μL	2μL	4.5μL	9.5μL	20μL
9	2μL	2μL	2μL	4.0μL	10.0μL	20μL
10	2μL	2μL	2μL	4.5μL	9.5μL	20μL
12	2μL	2μL	2μL	3.0μL	11.0μL	20μL
22	2μL	2μL	2μL	2.5μL	11.5μL	20μL
24	2μL	2μL	2μL	2.5μL	11.5μL	20μL




Incubated at 37°L for 45 minutes

Transformed to 80°L heat block for 1 hour


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07/22/13

Transformation of Terminators/Promoters




Transforming the following parts from the iGEM distribution for use in shuttle vector




BBa_B1006 from 2013 Plate 3, Well 6D (in C3)

BBa_Bl003 from 2013 Plate 3, Well 4P (in C3)




Punctured wells with pipette tip

Re suspended DNA in 10μL of PCR water




1μL of DNA added to 50μL of competent cells

100μL of cells  Used 2μL of DNA




Incubated on ice for 30 minutes




Heat shocked cells for 1 minutes




Incubated on ice for 5 minutes




Add 600μL of Psi broth




Incubated at 37°C with shaking for 2 hours





Also Xformed

• BBa_K148012 Pveg
• BBa_K823000 P?
• BBa_K823002 P?
• BBa_K823003 Pveg
• BBa_K090504 ?



Plated 90μL of undiluted 1:10, 1:100, 1:1000 of BBa_B1006 and BBa_B1003


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07/22/13

PCR Cleanup of Cs Adh PCR Products


----------------------------pic---------------------------------
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07/22/13

Plasmid Extraction of Xformants




mlsR library colonies 5, 9, 10, 12, 22, 24

Extracted using Qiagen plasmid prep kit


------------------------pic----------------------------------------
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07/19/13

PCR of Ccel Adh




Preformed PCR to amplify Adh out of C. cellulolyticum


---------------------------pic---------------------------------------
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07/19/13

PCR of Cb SAdh




Choose lower temperature (50°C) for annealing.


-------------------------pic--------------------------------------


Still have primer dimer, or primers may be sitting down at another place on the genomic DNA


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07/19/13

Promoter and Transformation Trouble Shooting




No colonies have grown on the plates incubated with transformed Top 10 cells with pSB1C3 and various promoters plated on July 16 2013. We did a nanodrop quantification of the ligationproducts.


-----------------------PIC----------------------------------------------------


While the 260/280 ratios seem high, Dr. wawrick says that they aren’t troubling. However, the amount of DNA quantified is unrealistic. Since, we only started with 100ng of insert and 200ng of vector. We ran a gel to see if what we quantified is DNA and not RNA.




Well 1-Ladder

Well 2-Aadc – RBS

Well 3- thl + RBS





From this gel, it doesn’t look like we actually have any DNA (although there may be some really faint bands near the bottom). This means that the ligation didn’t actually occur (or we would have seen a band at 2000bp + 125-75bp). However, if the ligation didn’t work we would still expect to see 2 bands. One is the size of the vector and the other is the size of the insert. Perhaps, something went wrong in the phenol/chloroform extraction step.


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07/19/13

Restreak of mlsR + pSB1C3 Top 10




Transferred six colonies from library to individual plates. Numbers 5, 9, 10, 12, 22, and 24 from library.




PCR Purification of July 18, 2013 Cs

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07/18/13

PCR of C sacch and Cs Adh




Row 1
Lane Number	Sample	Dilution	Temperature (°C)
1	Ladder
2	Cs	Undiluted	55
3	Cs	Undiluted	62
4	Cs	Undiluted	65
5	Cs	1:10	55
6	Cs	1:10	62
7	Cs	1:10	65
8-16	Empty
Row 2
Lane Number	Sample	Dilution	Temperature (°C)
1	Ladder
2	Cb	Undiluted	55
3	Cb	Undiluted	62
4	Cb	Undiluted	65
5	Cb	1:10	55
6	Cb	1:10	62
Cb	Cs	1:10	65
8-16	Empty

