Team:UCLA/Notebook/Spider Silk Genetics

	1x Reaction (uL)	2x Reaction (uL)
5x Q5 Buffer	5	10
10 mM dNTPs	0.5	1
10 uM For	1.25	2.5
10 uM Rev	1.25	2.5
Template (110 pg)	0.5	1
5x GC Buffer	5	10
Q5 Polymerase	0.25	0.5
ddH2O	11.25	22.5
Total	25	50

98 C	30 sec
98 C	10 sec
66 C	15 sec
72 C	15 sec
repeat from step 2	17x/15x
72 C	2 min
12 C	hold

∙ We used 66 C as annealing because that temperature worked for previous amplification with the post-elution primers. In addition, NEB Tm calculator indicates 66 C annealing for the VF2/VR primer pair.

2 Results

∙ Cast 2% TAE gel to visualize results. Used 2 uL of NEB 50 bp ladder.

Figure 1: P/E indicates reactions with post-elution primer pair. VF/R indicates reactions with VF2 and VR primers. The VF/R primers successfully amplify with a clean band. P/E does not appear to amplify. Primer dimers are present in all lanes. Imaged by Danny. Don’t know why the image is orange.

Figure 2: P/E indicates reactions with post-elution primer pair. VF/R indicates reactions with VF2 and VR primers. The VF/R primers successfully amplify with a clean band. P/E does not appear to amplify. Primer dimers are present in all lanes. This image taken several hours after previous image. Some band smearing is present.

∙ The VF/R primer pair amplifies our plasmid as expected, but the post-elution primers do not. The expected size for VF/R amplification is 443 bp.

∙ May be due to bad primers for post-elution. Re-order or remake? We need to find more of the post-elution primers.

5/10/2015 - 5/16/2015

5/13/2015 - NotI Concatemer Ligation

5/13/2015

1 NotI Concatemers

Used yesterday’s gel purified fragments. We performed ligation using a 5:1 ratio of insert to vector. Our vector size is 2046 bp. Our insert is 143 bp. For 1121 ng of vector, we need 391.75 ng of insert. We set up the ligation reaction as below:

	Amount (uL)
Vector (112.12 ng/uL)	10
Insert (52.63 ng/uL)	10
10X T4 Ligase Buffer	2.5
T4 Ligase	3
Total	25.5

We used excess insert because the recorded concentration from the nanodrop was higher than the theoretical yield. In addition, we used more T4 Ligase than in typical reactions due to the reaction having three times as many sticky ends to ligate.

2 Results

We cast a 0.8% TAE agarose gel to separate the bands. We ran this particular gel at 90 V for 2 hours. We excised four bands indicated for gel purification (Qiagen). The bottom most band may be due to vector self-ligation. The second band at 2.2 kb is probably singly ligated product.

Figure 1: Ligation of 2CA NotI digested vector and insert. We excised the 4 indicated bands for purification.

In the future, we should CIP treat the vector so it doesn’t re-ligate.

5/12/2015 - T4/T7 Ligation Tests continued

5/12/2015

Vinson Lam

1 Gel Extraction

Performed gel extraction (Qiagen kit) of bands excised from yesterday’s gel. Results below:

	Concentration (ng/uL)	A 260/280
2AB	41.5	4.47
2BC	54.3	3.96
2CA	17.6	3.21
2CA NotI ins.	52.63	3.70
2CA NotI vec.	112.12	2.50

2 2AB, 2BC, 2CA BsaI Ligation Test

Set up ligation tests for remaining conditions (see entry for 5/8/2015)for other tests. For each reaction, we used 50 ng of each DNA species.

2.1 T4 Ligase Reactions

	2AB + 2BC	2BC +2CA
10X T4 Ligase Buffer	1.5 uL	1.5 uL
2AB (6.82 ng/uL)	7.3 uL	0 uL
2BC (54.3 ng/uL)	1 uL	1 uL
2CA (17.6 ng/uL)	0 uL	2.84 uL
T4 Ligase	1 uL	1 uL
ddH2O	4.2 uL	8.66 uL
Total	15 uL	15 uL

Reactions were incubated at 25 C for 10 min.

2.2 T7 Ligase Reactions

	2CA + 2AB	2BC + 2CA
2X T7 Ligase Buffer	7.5 uL	7.5 uL
2AB	1.2 uL	0 uL
2BC	0 uL	1 uL
2CA	2.84 uL	2.84 uL
T7 Ligase	0.5 uL	0.5 uL
ddH2O	2.96 uL	3.16 uL
Total	15 uL	15 uL

Reactions were incubated at 25 C for 10 min.

