8/6/16

The vector plasmids pSB1A3 and pSB1C3 were miniprepped, and a verification gel was run. The miniprep of the pSB1C3 plasmid was successful, while the one for pSB1A3 was not. The genes to be inserted were also designed using the BioBrick standard assembly and ordered from IDT.

[gel not accounted for]

8/11/16

The pSB1A3 plasmids were miniprepped again, and a gel was run on them using 5uL of each sample. This time the miniprep was successful.

Gel Lanes (left to right with lanes at the top)

Lane 1	Lane 2	Lane 3	Lane 4
ladder	tube 1	tube 2	tube 3

8/13/16

The forward and reverse primers were re-suspended:

• The tubes with the dried primers in them were spun down
• 786 uL of dI water was added to to the forward primer
• 860 uL of dI water was added to the reverse primer
• The primers were vortexed for 3-4 seconds
• Both tubes were centrifuged for 1 min at full speed

and both vectors were PCR amplified using the following amounts:

• 67.6 uL water
• 10 uL 5x Taq buffer
• 7.5 uL DNTPs (2.3 uM)
• 6.7 uL forward primer (20 uM)
• 6.7 uL reverse primer (20 uM)
• 5 uL Taq enzyme
• 1 uL DNA (10 ng/uL)

8/18/16

Both vectors were PCR purified and a gel was run afterwards. Three samples of each vector were run on the gel: the vector plasmids after miniprep (1); the vectors after PCR (2); and the vectors after being PCR purified (3)

The six samples were prepared as follows

• Samples A3 (1) and C3 (1):
• 1 uL DNA
• 2 uL loading buffer
• 7 uL dIH2O
• Samples A3 (2) and C3 (2):
• 2 uL DNA
• 2 uL loading buffer
• 6 uL dIH2O
• Samples A3 (3) and C3 (3):
• 5 uL DNA
• 2 uL loading buffer
• 3 uL dIH2O

10 uL of each sample was loaded into the gel

Gel Lanes (left to right with lanes at top)

Lane 1	Lane 2	Lane 3	Lane 4	Lane 5	Lane 6	Lane 7
ladder	A3 (1)	C3 (1)	A3 (2)	C3 (2)	A3 (3)	C3 (3)

[gel not accounted for]

The results indicated that the PCR was unsuccessful.

Meanwhile, an amplification PCR was set up for the Chlorogenate Esterase gene. The gene was resuspended in 100 uL of dIH2O for a final concentration of 10 ng/uL. The solution was incubated at 50ºC for 20 min and then centrifuged. The forward and reverse primers were also resuspended. The forward primer was resuspended in 1500 uL of dIH2O, and the reverse primer was resuspended in 1600 uL of dIH2O. The gene was PCR amplified using the following amounts:

• 50 uL DNA (10 ng/uL)
• 20 uL 5x Taq buffer
• 12 uL dIH2O
• 7.5 uL dNTPs (2.3 uM)
• 5 uL forward primer (20 uM)
• 5 uL reverse primer (20 uM)
• 0.5 uL Taq enzyme

8/19/16

The vectors were PCR amplified a second time. This time some changes were made to the basic PCR settings. The annealing temperatures (cycle 2) were changed as follows:

Both of the vectors were divided into three samples: Sample 1 and a positive control (with VF2 and VR primers) were put at 54ºC; sample 2 was put at 56ºC; and sample 3 was put at 58ºC. All samples were run for 20 sec.

A gel was run for the results of the PCR:

Gel Key

Lane	Sample	PCR Temperature (℃)	Amount Loaded (uL)
1	ladder	X	5
2	pSB1A3	54	1
3	pSB1A3	56	5
4	pSB1A3	58	5
5	pSB1A3 (Control)	54	5
6	X	X	X
7	pSB1C3	54	1
8	pSB1C3	56	5
9	pSB1C3	58	5
10	pSB1C3 (Control)	54	5
11	X	X	X
12	Chlorogenate Esterase after resuspension	X	1
13	Chlorogenate Esterase amplified product	X	5

8/20/16

Both vectors were PCR purified, and restriction digest was done on both the vectors and the Chlologenate Esterase gene. The amounts of reactants used are as follows.

