iGEM 2019 — Team Bio_Without_Borders

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# Team:Bio Without Borders/Notebook

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TEAM

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PROJECT

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[Design](https://2019.igem.org/Team%3ABio_Without_Borders/Design)
[Experiments](https://2019.igem.org/Team%3ABio_Without_Borders/Experiments)
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PARTS

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HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3ABio_Without_Borders/Human_Practices)
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BwoB iGEM 2019 · Benchling

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# BwoB iGEM 2019 Notebook

**Project:** 2019 iGEM

**Authors:** Ellen Jorgensen

Friday, 5/24/19

**Mentor**: Craig

**PI**: Ellen

**Administrator**: Chris

●

Introductions of members

●

Biobuilder: download Riley

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iGEM Academy on Youtube

○

Collection of videos: parodies of molecular biology

○

Helpful for a basis

●

Introduce iGEM competition description to BEAM center students

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Restriction Digest

○

2017: targeted psyllid (vector) so it’d have trouble carrying virus

○

Talking Biotech Podcast 185 by Dr. Steven Savage

■

Reach out about citrus virus -- use Tristeza virus

○

Tristeza: 1960s virus mutated inside citrus plant

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DNA Learning Center: educational videos of crystallography

●

Intro to pipette usage -- hands-on practice

Friday, 5/31/19

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Watched video on recombinant DNA (dnalc-mechanisms of recombination)

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Cut plasmids with restriction enzymes

Friday, 6/7/19

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Showed video to introduce restriction enzyme

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Displayed process for agarose gel for electrophoresis (1% gel = 0.5 TAE Solution)

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Demonstrated how to prep gel, pour it, and add ethidium bromide

○

Restriction digest products still in the fridge

Friday, 6/14/19

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Introduced Google Classroom as main communication platform

○

Google Hangout & Discord when unable to attend in person -- useful video broadcast over channels

○

Four interns in lab to mentor students

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Safety lab forms: for iGEM and Biotech Without Borders

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Review Restriction Digest: enzymes = Bozeman Science video

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Polymerase Chain Reaction (PCR)

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Walk through and introduced basic lab

Friday, 6/21/19

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Safety agreements & forms (1): If under 18, need signatures of parents

○

Classroom link with online version available

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*Deliverables* on iGEM ~ test: answer *questions*

○

Team wiki (see 2017 fancy team wiki), team poster, team presentation, judging form, project attributors, safety, registry part pages, project inspiration

●

iGEM account: igem.org → fill out personal info as prompted → login to account → membership code to team: 327718

○

Pending queue -- need verification

●

Keep up with Google Classroom account (3); Biotech Without Borders (4)
 forms

●

Video call with Dr. Georgios Vidalakis of the University of California, Riverside

○

1998-2005: FL neglected severity of HLB insect - disease spread and threatens the citrus industry; FL has lost half the industry; 2008: CA industries jeopardized

○

Improve RATE of detection of bacteria in leaves; create a quiz that allows kids across the country to help diagnose millions of leaves (virtual game); trap the psyllids with traps (acetic acid); infrared cameras track disease

○

Localized immunity issues? signals are systemic; surrogate bacteria in mango (cultures that can be sent over) ALSO can send over actual bacteria Diaphorina citri
 after arranging the permits; california suspecting peptides & concern with consumers; smaller virus RNA -> easier to work with the tristeza virus

○

<https://news.ucr.edu/articles/2019/04/01/plant-pathologist-leads-research-stop-spread-citrus-destroying-disease>

●

See Craig for head start with lab work → increases expertise & familiarity

○

Start with scheduling with students -- genetic engineering tasks

Friday, 6/28/19

●

Established that the actual bacteria *Candidatus Liberibacter asiaticus*
 would not be easily culturable

●

Considered periwinkle as a cultured plant & discussed the surrogate bacteria as usable: *Liberibacter crescens*