3 Results

We cast a 2% TAE agarose gel to visualize the results. We used 2 uL of 50 bp ladder. See below.

Figure 1: T4 T7 Ligation Test

5/11/2015 - BsaI Digestion of MaSp Plasmids

5/11/2015

Vinson Lam

1 2AB, 2BC, 2CA Ligation Test

1.1 BsaI digestion of 2AB, 2BC, 2CA

Used 5 ug of each DNA.

	2AB (112 ng/uL)	2BC (205 ng/uL)	2CA (218.7 ng/uL)
BsaI	4 uL	4 uL	4 uL
Template	44.6 uL	24.39 uL	22.86 uL
Cutsmart	5 uL	5 uL	5 uL
ddH2O	0 uL	16.61 uL	18.14 uL
Total	53.6 uL	50 uL	50 uL

50 C	2 hr
65 C	20 min

We are conducting ligation tests between all possible combinations using T4 and T7 ligase

2 NotI Concatemers

Sri suggested using NotI to digest the plasmids and create a super plasmid that contains multiple copies of the monomers.
Testing NotI digestion today using 2CA with the protocol below.

	2CA (280 ng/uL)
NotI-HF	2 uL
DNA	7.2 uL
Cutsmart	5 uL
ddH2O	35.8 uL
Total	50 uL

37 C	1 hr
65 C	20 min

3 Results

Cast 200 mL of a 1.5% TAE agarose gel to visualize results of both BsaI and Not1 digestion.

Figure 1: Digestion Results: The 100 bp bands were excised as well as the second band in the NotI digestion.

5/3/2015 - 5/9/2015

5/8/2015 - T4/T7 Ligation Test

5/8/2015

1 T4/T7 Ligation Test

We tested the ability for the BsaI digested and gel purified monomers to ligate together. We ran these initial tests using both T4 and T7 DNA ligase using the following protocols (50 ng for each DNA species):

1.1 T4 Ligation

10x T4 Ligase Buffer	1.5 uL
2AB	4 uL
2CA	5 uL
T4 Ligase	1 uL
ddH2O	3.5 uL
Total	15 uL

25 C	10 min

1.2 T7 Ligation

2x T7 Ligase Buffer	7.5 uL
2AB	4 uL
2BC	5 uL
T7 Ligase	0.5 uL
ddH2O	0 uL
Total	17 uL

25 C	10 min

2 Results

We cast a 2% TAE gel to visualize the results. We use 2 uL of NEB 50 bp ladder. The gel was run at 150V for 35 min.

Figure 1: Preliminary ligation test with T4 and T7 polymerase. The band slightly larger than 200 bp is our desired ligation product.

Our ligation test indicates that the new sticky ends exhibit specific binding. In both reactions, the ligation was incomplete due to the presence of a band at 100 bp. This may be due to non-equal amounts of DNA.

We plan to conduct this same test next week using all possible combinations between the three monomers to verify specific binding. We plan to test both T4 and T7.

5/7/2015 - BsaI Digestion of MaSp plasmids

5/7/2015

1 BsaI Digestion of 2AB, 2BC, 2CA

∙ Gel purification of yesterday’s BsaI digested fragments (Qiagen).

∙ Obtained approx. 7 ng/uL for each monomer. A 260/280 for all three were around 1.8. Will use these as is for ligation test tomorrow.

5/6/2015 - DNA Maxiprep

5/6/2015

∙ Spun down yesterday’s cells in 50 mL tubes for 15 min @ 6000x g @ 4 C.

∙ Used Maxiprep (Qiagen) kit to prepare DNA. Eluted in 400 uL EB. Concentrations from each are listed below:

	Amount (ng/uL)
2AB	112
2BC	205
2CA	219

∙ The DNA amounts are low. We probably need to improve our technique.

5/5/2015 - DNA Miniprep

5/5/2015

∙ Miniprep (Zymo) yesterday’s overnight bacterial culture. We also created glycerol stocks from these cultures.

	Amount (ng/uL)
2AB	380
2BC	460
2CA	219

∙ Prepared 100 mL culture of 2AB, 2BC, 2CA for Maxiprep tomorrow.

