iGEM 2015 — Team BroadRun-NorthernVA

# Team:BroadRun-NorthernVA/Notebook

{{BroadRun-NorthernVA}}

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[* HOME](https://2015.igem.org/Team%3ABroadRun-NorthernVA)
* PROJECT
  [+ Problem](https://2015.igem.org/Team%3ABroadRun-NorthernVA/Problem)
  [+ Solution](https://2015.igem.org/Team%3ABroadRun-NorthernVA/Solution)
  [+ Methods](https://2015.igem.org/Team%3ABroadRun-NorthernVA/Methods)
  [+ Results](https://2015.igem.org/Team%3ABroadRun-NorthernVA/Results)
  [+ Applied Design](https://2015.igem.org/Team%3ABroadRun-NorthernVA/Design)
[* PARTS](https://2015.igem.org/Team%3ABroadRun-NorthernVA/Parts)

[* NOTEBOOK](https://2015.igem.org/Team%3ABroadRun-NorthernVA/Notebook)
[* SAFETY](https://2015.igem.org/Team%3ABroadRun-NorthernVA/Safety)
* HUMAN PRACTICES
  [+ Overview](https://2015.igem.org/Team%3ABroadRun-NorthernVA/Practices)
  [+ Education and Public Outreach](https://2015.igem.org/Team%3ABroadRun-NorthernVA/Education%20and%20Public%20Outreach)
  [+ Sustainability](https://2015.igem.org/Team%3ABroadRun-NorthernVA/Sustainability)
  [+ Further Applications](https://2015.igem.org/Team%3ABroadRun-NorthernVA/BasicResearch)
[* COLLABORATIONS](https://2015.igem.org/Team%3ABroadRun-NorthernVA/Collaborations)
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## Lab Notebook

Welcome to our Lab Notebook! Here, we have documented the work done in our project so we can see and keep track of how our project is progressing.

## **June**

**Week 1**
- Brainstormed ideas for projects. Decided on cells that could break down starch.
**Week 2-4**
- Researched the best way to go about this solution.
- Saccharomyces was decided as our organism, because of it ability to thrive in a variety of conditions, both aerobic and anaerobic.
- Used alpha amylase as our target gene to be cloned

## **July**

- More literature research and worked on developing protocols.
  Ordered materials for the project, enzymes, reagents, buffers, kits, agar plates, LB broth, etc.
- Experiment details were solidified and planned out in more detail.
- Ordered materials for the project, enzymes, reagents, buffers, kits, agar plates, LB broth, etc.

## **August**

**Week 1**

* Designed 3 amylase gene constructs to be synthesized through IDT’s offer. The final makeup of the gene constructs are listed below.

  Construct 1

  * Biobrick prefix
  * Promoterless
  * Kozak sequence (Part BBa\_K165002)
  * Native secretion sequence, from Bacillus amyloliquefaciens
  * Alpha amylase coding sequence from Bacillus amyloliquefaciens
  * ADH1 Terminator (Part BBa\_K392003)
  * Biobrick Suffix

  Construct 2

  * Biobrick prefix
  * Minimal cyc promoter (Part BBa\_K105027)
  * Kozak sequence (Part BBa\_K165002)
  * Native secretion sequence, from Bacillus amyloliquefaciens
  * Alpha amylase coding sequence from Bacillus amyloliquefaciens
  * ADH1 Terminator (Part BBa\_K392003)
  * Biobrick Suffix

  Construct 3

  * Biobrick prefix
  * Minimal cyc promoter (Part BBa\_K105027)
  * Kozak sequence (Part BBa\_K165002)
  * Native secretion sequence, from Bacillus amyloliquefaciens
  * Alpha amylase coding sequence from Bacillus amyloliquefaciens
  * ADH1 Terminator (Part BBa\_K392003)
  * Biobrick Suffix
  * No spacing was needed in between the composite parts, all constructs were optimized for S.cerevisiae, and an extra eight bases were added before the Ecor1 restriction site and after the pst1 restriction site, in order to increase the efficiency of the enzyme.
  * Designed primers for the three gene constructs, by hand using New England Biolabs Tm calculator. Primers were named p01, p02, p03, and p04.* p01- left primer for construct 1* p02 - right primer for construct 1* p03- left primer for construct 2 and 3* p01- right primer for construct 2 and 3