●

Reviewed restriction enzyme & bioengineering techniques with our team members

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Established that we will create a written protocol for new techniques for members away at Guatemala

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Introduced t-shirt design project & team project names

Monday, 7/8/19

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Practice lab techniques

○

Pipet

○

Ligation

○

Colony PCR

○

Gel Electrophoresis

○

Transformation

○

Restriction Digest

Tuesday, 7/9/19

●

PCR Polymerase Chain Reaction

○

Amplify a certain area of DNA

■

1. Break Apart Double Helix (Heat it up)- Denaturing

■

2. Primers sit on complementary base pair

■

3. Extend the areas after primers

●

1 X TAE contents:

○

C1V1=C2V2 (C is concentration, V is volume)

●

Gel Electrophoresis:

○

1. Pour out 50 mL 1 X TAE into flask

○

2. Add .35 g agarose

○

3. Cover Flask with Plastic Wrap

○

4. Put in microwave for 1 minute 15 seconds (check every 30 seconds)

○

5. Pour into gel mold and add 5 microliters of ethidium bromide

○

6. Put in combs and let sit for 30 minutes

Friday, 7/12/19

●

Remove syllids as they pick up RNAi + cloning project

●

Fluorescent protein genes picked out

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iGEM BioBrick

#####

Tuesday, 7/16/19

●

PCR Digestion (Restriction DIgest)

○

1 mL DNA

○

5 mL NEB Buffer 2.1

○

1 ml EcoR1 HF

○

1 mL Pst 1

○

50 mL MGBW

Thursday, 7/18/19

●

DIstribution Kit 2019

●

Transformation:

○

2 types of transformations: Electric Shock and Heat Shock

○

Wash cell in buffer solution to weaken the cell walls

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Mini Prep: (Mini Prep kit + Monarch)

○

Spin colony to get the pellet on the bottom. Pellet 1-5 mL

Monday, 7/22/19

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Overnight Prep For Mini Prep (Grow Colonies)

○

5000 microliters of LB

○

5 microliters Chlor

○

10 microliters colony PCR

■

1. Mix everything together

■

2. Parafilm the tube

■

3. Put in the Rotissirie

Thursday, 7/25/19

●

Transformation for Red Colonies

●

Plasmid RFP + AMP

●

DNA Distribution Kit

○

Linearized back bones, competent cell test kit, measurement kit

●

Reporter quantitizes how bright the colors are

●

Insert conatins ribosome, binding site

●

Bacterial Transformation: Method of foreign DNA being introduced into competent cells. Bacteria takes up the plasmids and expresses the specific gene in the DNA

●

Colony PCR:

○

Inside Thermocycler:

■

Denaturation- separation of double stranded DNA

■

Annealing- laying of primers on template DNA

■

Extension- polymerase synthesizes new strands of DNA using dNTP's

○

Check product by running a .7% gel

○

Combine colonies with LB broth and antibiotics

○

Rotate overnight

○

Mini prep

■

Method of extracting and purifying the plasmid DNA from bacterial cells. Results in large source of DNA that is pure and contains desired construction in a circulated form used for bacterial transformation
or PCR reaction

Tuesday, 7/30/19

●

Another transformation

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Overnight prep (we want more bacteria with the plasmid)

Wednesday, 7/31/19

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Transcription of DNA in bacteria

○

DNA to RNA to Protein

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Mini Prep (followed given instructions)

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Nanodropped the concentrations made

Thursday, 8/1/19

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Transformation (GFP + AMP) and Gel Electrophorises

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RNAi

○

Used to control genes

Monday, 8/12/19

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Transformed Ecoli cells with Gblock+Chlor plasmids

Tuesday, 8/13/19

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Looked at plates from transformation on 8/12

●

We had a few colonies that succesfully transformed

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Took colonies from each plate and suspended them in seperate tubes with 20 microliters of MBGW (template)

○

Tubes were labeled S1, S2, & S3, corresponding to plates that colonies were sourced from