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07/18/13

Gel of Isolated Plasmids




Row 1
Lane Number	Sample
1	Ladder
2	pSB1C3 + mlsR1
3	pSB1C3 + mlsR7
4	pSB1C3 + mlsR13
5	pSB1C3 + mlsR13
6	pSB1C3 + mlsR21
7	pSB1C3 + mlsR23
8	pSB1C3 + mlsR26
9	puc19 + KIVD

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07/18/13

Dual Digests of Isolated Plasmids (18 July)




500ng of plasmid for digest




puc19 + KIVD

$500ng\; X\; (\mu L/516ng)\; =\; 0.89\mu L$


pSB1C3 + mlsR21

$500ng\; X\; (\mu L/99.4ng)\; =\; 5.03\mu L$


pSB1C3 + mlsR13

$500ng\; X\; (\mu L/81.0ng)\; =\; 6.17\mu L$


pSB1C3 + mlsR1

$500ng\; X\; (\mu L/101.0ng)\; =\; 4.95\mu L$


pSB1C3 + mlsR7

$500ng\; X\; (\mu L/65.1ng)\; =\; 7.68\mu L$


pSB1C3 + mlsR23

$500ng\; X\; (\mu L/84.1ng)\; =\; 5.95\mu L$


pSB1C3 + mlsR26

$500ng\; X\; (\mu L/86.1ng)\; =\; 5.81\mu L$



Gene	EcoR1	Pst1	10x Buffer	DNA	PCR Water	Total
KIVD	2μL	2μL	2μL	1μL	13μL
mlsR21	2μL	2μL	2μL	5μL	9μL
mlsR13	2μL	2μL	2μL	6μL	8μL
mlsR1	2μL	2μL	2μL	5μL	9μL
mlsR7	2μL	2μL	2μL	7.5μL	6.5μL
mlsR23	2μL	2μL	2μL	6μL	8μL
mlsR26	2μL	2μL	2μL	6μL	8μL


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07/18/13

Plasmid Prep of Transformed Top10 Cells with pSB1C3 + mlsR and puc19 + KIVD


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07/17/13

DNA Extraction from Ca, Cs, Cb, and Cc using MoBio Power Soil DNA Extraction Kit


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07/10/13

C. acetobutylicum Shuttle Vector Design




The following sequences have been verified:

Clos Ori BBa_J238001

Pthl BBa_J238002

Paadc BBa_J238003

Ptb BBa_J238004

mlsR BBa_J238005





Use Terminators for test testing

Long artificial terminator (T>90%)

• BBa_B1006
• 39 base pairs
• 2013 p3w6D in C3

Small artificial terminator (T = 85%)

• BBa_B1002
• 34 base pairs
• 2013 p5w4D in Ak3

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07/10/13

1% Agarose Gel Electrophoresis of 9 July PCR Products




Procedure followed as on pg. 50

Row 1
Lane	Sample	Dilution	Temperature (°C)
1	Ladder
2		C. sacc	1:10	50.8
3	C. sacc	1:100	50.8
4	C. sacc	1:1000	50.8
5	mlsR	1:10	50.8
6	mlsR	1:100	50.8
7	mlsR	1:1000	50.8
8	C beij	1:10	50.8
9	C beij	1:100	50.8
10	empty
11	empty
12	empty
13	empty
14	empty
15	empty
16	empty
Row 2
1	Ladder
2	C. sacc	1:10	52.8
3	C. sacc	1:100	52.8
4	C. sacc	1:1000	52.8
5	mlsR	1:10	52.8
6	mlsR	1:100	52.8
7	mlsR	1:1000	52.8
8	C beij	1:10	52.8
9	C beij	1:100	52.8
10	empty
11	empty
12	empty
13	empty
14	empty
15	empty
16	empty
Row 3
1	Ladder
2		C. beij	1:10	54.1
3	C. beij	1:100	54.1
4	mlsR	1:10	54.1
5	mlsR	1:100	54.1
6	mlsR	1:1000	54.1
7	C. sacc	1:10	54.1
8	C. sacc	1:100	54.1
9	C. sacc	1:1000	54.1
10	empty
11	empty
12	empty
13	empty
14	empty
15	empty
16	empty
Row 2
1	Ladder
2	C. beij	1:10	58.8
3	C. beij	1:100	58.8
4	mlsR	1:10	58.8
5	mlsR	1:100	58.8
6	mlsR	1:1000	58.8
7	C. sacc	1:10	58.8
8	C. sacc	1:100	58.8
9	C. sacc	1:1000	58.8
10	empty
11	empty
12	empty
13	empty
14	empty
15	empty
16	empty