## Week 2

- The gene constructs and primers were first resuspended, according to protocol.- Primer resuspension:
    Amount of water added to reach 100uM concentration, water was added and pipetted up and down to resuspend.
    * p01: 293 µl of water* p02: 336 µl of water* p03: 269 µl of water* p04: 345 µl of water- 100uM concentration was then diluted to 10uM concentration.
      Gene construct resuspension: 100 µl of TE buffer was added to each gene construct and pipetted up and down and vortexed to resuspend.
      Tubes were then incubated at 30 degrees Celsius for 20 minutes.Then we amplified the DNA with the primers using PCR.
      **Amplification PCR**
      100 µl per reaction
      Reaction #1

      |  |  |  |  |  |
      | --- | --- | --- | --- | --- |
      | primer 01 primer 02 Gene construct #1 2x Master Mix water | | | | |
      | 5 5 2 50 38 | | | | |

      Reaction #2

      |  |  |  |  |  |
      | --- | --- | --- | --- | --- |
      | primer 03 primer 04 Gene construct #2 2x Master Mix water | | | | |
      | 5 5 2 50 38 | | | | |

      Reaction #3

      |  |  |  |  |  |
      | --- | --- | --- | --- | --- |
      | primer 03 primer 04 Gene construct #3 2x Master Mix water | | | | |
      | 5 5 2 50 38 | | | | |

      Negative Control #1

      |  |  |  |  |  |
      | --- | --- | --- | --- | --- |
      | primer 01 primer 02 Gene construct 2x Master Mix water | | | | |
      | 5 5 0 50 40 | | | | |

      Negative Control #2

      |  |  |  |  |  |
      | --- | --- | --- | --- | --- |
      | primer 03 primer 04 Gene construct 2x Master Mix water | | | | |
      | 5 5 0 50 40 | | | | |

      - Ran a gel to confirm size.
      Gel Electrophoresis* 10µl of each PCR reaction was added into ten different tubes and 2µl of loading dye added.* 11µl was then loaded into each of the wells and ran for 15 minutes.
        **Week 3**

 **PCR Purification**
- 30 µl of PCR product out of 100 µl was purified, using a Quiagen PCR purification kit.

 **Restriction Digest**
- Total reaction volume was 50 µl- Concentrations of plasmids and PCR products
  * pSB1c3 25ng/µl* pRS426 129ng/µl* pAG36 900ng/µl* PCR products 100ng/µl
        Restriction Digest of Plasmids

        |  |  |  |
        | --- | --- | --- |
        | pSB1c3 plasmid pAG36 yeast vector pRS426 yeast vector | | |
        | 4µl DNA 7.8 µl DNA 1.1 µl DNA | | |
        | 5 µl 10x NEB Cut Smart Buffer 5 µl 10x NEB Cut Smart Buffer 5 µl 10x NEB Cut Smart Buffer | | |
        | 1µl EcoR1 enzyme 1µl Kpn1 enzyme 1µl EcoR1 enzyme | | |
        | 1µl Pst1 enzyme 1µl Spe1 enzyme 1µl Pst1 enzyme | | |
        | 39 µl water 35.2 µl water 41.9 µl water | | |

        Restriction Digest of PCR Products

        |  |  |  |  |
        | --- | --- | --- | --- |
        | PCR product 1 (promoterless and native secretion sequence), cut with EcoR1 and Pst1 PCR product 2 (cyc promoter and native secretion sequence), cut with EcoR1 and Pst1 PCR product 3 (cyc promoter and mating factor alpha1 secretion sequence), cut with EcoR1 and Pst1 PCR product 3 (cyc promoter and mating factor alpha1 secretion sequence), cut with Kpn1 and Spe1 | | | |
        | 10 µl DNA 10µl DNA 10µl DNA 10 µl DNA | | | |
        | 5 µl 10x NEB Cut Smart Buffer 5 µl 10x NEB Cut Smart Buffer 5 µl 10x NEB Cut Smart Buffer | | |
        | 1µl EcoR1 enzyme 1µl EcoR1 enzyme 1µl EcoR1 enzyme 1µl Kpn1 enzyme | | | |
        | 1µl Pst1 enzyme 1µl Pst1 enzyme 1µl Pst1 enzyme 1µl Spe1 enzyme | | | |
        | 33 µl water 33 µl water 33 µl water 33 µl water | | | |- After ligation, we completed a bacterial transformation and plated cultures on agar plates.