●

Performed PCR with 1 micro templates from

○

12.5 microliters of Q5 Master Mix

○

1.5 microliters of VR Primer

○

1.5 microliters of VF2 Primer

○

1 microliter of the template

○

8.5 microliters of MBGW

●

Loaded test tubest with Colony PCR reagents into the Thermocycler

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Cast a 0.7% Agarose gel to perform Gel electrophoresis with products from our PCR

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Replenished 1x working stock of TAE from 50x stock

○

Performed C1V1 = C2V2

■

(50) V1 = (1) 1000 mL

■

V1 = 20

○

Added 20 mL of 50x TAE stock into 980 mL of dH2O to create 1000 mL of 1x TAE working stock

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Mixed PCR products with gel

●

Ran gel

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No bands

●

Was most likely due to the fact that we did not spin down the the PCR reagents in their tubes prior to loading into thermocycler

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Will Evaluate tonight

Wednesday, 8/14/19

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Performed mini preparation of plasmid DNA: GFP + C, RFP + A

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Repeatedly centrifuged and added wash buffers to DNA in order to isolate plasmid DNA from bacteria

○

Known as alkaline lysis

Thursday, 8/15/19

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Made and poured plates

○

Mixed 8.75 grams of agar with 250 milliters of distilled water

○

Put the mixture in a pressure cooker for 30 minutes

○

Let the mixture cool down for ten minutes

○

Add antibiotic to the mixture

○

Pour mixture onto plates

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Performed a transformation using Ecoli T7 Express cells

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Performed transformation on GFP + C made in mini prep on 8/14 to make sure we labled the tubes correctly

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Constitutive promoter: always displaying trait, inducible promoters, repressible promoters

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Initiation - Elongation - Termination (independent hairpin loop forms)

Monday, 8/19/19

●

We found out that while ligating out backbone folded onto itself. This prevented out insert from ligating with the backbone to form a full plasmid. To troubleshoot this we decided to use an enzyme called
"Antartic Phosphatase" to cut the ends of the backbone. This will dephophate the backbone and prevent it from folding onto itself.

●

Dephosphating Backbone - 2microliter buffer, 1 microliter ant(Antartic Phosphatase), 10 microliter backbone, 7 mucroliter mgbw

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Ligation- 2 microliter buffer, 1 microliter dephosphated DNA, 1 microliter ligase, 16 microliter insert

Tuesday, 8/20/19

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We are transforming out newly made DNA from 8/19. If our dephophatase worked then the cells should grow with a blue flourescent color.

○

Take out E-Coli(c2987) cells and defrost them from nitrogen.

○

Put 4 chlor pretri dishes in an incubator and make an ice bath.

○

Pipette 50uL of E-Coli and put them into tubeG-Block 1, G-Block 2, G-Block 3, and RFP control.

○

Pipette our designated plasmids onto each of our tube. 3uL for G-Block, 1uL for RFP.

○

Heat shock the tubes for 45secs at 42 ddegrees.

○

Take them out and put them in icebath for 10 min.

○

Add 950 uL of LB to the tubes.

○

Put parafilm on the tube and place in rotisserie for a hour.

Tuesday, 8/27/19

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Pasmid Prep (Mini Prep) for blue chromo protein

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Gel electrophoresis from the result of the plasmid prep

Tuesday, 9/3/19

Workflow for the day was to inoculate colonies from separate plasmid cassette transformation into overnight culture, that will be used to create individual plasmid preps for Wednesday 9/4.

●

First we counted all the colonies on the petre dishes, and got tubes for each colony (24).

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Next we placed the 3 millimeters of Lb +Chlor.

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Then we scraped each of the colonies off of the petre dishes using the loops and placed them into the tubes of Lb + Chlor.

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Then we used film to seal Oof the tubes and placed them into the shaker incubator to grow up overnight.