5/4/2015 - BsaI Digestion Tes

5/4/2015

1 BsaI Digestion Test for 2AB

Tested digestion of 5 ug of plasmid to see efficiency of digestion.

	Amount (uL)
BsaI	2 uL
2AB (287 ng/uL)	14.7 uL
Cutsmart	5 uL
ddH2O	28.28
Total	50 uL

50 C	2 hr
65 C	20 min

Results are shown below:

Figure 1: Results of BsaI digestion test. The band at about 100 bp corresponds to the target digested insert.

∙ The digestion process works, so we’ll be proceeding with this protocol.

∙ We gel extracted the digested insert band. The end result was 11.9 ng/uL, A (260/280): 4.4

2 BsaI Digestion for Post-elution primer amplified MaSp

We BsaI digested the PCR products from previous PCRs using the following protocol

2.1 BsaI Digest Protocol

	2AB(61.43 ng/uL)	2BC*	CA (56.65 ng/uL)
BsaI	2 uL	2 uL	2 uL
DNA (500 ng)	8.14 uL	18.01 uL	8.83 uL
10x Cutsmart	5 uL	5 uL	5 uL
ddH2O	34.86 uL	25 uL	34.17 uL
Total	50 uL	50 uL	50 uL

50 C	2 hr
65 C	20 min

*For 2BC, we combined 8.84 uL of 33.9 ng/uL solution and 9.17 uL and 33.9 ng/uL solution to get 500 ng total.

2.2 Purification

∙ PCR purified the post-elution PCR digestion products and got the following yields:

	Concentration (ng/uL)	A (260/280)
2AB	3.74	1.22
2BC	9.81	2.54
2CA	11.38	2.33

Table 1: PCR purification of BsaI digested fragments generated through PCR using post-elution primers.

∙ Yield and absorbance for the digested inserts are not very good. This might be a problem due to the PCR process, even though product was generated.

3 Bacterial Propagation

∙ Grew 7 mL each of AB, BC, CA from glycerol stock for DNA extraction tomorrow.

4/26/2015 - 5/2/2015

5/1/2015 - Primer Exhaustion Calculation

5/1/2015

1 Calculation for Primer exhaustion

∙ Vector+insert = 2205 bp (150 bp insert) (2055 bp vector)

∙ DNA: 649 g/mole/bp

∙ AB: 143105 g/mole

∙ 500 pg / 1431045 g/mol = 3.494 E -16 moles DNA

∙ 1.25 uL of 10 uM primers = 1.25 E -11 moles primers

∙ 1.25 E -11 / 3.494 E -16 = 35775.6

∙ log2(35775.6) = 15.1 cycles (assuming perfect PCR)

2 Modified PCR using post-elution primers

Redo PCR using same amounts of reagents, but with 20 cycles instead of 30. Made 2x 50 uL for each MaSp plasmid.

4/30/2015 - Post-elution PCR amplification of MaSp plasmids

4/30/2015

1 Post-elution primer PCR of MaSp plasmids

∙ The previous anomalous results may be due to a human factor. So we decided to have Olivia prepare the PCR today using the same protocol, but scaled to 3x 25 uL each for 2AB and 2BC. Results shown below:

Figure 1: Post-elution primer PCR of MaSp plasmids. This result is very strange.

4/29/2015 - Post-elution PCR amplification of MaSp plasmids

4/29/2015

1 Post-Elution primer PCR of MaSp 2 plasmids

∙ Performed same PCR reaction as yesterday, but with 2x 50 uL for each reaction. This was prepared as a master mix. Results shown below.

Figure 1: Post-elution amplification of MaSp plasmids. Results are inconsistent even though a master mix was used.

∙ We excised the bands from 2AB, 2BC (only one), and 2CA.

4/28/2015 - Post-elution PCR amplification of MaSp plasmids

4/28/2015

1 Post-Elution Primer PCR Amplification of MaSp2 Sequences

We used the post-elution primers to test amplify 2AB plasmid and to determine an appropriate annealing temperature. We used the following protocol:

	1x Reaction (uL)	4x Reaction (uL)
5x Q5 Buffer	5	20
10 mM dNTPs	0.5	2
10 uM For (F-03)	1.25	5
10 uM Rev (G-03)	1.25	5
2AB (500 pg)	1.74	6.96
5x GC buffer	5	20
Q5 Pol	0.25	1 uL
ddH2O	10.01	40.04
Total	25	100

98 C	30 sec
98 C	10 sec
69, 66.8, 65.4, 63 C	15 sec
72 C	15 sec
Repeat from step 2	30 x
72 C	2 min
12 C	hold

2 Results

Figure 1: Annealing temperature test with MaSp 2AB at different temperatures. Our expected product is 170 bp.