  #### **Week 4**

  ###### The PCR mix components were combined in a large microcentrifuge tube with:

  - 100µl of 2x master mix
  - 16µl of VF2 primer
  - 13 µl of VR primer
  - 71 µl of sterile water
  - 20µl of the mix was added into each of nine PCR tubes.

  ###### Three colonies were chosen from each of the pSB1c3 ligations that had been transformed into the E.coli, labeled 1A,1B,1C, 2A, so forth. Each colony was mixed into the PCR mix and then streaked onto a master plate.

  - 10µl of the finished PCR product was mixed with 2µl of loading dye and loaded into a agarose gel.

    ###### The results confirmed that our insert size was correct

    ###### Next, we mini-prepped our liquid cultures using Quiaprep mini prep kit. There were eighteen cultures total, three colonies for each of the five transformations.

    After the mini prep, we needed to check the size of the DNA and the DNA had not been lost during the miniprep.
    To linearize the DNA, 5µl was added to 45 µl of a master mix.

    ###### Two master mixes were used, one with EcoR1 and one with Spe1. The ligations with the pSB1C3 plasmid and yeast vector pRS426 contain a single EcoR1 restriction site and the pAG36 yeast vector contains a Spe1 restriction site. Master mix with Ecor1 was made for 15 reactions and the Spe1 master mix for 5 reactions.

    Master mix:

    Ecor1 mix:

    - 75 µl 10x New England Bio Labs Cut Smart Buffer- 15 µl EcoR1 enzyme- 585 µl distilled water- Spe1 mix:- 25 µl 10x New England Bio Labs Cut Smart Buffer- 5 µl Spe1 enzyme- 195 µl distilled water

                  ###### DNA was incubated at 37° Celsius for one hour.

                  ###### 10 µl of loading dye was added, the solution was pipetted up and down to mix thoroughly.

                  ###### 20µl was loaded into each well in addition to a 1kb DNA ladder.

                  #### **Week 5**

                  ##### Master Mix 1:

                  |  |  |  |  |  |  |
                  | --- | --- | --- | --- | --- | --- |
                  | Primer 1 Primer 2 std taq dntps taq h2o | | | | | |
                  | 10 10 20 4 2 134 | | | | | |

                  ##### Master Mix 2:

                  |  |  |  |
                  | --- | --- | --- |
                  | Primer 2 Primer 3 std taq | | |
                  | 10 10 20 | | |

                  ###### colony pcr gel

                  - 10 fold dilution(2a 2b 3a 3b 4a 4b 5a 5b 6a 6b)- 100 fold dilution (2a 2b 3a 3b 4a 4b 5a 5b 6a 6b)- prs426 positive control- pag36 pc- psb1c3 nc

                            ## Project Timeline

                            **Week 1**

                            - Colony PCR on bacterial cultures- Made liquid cultures of positive colonies- Miniprepped liquid cultures

                                  - Transformed DNA samples into  *S.cerevisiae*

                                    **Week 2**

                                    - Tested genetically engineered cells for ability to produce amylase and hydrolyze starch- Cells were tested in known concentrations of soluble starch dissolved in water
                                        Iodine was used to determine starch concentrations- Then the yeast cells were tested in a sample of Armstrong's factory water

                                        **Week 3**

                                        - Cells were heat killed at 80 degrees Celsius to inactivate enzymes.- Ran through a gel to verify the fragments- Ligation of gene after successful gel.

                                              **Week 4**

                                              - Today we ran colony PCR to confirm the insert size. Our bacterial transformations went well, and we had both liquid cultures and agar plates with the colonies. The synthesized primers from IDT for the yeast vectors have not arrived, so colony PCR was done only on the three transformations with the pSB1c3 vector.

                                                **Week 5**

                                                - Colony PCR- Starch Analysis- psb13 plasmid results