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07/9/13

DNA Extraction for C sacc, C aceto, and C beij




Procedure followed as on pg. 50

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07/9/13

Gradient PCR of C. scc, mlsR, and C. beij




Column Number	Temperature (°C)
3	50
5	52.8
6	54.1
10	58.8




Lane Number	Sample
1	C sacc 1:10
2	C sacc 1:100
3	C sacc 1:1000
4	mlsR 1:10
5	mlsR 1:100
6	mlsR 1:1000
7	C beij 1:10
8	C beij 1:100




Vf Preparation




Component	Amount
2x Mastermix	125μL
Forward	2μL
Reverse	2μL
PCR dH2O	121μL
Total	250μL


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07/3/13

Gel of (2 July) PCR Products




Row 1
Lane	Sample
1	Ladder
2		C. sacc 1:10
3	C. sacc 1:100
4	C. sacc 1:1000
5	C. sacc 1:10000
6	Negative
7	mlsR 1:10
8	mlsR 1:100
9	mlsR 1:1000
10	mlsR 1:10000
11	Negative
12	ladder
13	empty
14	empty
15	empty
16	empty
Row 2
1	Ladder
2	C. sacc 1:10
3	C. sacc 1:100
4	C. sacc 1:1000
5	C. sacc 1:10000
6	Negative
7	C. beij 1:10
8	C. beij 1:100
9	C. beij 1:1000
10	C. beij 1:10000
11	empty
12	empty
13	empty
14	empty
15	empty
16	empty


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07/2/13

PCR of mlsR and Redo of C. sacc and C. beij PCR


-------------------------pic----------------------------------------- Back to top

07/2/13

Ralstonia eutropha DNA Extraction




Due to previous low extraction efficiency

Attempted to extract higher [DNA]



1. For 5mL tubes, each containing 3mL of meat extract broth inoculated with R. eutropha cells, which were in stationary phase
2. 1mL aliquots transferred stepwise to two 15mL epi tubes, spinning at 14,000 rpm for five minutes to pellet cells
3. Supernatant discarded and additional 1mL aliquot added, spun until all cells pelleted
4. Cells re suspended in 500μL if 1x STE buffer in each of the two epi tubes
5. Vortex
6. 50μL of 10% SDS added to each tube
7. 500μL of TE saturated phenol pH8 added to each tube
8. Vortex well and centrifuge for 30 seconds
9. Aqueous recovered
10. Phenol extraction
11. Steps 7-9 repeated
12. 500μL chloroform added to each tube and vortexed
13. Aqueous discarded
14. Chloroform extraction repeated
16. 400μL split into each of four tubes
17. 100μL of 3M NaOH added to each tube
18. 350μL isopropanol added to each tube
19. Centrifuge at 14000 rpm for 25 minutes
20. Liquid discarded
21. Tubes places on 80°C heating block for 15 minutes
22. Contents of each tube re suspended in 500μL of PCR dH₂O

----------------------------4PIC--------------------------------------------------
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07/2/13

Gel Electrophoresis of PCR Product (1 July)




Lane	Sample
1	Ladder
2		C. sacc Undiluted
3	C. sacc 1:10
4	C. sacc 1:100
5	Negative
6	C. cell undiluted
7	C. cell 1:10
8	C. cell 1:100


---------------------------------------pic-----------------------------

Lane	Sample
1	Ladder
2		Negative Control
3	1:10 Diluted C. beij
4	C. beij 1:100
5	C. beij 1:1000
6	C. beij 1:10000
7	Empty
8	Empty