Wednesday, 9/4/19

Plasmid Prep

●

take out 24 large tubes of Lb + Chlor AK and SOB

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Pipette 2 microliter for each of the Lb + Chlor AK and SOB into the microcentrifuge tubes

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place them balanced and spaced eqally in centrifugation for 30 seconds

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Discard by pipetting liquid out and leave the cells at the bottom

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pipette 2 microliter of Lb + Chlor back inside the tube. Then repeat the process of placing in the centrifugation for 30 seconds

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Pipette 200 microliter of Plasmid Resuspension Buffer into the tube and slowly break or mix with the cells. (until the solid is gone)

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Add 200 microliter of Plasmid Lysis Buffer in the tube and shake it for 5 times. Set for 1 minute at a room temperature.

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Add 400 microliter of Plasmid Neutralization Buffer in the tube and shake it until the color completely changes. Set for 2 minutes at room temperatur

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Back to centrifugation (balanced and spaced equally) for 5 minutes.

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Transfer the supernatant to the plasmid column and place it into the centrifugation for 4 minutes.

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Transfer the column into a new tube and add 200 microliter of Plamid Wash Buffer 1. Then place it into the centrifugation for 1 minute.

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Add 400 microliter of Plasmid Wash Buffer 2 into the column and place it into the centriguation for 1 minute.

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Transfer the columns to a new microfuge tube and add 50 microliters of DNA Elution Buffer in the tube. Wait for 1 minute and place it into the centrifugation for 1 minute.

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Separate the columns. The leftover liquid is the DNA sample.

Thursday, 9/5/19

Plasmid Prep through Gel

●

Put the weigh paper on a gram scale. Then measure 0.35 g of agarose.

●

50mL of 1X TAE and put it into a erlenmeyer flask. Put the 0.35 g of agarose in the flask and slowly swirl / mix it.

●

cover the top of the elenmeyer flask with a clean wrap foil and poke the top.

●

Put it into a microwave for 30 seconds.

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Then slowly swirl it and put it in the microwave again for 30 seconds.

●

Then slowly swril it and put it in the microwave for 15 seconds. Cool it after it's done.

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Prepare the Gel electrophoresis / Gel box. Then pour the agarose liquid into the Gel box and cool it for a while.

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After cooling, put 5 microliter of Bromide in the gel liquid and swirl / mix it.

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Place the comb and make the wells. Wait for 30 minutes.

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Replace the Gel and put a black piece of paper under the gel box for better observations.

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pour the 1X TAE and fill the gel box until it covers the gel.

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prepare the parafilms, ladder, purple dye, and the AK and SOB DNA samples.

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Then pipette 6 microliter of ladder and slowly + gently place it in one of the wells.

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Then pipette 1 microliter of purple dye and place it on the parafilm.

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add 5 microliter of one of the DNA samples and mix it with the purple dye.

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Then pipette as 6 microliter of the mixed purple dye and DNA sample and place it to the next well of the ladder.

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\*repeat the process for each of the DNA samples.

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After filling the wells, carry it to the power source and set as 80V. Put the cover on top of the gel box and set it as 'run' for 65minutes.

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After 65 minutes, take the gel out and pour out the liquid.

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Put the gel in the UV transilluminator and turn the power on.

# Sequential Double Digest

## Introduction

This is the Sequential Double Digest Protocol with Standard Restriction Enzymes. If there is no buffer in which the two enzymes exhibit > 50% activity, this sequential digest can be performed. More information
from NEB can be found [here.](https://www.neb.com/tools-and-resources/usage-guidelines/double-digests) Double Digests can be designed using
[NEB's Double Digest Finder](https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder).
[NEBcloner](http://nebcloner.neb.com/#!/) will help guide your reaction buffer selection when setting up double digests. See the [NEBuffer Activity/Performance Chart with Restriction Enzymes](https://www.neb.com/tools-and-resources/usage-guidelines/nebuffer-performance-chart-with-restriction-enzymes) for the incubation temperatures.