∙ Base on these results, we decided to scale up to 3x 50 uL reactions for each monomer, and would use 66 C as the annealing temperature.

3 Amplification Again

We scaled up the above protocol for 3x 50 uL reactions for each monomer.

	AB (287 pg/uL)	BC (260 pg/uL)	CA (223 pg/uL)
5x Q5 Buffer	30 uL
10 mM dNTPs	3 uL
10 uM For (F-03)	7.5 uL
10 uM Rev (G-03)	7.5 uL
Template (500 pg)	5.22 uL	5.76 uL	6.73 uL
Q5 Polymerase	1.5 uL
5x GC enhancer	30 uL
ddH2O	65.28 uL	64.74 uL	63.77 uL
Total	150 uL

98 C	30 sec
98 C	10 sec
66 C	15 sec
72 C	15 sec
repeat from step 2	30x
72 C	2 min
12 C	hold

4 Results for second PCR

∙ Visualized on 2% gel. Used 2 uL 50 bp ladder.

Figure 2: Results from large scale amplification of MaSp using post-elution primers. The amplification is inconsistent.

4/27/2015 - Initial ICA Ligation Test

4/27/2015

1 2AB, 2BC, 2CA Sticky End Ligation Test

	AB+BC	BC+CA	CA+AB	AB+BC+CA
2x T7 Ligase Buffer	5 uL	5 uL	5 uL	5 uL
ddH2O	1.5 uL	1.5 uL	0.5 uL	0 uL
DNA 1	2 uL	1 uL	2 uL	2 uL
DNA 2	1 uL	2 uL	2 uL	1 uL
DNA 3	n/a	n/a	n/a	2 uL
T7 Ligase	0.5 uL	0.5 uL	0.5 ul	0.5 uL
Total	10 uL	10 uL	10 uL	10 uL

25 C	5 min
65 C

∙ The ligation test was run on 2% gel with 2 uL of 50 bp ladder. Results shown below.

Figure 1: T7 Ligation Test with MaSp2 fragments. BC was used as a negative ligation control. The presence of ladders may indicate non-specific binding.

4/19/2015 - 4/25/2015

4/24/2015 - Sending DNA for sequencing

4/24/2015

1 Sequencing of MaSp2 constructs

∙ Prepared each sample from yesterday for sequencing.

∙ Diluted 500 ng of each DNA species and 2.5 uL of VF2 primer into 15 uL in a PCR strip.

2 Results: 4/25/2015

∙ Used Clustal W2 to align sequencing results.

∙ Constructs 2AB-2, 2BC-1, and 2CA-2 are sequenced properly.

4/23/2015 - Miniprep DNA for sequencing

4/23/2015

1 Miniprep

∙ Prepared Glycerol stocks of each culture: 500 uL of bacteria + 500 uL 50% v/v glycerol.

∙ Used zymo miniprep kit to extract DNA from yesterday’s cells.

	Concentration (ng/uL)
2AB-1	640
2AB-2	287
2BC-1	261
2BC-2	275
2CA-1	469
2CA-2	223

4/22/2015 - Colony PCR

4/22/2015

1 Bacterial Transformation

∙ Observed many colonies growing on the plates. We will do colony PCR to verify proper insertion.

2 Colony PCR

∙ Picked four colonies from each plate. Resuspended each each colony in 100 uL water. Used protocol below:

	1x Reaction (uL)	12x Reaction (uL)
5x Q5 buffer	5	60
10 mM dNTPs	0.5	6
10 uM For (VF2)	1.25	15
10 uM Rev (post-elution G-03)	1.25	15
Colony (Resuspended)	1 uL	1 uL
5x GC Enhancer	5	60
Q5 polymerase	0.25	3
ddH2O	10.75	129
Total	25	300

98 C	3 min
98 C	10 sec
66 C	15 sec
72 C	15 sec
repeat from step 2	25 x
72 C	2 min
12 C	hold

3 Results

∙ Cast 1.0% gel to visualize.

Figure 1: Colony PCR to verify incorporation of insert.