----------------------------PIc-------------------------------------------
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07/1/13

16S Gel Repeat




16S gel of C. acetobutylicum, R. eutropha, C. sacchaolyticum, and C. beijerinki to confirm wheather or not we have what we think we have




16S Gel
Lane Number	Sample
1	Ladder
2	C. beijerinki
3	R. eutropha
4	C. sacchrolyticum
C. acetobutylicum


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06/28/13

1:4 Loading Dye




1 part loading dye

2 parts 40% glycerol

2 parts PCR DI water

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06/27/13

DNA Extraction of R. eutropha, C. saccharolyticum, and C. beijerinki




Procedure performed as outline on pg. 42


---------------------------3 pics------------------------------------------


16S PCR of C. acetobutylicum, R. eutropha, C. sacchrolyticum, and C. beijerinki to confirm wheather or not we have what we think




PCR Solution
Component	Amount (μL)
2x PCR Mastermix	350
Forward primer (27F)	Reverse primer (1525R
PCR water	344
Total	700

50μL for each reaction, three reactions for each organism with different template dilutions
• Undiluted
• 1:10
• 1:100

Place 1μL template into each tube

Negative control won’t have a template




Row Number	Colum Number	Sample
3	7	Negative Control
4	4	C. beijerinki
4	5	C. beijerinki 1:10
4	6	C. beijerinki 1:100
4	7	C. sacchrolyticum
4	8	C. sacchrolyticum 1:10
4	9	C. sacchrolyticum 1:100
5	4	C. acetobutylicum
5	5	C. acetobutylicum 1:10
5	6	C. acetobutylicum 1:100
5	7	R. eutropha
5	8	R. eutropha 1:10
5	9	R. eutropha 1:100




Gel of PCR product
Row Number	Colum Number	Sample
1	1	Ladder
1	2	C. beijerinki
1	3	C. beijerinki 1:10
1	4	C. beijerinki 1:100
1	5	C. sacchrolyticum
1	6	C. sacchrolyticum 1:10
1	7	C. sacchrolyticum 1:100
2	1	Ladder
2	2	C. acetobutylicum
2	3	C. acetobutylicum 1:10
2	4	C. acetobutylicum 1:100
2	5	R. eutropha
2	6	R. eutropha 1:10
2	7	R. eutropha 1:100
2	8	Negative Control


-------------------------------------------pic---------------------------

We have bands of the correct length for Cb, Cs, and Ca, they are too faint for sequencing. So, we are going to redo the PCR without template dilutions and do 35 cycles instead of 30 cycles


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06/25/13

Modified VM Medium




C. cellulolyticum may be grown at 37°L

VM medium to reduce precicpitation

AEM: 77, 2727-2733 (2011)


0.15g

Modified ? (per liter)
Component	Amount
KH2PO4	1g
KH2PO4	3.4g
Urea	2.14g
MgCl2 - 6H2O	1g
CaCl2 - 2H2O
FeSO2 - 6H2	1.25g
MOPS 3-(N-morpholino)propanesulfuric acid	10g
Rezazurin	2mg
Vitamin solution	10mL
Yeast Extract	2g
Oligoelement solution	1mL
Cysteine – HCl	1g
Cellobiose	5.1345




100x Vitamin Solution
ComponentAmount
Biotin	0.08μM
Pyridoxamine	0.02μM
Cyanocobalamine	0.001μM
p-aminobenzoic acid	0.15μM
Thiamine	0.9μM
L-alaning	0.22μM




1000x Oligoelement Solution (per liter)
Component	Amount
FeSO4 - 7H2O	5g
ZnSO4 - 7H2O	1.44g
MnSO4 - 7H2O	1.12g
CuSO4 - 5H2O	0.25g
Na2B4O7	0.2g
(Mo)7(NH4)6O24 - 4H2O	1g
NiCl2	0.04g
CoCl2	0.02g
HBO3	0.03g
Na2SeO3	0.02g
10M HCl	50mL