## Materials

* DNA **1 µg**
* [NEBuffer](https://www.neb.com/tools-and-resources/usage-guidelines/nebuffer-performance-chart-with-restriction-enzymes)

  + 1X
* [NEB Restriction Enzymes](https://www.neb.com/products/restriction-endonucleases/restriction-endonucleases?&gclid=CLjO8djMx80CFYRrfgodvKsNkw)
* Deionized Water

## Procedure

* Sequential Double Digest

1. Set up the following reaction using the restriction endonuclease that has the lowest salt concentration in its recommended buffer (total reaction volume **50 µl**).

1. Set up the following digest reaction on ice

|  |  |
| --- | --- |
| **Component** | **Volume (µl)** |
| Buffer (10x) | 5 |
| DNA \* | Input Volume for ng |
| Restriction Enzyme #1 \*\* | 1 |
| Deionized Water (μl) | 1 |
| Total Volume (μl) |  |

|  |
| --- |
|  |
|  |
|  |
|  |
|  |
|  |
|  |

* \*A 50 µl reaction volume is recommended for digestion of 1 µg of substrate.

* \*\* Restriction Enzyme, 10 units is sufficient, generally **1 µl** is used

* \*\*\*The enzyme should be the last component added to reaction

1. Mix components by pipetting the reaction mixture up and down, or by "flicking" the reaction tube.

1. Quick ("touch") spin-down in a microcentrifuge. Do not vortex the reaction.

1. Incubate for 1 hour at the enzyme-specific appropriate temperature.

* Can be decreased to 5-15 minutes by using a [Time-Saver™ Qualified Restriction Enzyme](https://www.neb.com/products/restriction-endonucleases/hf-nicking-master-mix-time-saver-other/time-saver-qualified-restriction-enzymes/time-saver-qualified-restriction-enzymes)

* See the [NEBuffer Activity/Performance Chart with Restriction Enzymes](https://www.neb.com/tools-and-resources/usage-guidelines/nebuffer-performance-chart-with-restriction-enzymes) for the incubation temperatures.

1. Adjust the salt concentration of the reaction (using a small volume of a concentrated salt solution) to approximate the reaction conditions of the second restriction endonuclease.

* Alternatively, a spin column can be used to isolate the DNA prior to the second reaction.

1. Add the second enzyme.

* \*\* Restriction Enzyme #2, 10 units is sufficient, generally **1 µl** is used

1. Mix components by pipetting the reaction mixture up and down, or by "flicking" the reaction tube.

1. Quick ("touch") spin-down in a microcentrifuge. Do not vortex the reaction.

1. Incubate for 1 hour at the enzyme-specific appropriate temperature.

* Can be decreased to 5-15 minutes by using a [Time-Saver™ Qualified Restriction Enzyme](https://www.neb.com/products/restriction-endonucleases/hf-nicking-master-mix-time-saver-other/time-saver-qualified-restriction-enzymes/time-saver-qualified-restriction-enzymes)

* See the [NEBuffer Activity/Performance Chart with Restriction Enzymes](https://www.neb.com/tools-and-resources/usage-guidelines/nebuffer-performance-chart-with-restriction-enzymes) for the incubation temperatures.

# Ligation Protocol WITH T4 DNA Ligase (M0202)

## Introduction

*Please see the* [*NEB website*](https://www.neb.com/protocols/1/01/01/dna-ligation-with-t4-dna-ligase-m0202) *for supporting information on this protocol.*

## Materials

* [10X T4 DNA Ligase Reaction Buffer](https://www.neb.com/products/b0202-t4-dna-ligase-reaction-buffer)
* [T4 DNA Ligase](https://www.neb.com/products/m0202-t4-dna-ligase)
* Vector DNA (4kb)
* Insert DNA (1kb)
* Nuclease-free water