∙ We picked colonies 3 and 4 from each construct and grew 5 mL liquid culture for miniprep and sequencing tomorrow. Renumbered the cultures to 1 and 2 respectively.

4 Column purification of MaSp2 PCA

∙ Used zymo kit to purify yesterday’s MaSp2 PCA reactions. Eluted in 10 uL EB. Results below:

	Concentration (ng/uL)
2AB	279.34
2BC	154.57
2CA	236.36

5 BsaI Digestion of PCA products from 4/21/2015

∙ Set up 2x 25 uL reactions for each:

	2AB(279 ng/uL)	2BC (155 ng/uL)	2CA (237 ng/uL)
DNA	2 uL ( 560 ng)	5 uL ( 770 ng)	2.5 uL ( 590 ng)
10x Cutsmart	2.5 uL:	2.5 uL	2.5 uL
ddH2O	18.5 uL	15.5 uL:	18 uL
BsaI	2 uL	2 uL	2 uL
Total	25 uL	25 uL	25 uL

50 C	2 hr
65 C	20 min

∙ The digestion products were column purified using zymo kit with the following yields:

	Concentration (ng/uL)	A 260/280
2AB	24.7	2.09
2BC	55.17	1.9
2CA	22.38	2.14

4/21/2015 - Ligation of MaSp sequences into pSB1C3 and Transformation

4/21/2015

1 Ligation of MaSp 2AB, 2BC, 2CA in to pSB1C3

∙ Used ligation calculator to determine amounts of each insert to use to ligate into 50 ng of vector for a 3:1 ratio of insert to vector.

∙ 2000 bp vector, 170 bp insert.

∙ Determined 12.75 ng of insert to 50 ng vector. Proceeded with ligation using protocol below:

	2AB (43.28 ng/uL)	2BC (34.56 ng/uL)	2CA (34.9 ng/uL)
T4 Ligase	1 uL	1 uL	1 uL
Insert (12.75 ng)	0.30 uL	0.370 uL	0.365 uL
Vector (50 ng)	1.484 uL	1.484 uL	1.484 uL
10x T4 Ligase Buffer	2 uL	2 uL	2 uL
ddH2O	15.22 uL	15.15 uL	15.16 uL
Total	20 uL	20 uL	20 uL

25 C	20 min
65 C	10 min

2 Bacterial Transformation

∙ Used 2 uL of each reaction to transform 50 uL competent cells.

∙ Incubate on ice 5 min.

∙ Add 200 uL of pre-warmed SOC to each tube.

∙ Rescue for 10 min.

∙ Plate 50 uL onto chloramphenicol plates. Incubate at 37 C overnight.

3 MaSp2 PCA

∙ Set up 2x 50 uL reactions for AB, BC, CA each using protocol from 4/13/2015 scaled to 50 uL with the following modifications:

∙ Annealing temp: 64 C

∙ 25 cycles instead of 20.

4/20/2015 - PCR amplification of pSB1C3 and EcoRI, PstI digestion

4/20/2015

1 PCR Amplification of pSB1C3

∙ Due to poor amplification of pSB1C3 from friday, we are doing a temperature test today.

∙ Followed same protocol as 4/17/2015, but scaled to 3x 25 uL reactions.

∙ Decided to take nanodrop reading of the template Julian provided. It was 230 ng/uL. Instead of using stock concentration of template, we will use a 1:100 dilution. Results shown below.

Figure 1: pSB1C3 amplification temperature test

∙ We gel extracted the bands together. 26.4 ng/uL, A 260/280: 1.56.

2 EcoRI and PstI Digestion of MaSp 2AB, 2BC, 2CA, and pSB1C3

∙ We pooled together all the linearized pSB1C3 that we had. Final concentration: 16.5 ng/uL.

∙ Set up digestion as below:

	Concentration (ng/uL)
2AB	37.58
2BC	42.8
2CA	79.11
pSB1C3	16.5

	2AB	2BC	2CA	pSB1C3
EcoRI	1 uL	1 uL	1 uL	1 uL
PstI	1 uL	1 uL	1 uL	1uL
10x NEbuffer 2.1	5 uL	5 uL	5 uL	5 uL
DNA (500 ng)	16 uL	11 uL	6 uL	19 uL
ddH2O	27 uL	32 uL	37 uL	24 uL
Total	50 uL	50 uL	50 uL	50 uL

37 C	1 hr
65 C	20 min
12 C	hold

∙ Column purified all DNA after digestion using Zymo kit. Eluted in 10 uL of EB. Results shown below:

	Concentration (ng/uL)	A 260/280
2AB	43.28	1.9
2BC	34.56	1.7
2CA	34.9	1.7
pSB1C3	33.7	1.61

4/12/2015 - 4/18/2015

4/17/2015 - Amplification of pSB1C3

4/17/2015

1 PCR amplification of pSB1C3

We set up a 50 uL reaction using the following protocol from Julian’s notebook.