VM Medium placement

• 100mL in bottles
• 20mL in vials
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06/25/13

Re constituting Strains in Anaerobic Chamber



• Clean glass vial with acetone
• In aerobic Chamber:
• Chemwipes
• Sterile Needles
• Bring into chamber
• Heavy duty aluminum foil to work over
• Glass cutter
• File
1. Cycle chamber airlock; twice
2. Clean gloves and chamber with 1N HCl (x2)
3. ???????????????????
4. Use sterile syringe to add 0.5mL media to inner vial
5. Mix with syringe
6. Add 0.5 of inoculums to each of the two media vials
7. Transfer an inoculum from these vials to a second vial to get one heavy and one light inoculum

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06/24/13

Media Preparation for C. acetobutylicum




Media components were added to a 2000mL Erlenmeyer flask




0.1mL of 1g/L Rezarin was added to the medium and was degassed under N₂ for 45 minutes


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06/21/13

Media Preparation for C. acetobutylicum




C. acetobutylicum will grow on defined media:

AEM 50:1238 (1985) or the following




Dr. Tanner’s recipe of C .acetobutylicum (per liter)
Component	Amount
Yeast Extract	1g
Glucose/Dextrose	20g
RST minerals	10mL
RST vitamins	10mL
RST metals	5mL




RST Minerals (per liter)
Component	Amount
NaCl	80g
NH4Cl	100g
KCl	10g
KH2PO4	10g
MgSO4 - 7H2O	20g
CaCl2 - 2H2O	4g




RST Minerals (per liter)
Component	Amount
Nitriloacetic Acid	2g
MnSO4 - H2O	1g
Fe(NH4)2(SO4)2 - 6H2O	0.8g
CoCl2 - 6H2O	0.2g
2nSO4 - 7H2	0.2g
CuCl2 - 2H2O	0.02g
NiCl2 - 6H2O	0.02g
Na2MoO4 - 2H2O	0.02
Na2SeO4	0.02g
Na2WO4	0.02g




RST Vitamins
Components	Amount	Light Sensitivity (Y/N)
Pyridoxine HCl	10mg	Y
Thiamine HCl	5mg	Y
Riboflavin	5mg	Y
Calcium Pantothenate	5mg	Y
Thioctic Acid	5mg	N
p-Aminobenzoic Acid	5mg	Y
Nicotinic Acid	5mg	N
B12 = Cyanocobalamine	5mg	N
Biotin	2mg	N
Folic Acid	2mg	Y
Mercaptopethane Sulfuric Acid	10mg	N

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06/17/13

Competent pAN1 containing DH5 E. coli




Suspended a unit sized inoculums of pAN1 containing DH5 E. coli in LB with chloramphenicol resistance.




Incubated overnight at 37°C and at 220 rpm for 2.5 hours




One culture has an OD600 of 0.38 and another is 0.475



• Spun at 12000 g for 20 minutes
• Discarded supernatant and re suspended in 15mL of TFBl
• Incubated on ice for 60 minutes
• Centrifuged at 12000 g for 20 minutes

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06/14/13

Gel of puc19 with KIVD Digest (EcoR1 and Pst1)



----------------------------------pic------------------------------------------------- Back to top

06/14/13

Digestion of pUC19 containing KIVD with EcoR1-Hf and Pst1-Hf





Component	Amount (μL)
EcoR1	1
Pst1-Hf	1
10x Buffer	2
DNA	2.5 (500ng)
PCR H2	13.5
Total	20




Place in 37°C water bath for 45 minutes


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06/13/13

Gel of PCR





Row 1
Well Number	Sample	Temperature (°C)
1	Ladder
2	AdhE1	50.8
3	AdhE1	52.8
4	AdhE1	54.1
5	AdhE1	58.8




Row 2
Well Number	Sample	Temperature (°C)
1	Ladder
2	AdhE	50.8
3	AdhE	52.8
4	AdhE	54.1
5	AdhE	58.8




The gel results of this PCR are similar to the last one. The bands indicate smaller than 250 base pairs, which is smaller than the size of the genes we intended to amplify (2000 base pairs).