For each vector:

• 33 uL dIH2O
• 10 uL DNA
• 5 uL CutSmart
• 1 uL EcoR1
• 1 uL Pst1

For the Esterase gene:

• 28 uL dIH2O
• 15 uL DNA
• 5 uL CutSmart
• 1 uL EcoR1
• 1 uL Pst1

The Chlorogenate Esterase construct was ligated into each of the vectors, and the new A3+ChEst and C3+ChEst plasmids were transformed into E.coli. There were six transformations made, each using 50 uL of competent cells mixed with the specified amount of the specified vector, with the addition of a positive control and a negative control:

1. 3 uL A3
2. 5 uL A3
3. 3 uL C3
4. 5 uL C3
5. + control
6. - control

The cells were plated and allowed to grow overnight.

8/25/16

The Lipase gene fragment arrived by mail and was resuspended in 100 uL dIH2O for a final concentration of 10 ng/uL. Restriction digest was done on the Lipase gene before it was ligated with each vector and transformed into E.coli. The cells were plated and allowed to grow overnight.

8/27/16

All of the plates with the transformed cells were screened with colony PCR.

PCR Settings:

Cycle 1 (repeat 1 time)

• 98ºC - 30 sec

Cycle 2 (repeat 30 times)

• 98ºC - 10 sec
• 55ºC - 30 sec
• 72ºC - 40 sec

Cycle 3 (repeat 1 time)

• 72ºC - 5 min
• 4ºC - infinity

There were too many samples to be run on one gel, so two gels were used (a large one and a smaller one).

The results of the colony PCR were run on a gel. The samples were prepared as follows:

• 2 uL DNA
• 1 uL loading dye (7.5 uL of extra dye was accidentally added to tubes CL1 - CL2, and 7 uL extra dye was added to tubes CL3 - CL5)
• 7 uL dIH2O
• The tubes were mixed and spun down
• 10 uL of each sample and 5 uL of each ladder were loaded

Gel Key for 1st Gel

Lane	Sample
1	ladder
2	ACE 1
3	ACE 2
4	ACE 3
5	ACE 4
6	ACE 5
7	CCE 1
8	CCE 2
9	CCE 3
10	CCE 4
11	CCE 5
12	AL 1
13	AL 2
14	AL 3
15	AL 4

Gel Key for 2nd Gel

Lane	Sample
1	ladder
2	AL 5
3	CL 1
4	CL 2
5	CL 3
6	CL 4
7	CL 5

The gels indicated that only the AL 1 and CL 1 colonies contained the gene. The Chlorogenate Esterase gene was not present in any of the screened colonies, so the PCR had to be partially re-done.

9/01/16

The colony PCR for the Chlorogenate Esterase transformations was done over.

PCR Settings:

Cycle 1 (repeat 1 time)

• 98ºC - 30 sec

Cycle 2 (repeat 30 times)

• 98ºC - 10 sec
• 55ºC - 30 sec
• 72ºC - 40 sec

Cycle 3 (repeat 1 time)

• 72ºC - 5 min
• 4ºC - infinity

This gel also indicated that the cells did not have the Chlorogenate Esterase gene.

9/3/16

A restriction digest was done on the Chlorogenate Esterase gene fragment directly from the IDT tube. A gel was run on this digested Chlorogenate Esterase gene, as well as the two digested vectors. The samples were prepared as follows:

• 7 uL vector/insert
• 2 uL loading dye
• 1 uL dIH2O

Gel Key

Lane	Sample	Amount Loaded (uL)
1	ladder	3 uL
2	pSB1A3	10 uL
3	pSB1C3	10 uL
4	ChEst gene	10 uL

The gel results indicated that the restriction digests for both the insert and the vectors were successful.

The Chlorogenate Esterase gene was ligated with both vectors. The following ingredients were mixed in a clean microcentrifuge tube for each vector:

• 9 uL dIH2O
• 2 uL vector
• 6 uL insert
• 2 uL Ligase Buffer
• 1 uL T4 Ligase enzyme

The ligation was incubated in a PCR machine.

PCR Settings:

Cycle 1

• 20ºC - 1h
• 55ºC - 10 min
• 4ºC - infinity

9/08/16

The new Chlorogenate Esterase gene in both separate vectors was transformed into E.coli.

After realizing that our samples used in the verification gel for the first colony PCR were too concentrated to show results we decided to run the gel again, this time with the samples diluted. Forty samples were used: samples of every transformation from that first colony PCR, each one diluted to both 1/10 and 1/100. This gel also indicated that the PCR was not successful.

9/15/16