Team:UCLA/Notebook/Spider Silk Genetics

iGEM UCLA




Spider Silk Genetics Notebook

7/5/2015 - 7/11/2015
  • 7/6/2015 - Assembly of MaSp2 Sequencing Core, and ICA testing using M-270 Beads
    •
    6/28/2015 - 7/4/2015
  • 7/2/2015 - 6-mer Amplification using Post-elution Primers
    •
  • 7/1/2015 - 6-mer Ligation without beads
    •
    6/21/2015 - 6/27/2015
  • 6/26/2015 - ICA Troubleshooting with reduced initiator
    •
  • 6/25/2015 - ICA Troubleshooting to assess bead amplification
    •
  • 6/24/2015 - ICA Troubleshooting to assess bead binding: results
    •
  • 6/23/2015 - ICA Troubleshooting to assess bead binding
    •
    6/14/2015 - 6/20/2015
  • 6/19/2015 - Initial ICA Testing
    •
  • 6/18/2015 - BsaI Digestion of VF/R Amplified MaSp
    •
  • 6/17/2015 - PCR amplification of MaSp using VF2, VR
    •
    5/31/2015 - 6/6/2015
  • 6/5/2015 - ICA Oligos Characterization
    •
  • 6/3/2015 - ICA Oligos Characterization
    •
  • 6/2/2015 - Gel purification of BsaI digested MaSp
    •
  • 6/1/2015 - Preparing to test Initiator, Terminator, and Capping Oligos: PCR amplification and BsaI Digestion
    •
    5/24/2015 - 5/30/2015
  • 5/29/2015 - T7 Ligation Test for VF/R amplified MaSp
    •
  • 5/28/2015 - BsaI Digestion of VF/R amplified MaSp
    •
  • 5/27/2015 - Temperature Test for VF/R amplification of MaSp, Large-scale amplification of MaSp 2AB, 2BC, 2CA
    •
  • 5/26/2015 - BsaI Digestion of VF/R amplified MaSp
    •
    5/17/2015 - 5/23/2015
  • 5/22/2015 - PCR amplification of MaSp using VF2, VR
    •
  • 5/21/2015 - PCR amplification of MaSp using VF2, VR Cycle Testing
    •
  • 5/20/2015 - PCR amplification of MaSp using VF2, VR
    •
    5/19/2015 - PCR amplification test for MaSp plasmid 5/19/2015

    5/19/2015


    1 PCR Amplification of MaSp Plasmids

    ∙ Set up four 25 uL PCR reactions to test amplification. Used two pairs of primers and two different cycling conditions.

    ∙ Used MaSp2 2CA as template. Diluted 1:1000 to get 219 pg/uL. Created 2 master-mixes, one with each primer pair.

    ∙ Tested VF2/VR primers and post-elution primers using 17 cycles and 15 cycles using protocol below:

    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 217x/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.

    PIC

    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.

    PIC

    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

    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.

    PIC

    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

    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 + 2BC2BC +2CA
    10X T4 Ligase Buffer1.5 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 + 2AB2BC + 2CA
    2X T7 Ligase Buffer7.5 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.

    PIC

    Figure 1: T4 T7 Ligation Test

    5/11/2015 - BsaI Digestion of MaSp Plasmids 5/11/2015

    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
    Template44.6 uL 24.39 uL 22.86 uL
    Cutsmart5 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
    Cutsmart5 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.

    PIC

    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

    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 Buffer1.5 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 Buffer7.5 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.

    PIC

    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

    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

    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)
    2AB112
    2BC205
    2CA219

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

    5/5/2015 - DNA Miniprep 5/5/2015

    5/5/2015


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

    Amount (ng/uL)
    2AB380
    2BC460
    2CA219

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

    5/4/2015 - BsaI Digestion Tes 5/4/2015

    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:

    PIC

    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 uL8.83 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)
    2AB3.74 1.22
    2BC9.81 2.54
    2CA11.38 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

    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

    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:

    PIC

    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

    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.

    PIC

    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

    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 C15 sec
    72 C 15 sec
    Repeat from step 230 x
    72 C 2 min
    12 C hold

    2 Results

    PIC

    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 230x
    72 C 2 min
    12 C hold

    4 Results for second PCR

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

    PIC

    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

    4/27/2015


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

    AB+BCBC+CACA+ABAB+BC+CA
    2x T7 Ligase Buffer5 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 C5 min
    65 C

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

    PIC

    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

    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

    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-1640
    2AB-2287
    2BC-1261
    2BC-2275
    2CA-1469
    2CA-2223
    4/22/2015 - Colony PCR 4/22/2015

    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 225 x
    72 C 2 min
    12 C hold

    3 Results

    ∙ Cast 1.0% gel to visualize.

    PIC

    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)
    2AB279.34
    2BC154.57
    2CA236.36

    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 Cutsmart2.5 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 C2 hr
    65 C 20 min

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

    Concentration (ng/uL)A 260/280
    2AB24.7 2.09
    2BC55.17 1.9
    2CA22.38 2.14
    4/21/2015 - Ligation of MaSp sequences into pSB1C3 and Transformation 4/21/2015

    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 Buffer2 uL 2 uL 2 uL
    ddH2O 15.22 uL 15.15 uL 15.16 uL
    Total 20 uL 20 uL 20 uL

    25 C20 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

    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.

    PIC

    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
    pSB1C316.5

    2AB 2BC 2CA pSB1C3
    EcoRI 1 uL 1 uL 1 uL 1 uL
    PstI 1 uL 1 uL 1 uL 1uL
    10x NEbuffer 2.15 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 uL50 uL50 uL50 uL

    37 C1 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
    pSB1C333.7 1.61
    4/12/2015 - 4/18/2015
    4/17/2015 - Amplification of pSB1C3 4/17/2015

    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 230 x
    72 C 2 min
    12 C hold

    2 Results

    ∙ Cast a 1% gel to visualize.

    PIC

    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

    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.

    PIC

    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-146.28 2.41
    2AB-224.97 4.29
    4/15/2015 - PCA of MaSp2 Pieces 4/15/2015

    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
    2AB12.73 2.01
    2BC42.8 1.93
    2CA79.11 1.91
    4/14/2015 - PCA of MaSp2 Pieces 4/14/2015

    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.

    PIC

    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

    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.

    PIC

    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.

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    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
    • 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 troubleshooting ICA to get it to work just right!

    Raw lab notebook entries

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