AdhE = 2589 base pairs

AdhE1 = 2577 base pairs

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06/12/13

PCR redo of AdhE and AdhE1





Temperature gradient and dilution up to 1:10000

Column Number	Temperature (°C)
3	50.8
5	52.8
6	54.1
10	58.8


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06/12/13

Plasmid Isolation of pUC19 from BBa_k273006



----------------------2pic-------------------------
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06/10/13

Amplification of AdhE and AdhE1 in C. acetobutylicum





Testing our primers using the template prepared on June/4




Forward primer used (AdhE)

Forward primer sequence




Reverse primer used (AdhE)

Reverse primer Sequence




Forward primer used (AdhE1)

Forward primer sequence




Reverse primer used (AdhE1)

Reverse primer sequence




Preparation of solution 1:

Into two separate 1.5mL centrifuge tubes

Component	Amount (μL)
PCR DI Water	146
2x PCR Mastermix	150
Forward primer	2
Reverse primer	2
Total	300




50μL of solution 1 were pipette into 5 PCR tubes for each solution (AdhE and AdhE1)




Template dilutions were prepared for 1:10, 1:100, 1:1000, and 1:10000




1mL of template was loaded into 2 each of 4 PCR tubes containing 50μL of solution 1




The 5th PCR tube containing solution 1 will serve as negative control


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06/4/13

Place pxy plates in glove box to degrass




Extracted Ca DNA

1. Re suspend cells in 500μL 1x STE buffer
2. Vortex
3. Add 1/10 volume of 10% SDS
4. 2 minutes in bead beater
5. Add 500μL TE saturated phenol pH 8
• Be sure to go down far enough in bottle to reach phenol
• Put directly back in fridge when done
6. Vortex
7. Centrifuge for 30 seconds
8. Recover aqueous phase
• Don’t recover white material at interphase
• Throw away
9. Add 500μL Chloroform: Isoamyl acetate
10. Vortex
11. Centrifuge for 30 seconds
12. Recover and throw away aqueous phase
13. Repeat Steps 8-12
15. Add 2/10 volumes 3M sodium acetate
16. Add 7/10 volumes isopropanol
17. Mix well
18. Centrifuge for 25 minutes
19. Remove liquid and don’t disturb invisible pellet
20. Dry pellet at 60°C in heat block for 10 minutes
• Leave caps off to allow isopropanol to evaporate
21. Re suspend pellet in 50μL of dH₂

-------------------------------pic-------------------------------------
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05/31/13

Plasmid Extraction/Purification of Top 10 E. coli Transformed with pSB1C3 and Clostridial Origin


-----------------------------11 PICS----------------------------------


Digestion as preformed on pg. 47




Gel of Digestion
Row 1
Well Number	Sample
1	Ladder
2	1
3	3
4	5
5	8
6	11
7	12
Row 2
Well Number	Sample
1	Ladder
2	16
3	18
4	20
5	22
6	24




Wells 2 and 3 like like what we need.

Sequencing primers are in the process of being made to comfirm


2 AdhE1 50.8 3 AdhE1 52.8 4 AdhE1 54.1 5 AdhE1 58.8


Row 2
Well Number	Sample	Temperature (°C)
1	Ladder
2	AdhE	50.8
3	AdhE	52.8
4	AdhE	54.1
5	AdhE	58.8




The gel results of this PCR are similar to the last one. The bands indicate smaller than 250 base pairs, which is smaller than the size of the genes we intended to amplify (2000 base pairs).