	Volume (uL)
5x Q5 Buffer	10
10 mM dNTPs	1
10 uM For (SB-prep 2EA)	2.5
10 uM Rev (SB-prep 3P1)	2.5
Template (Spycatcher from Julian)	1
Q5 Polymerase	.0.5
ddH2O	32.5
Total	50

98 C	30 sec
98 C	10 sec
66 C	20 sec
72 C	30 sec
repeat from step 2	30 x
72 C	2 min
12 C	hold

2 Results

∙ Cast a 1% gel to visualize.

Figure 1: PCR amplification of pSB1C3

∙ Gel extraction (Qiagen) yielded 36.47 ng/uL, A 260/280: 1.91.

4/16/2015 - PCA of MaSp2 Pieces

4/16/2015

1 Polymerase Chain Assembly of MaSp 2AB sequence.

Repeated PCA of 2AB using protocol from 4/13/2015 with the follow modifications.

∙ Annealing Temp: 64 C

∙ 25 cycles instead of 20.

∙ We cast a 1.5% gel to visualize the results shown below.

Figure 1: PCA of MaSp 2AB. Our target product is at 170 bp.

2 Gel Extraction

Fasih gel extracted the two 2AB bands.

	Amount (ng/uL)	A 260/280
2AB-1	46.28	2.41
2AB-2	24.97	4.29

4/15/2015 - PCA of MaSp2 Pieces

4/15/2015

1 MaSp2 Polymerase Chain Assembly

Set up two 50 uL PCR reactions each for 2BC and 2CA using the same guidelines on 4/13/2015 with the following modifications:

∙ Annealing Temp: 64 C

∙ 25 cycles instead of 20

2 Gel Purification of MaSp2 Monomers

∙ Visualized with 1.5% agarose gel. Similar result to yesterday’s gel.

∙ Used gel purification kit (zymo) to extract DNA from 2AB, 2BC, and 2CA.

	Amount (ng/uL)	A 260/280
2AB	12.73	2.01
2BC	42.8	1.93
2CA	79.11	1.91

4/14/2015 - PCA of MaSp2 Pieces

4/14/2015

1 MaSp2 Polymerase Chain Assembly

Set up 2 50 uL reactions following the same protocol from yesterday scaled to 50 uL for 2AB. We included the following modifications:

∙ Annealing temperature: 64 C

∙ 25 cycles instead of 20

∙ Used primers 2A-F, 2B-R

2 Results

Cast 1.5% TAE agarose gel. Used 3 uL of NEB 50 bp ladder.

Figure 1: Each band corresponds to 25 uL reaction of MaSp2.

This protocol works better than the one previously. The target product at 170 bp is clearly more intense. While there is still some primer dimer formation, we will stick with this protocol.

4/13/2015 - PCA of MaSp2 Pieces

4/13/2015

1 Polymerase Chain Assembly

The primers corresponding to the new MaSp2 sequences with a valine added to the end came today. We conducted polymerase chain assembly to place the biobrick prefix/suffix and the BsaI restriction enzyme recognition sites with the designed sticky ends.

We used NEB’s Q5 PCR annealing temperature calculator to determine that an annealing temperature of 65 C was optimal. We also decided to test annealing at 61.4 C and 59 C. In addition, we planned to use the GC enhancer because our sequence is GC-rich. We prepared reactions as follows:

	1x (uL)	3x (uL)
5x Q5 Buffer	5 uL	15 uL
10 mM dNTPs	0.5 uL	1.5 uL
10 uM For	1.25 uL	3.75 uL
10 uM Rev	1.25 uL	3.75 uL
MaSp core (2.7 pg/uL)	3.7 uL	11.1 uL
5x GC enhancer	5 uL	15 uL
Q5 Polymerase	0.25 uL	0.75 uL
ddH2O	8.05 uL	24.15 uL
Total	25 uL	75 uL
98 C	30 s
98 C	10 s
65/61.4/59 C	15 s
72 C	15 s
Repeat from step 2	20 x
72 C	2 min
12 C	hold

2 Results

We cast a 1.5% TAE gel to visualize the results. We used 2 uL of NEB 50 bp ladder.