## Procedure

* Set up the T4 DNA Ligase Reaction

* *Note:* T4 DNA Ligase should be added last. The table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.  *Use* [*NEB calculator*](http://nebiocalculator.neb.com/#!/) *to calculate molar ratios.*

1. Thaw the T4 DNA Ligase Buffer and resuspend at room temperature.

* *Tip: Alicuote the 10x buffer less concentrated so when thawing, the DTT gets soluble more easily.*

1. Set up the following reaction in a microcentrifuge tube on ice:

|  |  |
| --- | --- |
| **Component** | **Volume (μl)** |
| 10X T4 DNA Ligase Buffer | 2 |
| Vector DNA: 50 ng (0.020 pmol) |  |
| Insert DNA: 37.5 ng (0.060 pmol) |  |
| Nuclease-free water | 17 |
| T4 DNA Ligase | 1 |
| Total | 20 |

|  |
| --- |
|  |
|  |
|  |
|  |
|  |
|  |
|  |
|  |

1. Gently mix the reaction by pipetting up and down and microfuge briefly.

1. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours.

1. Heat inactivate at 65 degrees C for 10 minutes.

1. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.

* Use 25 uL DH5α cells, and add 2 uL of reaction mixture.

# Transformation

## Introduction

A process in which we stress competent cells in a series of times heat shocks and ice baths. This allows the cells to take in the plasmids we want them to have.

## Materials

* 100 μL aliquots of competent cells
* ice block
* DNA ligation mix
* micropipettes and sterile pipette tips
* Glass beads
* control plasmid DNA
* water bath at 42 °C (or 37 °C)
* Luria Broth
* Incubator 37 °C with a rotisserie
* Luria Broth agar plates (with desired antibiotic)
* Heatblock

## Procedure

* Transformation Protocol

1. Obtain competent cells from a -80°C freezer and let it thaw on ice for 5 minutes .

1. After labeling each tube, use a sterile tip to add 5 μL of the ligation mix to the competent cells and leave them on ice for one hour.

1. Using a Heatblock for 45 sec. at 42 degrees C. (Remember to turn off the heatblock.)

1. Immediately after heat schocking the cells, place them back on ice for 5 minutes.

1. Using a sterile tip add 950 μL Luria broth to the cells and parafilm the tubes

1. Place the parafilmed tubes into a rotisserie for 1 hour.

1. Once the tubes are done incubating take off the parafilm.

1. Warm the Luria broth agar plates with the desired antibiotic resitstance at room temperature.

1. Label the plates with the desired product, name, and date.

1. Plate 100 μL of the transformed cells in to seperate plates.

1. Place 3 to 5 glass beads into the plate to spread the cells evenly.

1. Shake the plates in an up and down motion for 2 minutes.

1. Remove beads and dispose of them into a waste bin.

1. Parafilm the plates and place in the rotisserie at 37°C for 16 hours.


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# Team:Bio Without Borders/Experiments

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[HOME](https://2019.igem.org/Team%3ABio_Without_Borders)

TEAM

[Team Members](https://2019.igem.org/Team%3ABio_Without_Borders/Team)
[Collaborations](https://2019.igem.org/Team%3ABio_Without_Borders/Collaborations)

PROJECT

[Description](https://2019.igem.org/Team%3ABio_Without_Borders/Description)
[Design](https://2019.igem.org/Team%3ABio_Without_Borders/Design)
[Experiments](https://2019.igem.org/Team%3ABio_Without_Borders/Experiments)
[Notebook](https://2019.igem.org/Team%3ABio_Without_Borders/Notebook)
[Contribution](https://2019.igem.org/Team%3ABio_Without_Borders/Contribution)
[Results](https://2019.igem.org/Team%3ABio_Without_Borders/Results)

[Attributions](https://2019.igem.org/Team%3ABio_Without_Borders/Attributions)

PARTS

[Parts Overview](https://2019.igem.org/Team%3ABio_Without_Borders/Parts)