AdhE = 2589 base pairs

AdhE1 = 2577 base pairs

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06/12/13

PCR redo of AdhE and AdhE1





Temperature gradient and dilution up to 1:10000

Column Number	Temperature (°C)
3	50.8
5	52.8
6	54.1
10	58.8


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06/12/13

Plasmid Isolation of pUC19 from BBa_k273006



----------------------2pic-------------------------
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06/10/13

Amplification of AdhE and AdhE1 in C. acetobutylicum





Testing our primers using the template prepared on June/4




Forward primer used (AdhE)

Forward primer sequence




Reverse primer used (AdhE)

Reverse primer Sequence




Forward primer used (AdhE1)

Forward primer sequence




Reverse primer used (AdhE1)

Reverse primer sequence




Preparation of solution 1:

Into two separate 1.5mL centrifuge tubes

Component	Amount (μL)
PCR DI Water	146
2x PCR Mastermix	150
Forward primer	2
Reverse primer	2
Total	300




50μL of solution 1 were pipette into 5 PCR tubes for each solution (AdhE and AdhE1)




Template dilutions were prepared for 1:10, 1:100, 1:1000, and 1:10000




1mL of template was loaded into 2 each of 4 PCR tubes containing 50μL of solution 1




The 5th PCR tube containing solution 1 will serve as negative control


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06/4/13

Place pxy plates in glove box to degrass




Extracted Ca DNA

1. Re suspend cells in 500μL 1x STE buffer
2. Vortex
3. Add 1/10 volume of 10% SDS
4. 2 minutes in bead beater
5. Add 500μL TE saturated phenol pH 8
• Be sure to go down far enough in bottle to reach phenol
• Put directly back in fridge when done
6. Vortex
7. Centrifuge for 30 seconds
8. Recover aqueous phase
• Don’t recover white material at interphase
• Throw away
9. Add 500μL Chloroform: Isoamyl acetate
10. Vortex
11. Centrifuge for 30 seconds
12. Recover and throw away aqueous phase
13. Repeat Steps 8-12
15. Add 2/10 volumes 3M sodium acetate
16. Add 7/10 volumes isopropanol
17. Mix well
18. Centrifuge for 25 minutes
19. Remove liquid and don’t disturb invisible pellet
20. Dry pellet at 60°C in heat block for 10 minutes
• Leave caps off to allow isopropanol to evaporate
21. Re suspend pellet in 50μL of dH₂

-------------------------------pic-------------------------------------
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05/31/13

Plasmid Extraction/Purification of Top 10 E. coli Transformed with pSB1C3 and Clostridial Origin


-----------------------------11 PICS----------------------------------


Digestion as preformed on pg. 47




Gel of Digestion
Row 1
Well Number	Sample
1	Ladder
2	1
3	3
4	5
5	8
6	11
7	12
Row 2
Well Number	Sample
1	Ladder
2	16
3	18
4	20
5	22
6	24




Wells 2 and 3 like like what we need.

Sequencing primers are in the process of being made to comfirm


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05/30/13

Plasmid Extraction/Purification of Top 10 E. coli Transformed with pSB1C3 and Clostridial Origin





Purified six tubes of DNA (plasmid)

Quantification with Nanodrop


------------------------------------6 PICS-----------------------------
DNA

Digestion
Component	Amount (μL)
EcoR1	2
Pst1	2
10x Buffer	1
5




Incubate in 37°C water bath for 1 hour

Load all 10μL onto gel




Gel of Digestion
Well Number	Sample
1	Ladder
2	Screen #3
3	Screen #6
4	Screen #10
5	Screen #19
6	Screen #19
7	Screen #26

Note: Screen # corresponds to quantification Id




Since our insert is about 700 base pairs and our vector is about 2000 base pairs. Well 2 looks like what we want. We are going to re streak colonies isolated from plate with screen #3 , make a freezer stock, and take more colonies from screen #10




Freezer stocks of Top 10, pAN1, pIKM1, and p34KM were made by suspending cells in 1mL of 10% glycerol.


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05/29/13

Made 80 stocks of C. acetobutylicum (2mL of culture into 2mL of glycerol)

Streaked three more Top 10 pSB1C3 and Clostridial Origin plates


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05/28/13

Inoculated cultures of C. acetobutylicum and a transfer was made

Six plates (LB + Chloramphenicol) were streaked with transformation of pSB1C3 and Clostridial Origin in Top 10 E. coli cells in preparation for plasmid extraction.


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05/23/13

b>