Figure 1: Annealing Temperature Test for Polymerase Chain Assembly of MaSp2. Our target product is 170 bp, and is located just under the 200 bp ladder band.

Our results indicate that 65 degrees is too high, and does not have good yield, but 61.4 is too low and has non-specific amplification as shown by the smears. The band at 50 bp is attributed to primer dimer formation.

Based on our results, tomorrow, we will perform polymerase chain assembly with annealing at 64 C and with 25 cycles.

Goals

Our goal is to be able to implement Iterative Capped Assmebly (ICA) for use in designing custom spider silk genes. The spider silk protein has a highly repetitive primary structure, and its properties can be changed by changing the sequence and identities of these repeats. We want to be able to fully control the primary sequence of our silk gene, and develop a quick, efficient method of customizing assembly of the spider-silk genes.

Achievements

We started testing ICA on 6/19/2015
We successfully constructed biobricks that contain our silk genes and the sticky overhangs. 4/24/2015
We proved that our designed sticky ends work as expected, and exhibit specific binding, showing that ICA can work in spider silk genes. 5/8/2015 and 5/12/2015
We have established a protocol generating fragments to be used in ICA.
We successfully characterized the use of ICA oligos with MaSp AB, BC, CA and showed that they could work in ICA! 6/3/2015
We successfully made a 3-mer using the ICA protocol on M-270 Streptavidin Beads! 7/7/2015
Find a list of our designed protocols here!

List of Parts

[http://parts.igem.org/Part:BBa_K1763002 MaSp2 2AB]
[http://parts.igem.org/Part:BBa_K1763003 MaSp2 2BC]
[http://parts.igem.org/Part:BBa_K1763004 MaSp2 2CA]

What to accomplish next

~~We are working on optimizing PCR amplification of MaSp and BsaI digestion so as to get the most amount of working monomers for ICA.~~--DONE! see here
~~We need to characterize our MaSp monomers and how they behave when used with the initiators, terminators, and capping oligos that are also used in ICA.~~--DONE! 6/3/2015
~~We are going to start actual ICA soon.~~--STARTED! see 6/19/2015
We are optimizing ICA to get it to work just right!
Our final goal is to make a several different 15-mers that have different rations of MaSp1 and MaSp2 that we can express and characterize.

Raw lab notebook entries

April
M	T	W	T	F	S	S
		1	2	3	4	5
6	7	8	9	10	11	12
13	14	15	16	17	18	19
20	21	22	23	24	25	26
27	28	29	30

May
M	T	W	T	F	S	S
				1	2	3
4	5	6	7	8	9	10
11	12	13	14	15	16	17
18	19	20	21	22	23	24
25	26	27	28	29	30	31

June
M	T	W	T	F	S	S
1	2	3	4	5	6	7
8	9	10	11	12	13	14
15	16	17	18	19	20	21
22	23	24	25	26	27	28
29	30

July
M	T	W	T	F	S	S
		1	2	3	4	5
6	7	8	9	10	11	12
13	14	15	16	17	18	19
20	21	22	23	24	25	26
27	28	29	30	31

August
M	T	W	T	F	S	S
					1	2
3	4	5	6	7	8	9
10	11	12	13	14	15	16
17	18	19	20	21	22	23
24	25	26	27	28	29	30
31

September
M	T	W	T	F	S	S
	1	2	3	4	5	6
7	8	9	10	11	12	13
14	15	16	17	18	19	20
21	22	23	24	25	26	27
28	29	30

May
M	T	W	T	F	S	S
				1	2	3
4	5	6	7	8	9	10
11	12	13	14	15	16	17
18	19	20	21	22	23	24
25	26	27	28	29	30	31

May
M	T	W	T	F	S	S
				1	2	3
4	5	6	7	8	9	10
11	12	13	14	15	16	17
18	19	20	21	22	23	24
25	26	27	28	29	30	31

May
M	T	W	T	F	S	S
				1	2	3
4	5	6	7	8	9	10
11	12	13	14	15	16	17
18	19	20	21	22	23	24
25	26	27	28	29	30	31