[SAFETY](https://2019.igem.org/Team%3ABio_Without_Borders/Safety)

HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3ABio_Without_Borders/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3ABio_Without_Borders/Public_Engagement)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Bio_Without_Borders)

# Experiments

We improved the empty cassette of TecCEM 2017 team to include BamHI and HincII sites and spacer DNA for the siRNA. We worked on cloning the empty cassette, BBa\_K3277000 with a linearized chloramphenicol backbone from the iGEM Distribution kit, and an ampicillin backbone from a J04450-pSB1A3 plasmid. The empty cassette is a vector for our siRNAs targeting the chitin synthase and superoxide dismutase genes. Then, we ligated the BBa\_3277000 + pSB1C3 plasmid with the oligos of the siRNAs. We also attempted to improve upon the WNT siRNA of the previous team.

Digestion of Linearized Backbones · Benchling

# Digestion of Linearized Backbones

## Introduction

The linearized backbones from the iGEM Distribution Kits have to digestion with EcoRI and PstI and demethylated with DpnI before ligating with the BBa\_K3277000 part.

## Materials

* BSA
* EcoRI-HF
* PstI
* DpnI
* dH2O or Nuclease Free Water
* Linearized backbone

## Procedure

* Enzyme Mastermix for Digestions and Demethylation

1. 5μL NEB Buffer 2

1. 0.5μL of BSA (or nuclease free water).

1. 0.5μL of EcoRI-HF

1. 0.5μL of PstI

1. 0.5μL of DpnI

1. up to 25μL with dH2O or nuclease free water

* Digest of Linearized Backbone

1. 4μL of linearized backbone (25ng/μL for 100ng)

1. 4μL of Enzyme mastermix

1. Digest 37℃ for 30 minutes and heat inactivate at 80℃ for 20 minutes.

Ligation with T4 DNA Ligase · Benchling

# Ligation with T4 DNA Ligase

## Introduction

This protocol ligates an insert fragment of DNA to a vector backbone. We ligated our BBa\_K3277000 g-bloxk with linearized pSB1C3 backbone and the pSB1A3 backbone from the J04450-pSB1A3 plasmid (in two separate reactions). The same procedure was followed to ligate the annealed siRNA oligos to the BBa\_K3277000 + pSB1C3 plasmid.

## Materials

* T4 Ligase
* T4 Ligase buffer
* Backbone DNA
* Insert DNA
* Nuclease Free Water
* Thermocycler
* Micropipetter
* PCR Tubes

## Procedure

* Ligation with T4 Ligase

1. Add 2μL of T4 ligase buffer to pcr tube.

1. Add 1μL of backbone DNA

1. Add 7μL insert DNA

1. Add 1μL T4 Ligase

1. Fill up to 20μL with nuclease free water.

1. Inucbate overnight at 16℃ or at room temperature (25℃) for 10 minutes

Restriction Digest using EcoR1 and Pst1 · Benchling

# Restriction Digest using EcoR1 and Pst1

## Introduction

A restriction digest allows a plasmid to be cut at specific sites. Using EcoR1 and Pst1, the insert can removed from the backbone and can be reinserted in another backbone or vice versa. In this case, the ampicllin backbone from J04450-pSB1A3 were cut with EcoRI-HF and PstI to serve as a vector for the BBa\_K3277000 g-block fragment.

## Materials

* EcoRI-HF
* PstI
* Template DNA
* NEB Buffer 3.1
* Nuclease Free Water
* Thermocycler
* PCR Tubes
* Micropipettor

## Procedure

* Double Digest with EcoRI-HF and PstI

1. Add 2μL of NEB Buffer 3.1 to PCR tube.

1. Add 1μL of EcoRI-HF

1. Add 1μL of PstI

1. Add 5μL of plasmid

1. Fill up to 20μL with nuclease free water

1. Digest at 37℃ for 60 minuts and heat inactivate at 80℃ for 20 minutes.

Restriction Digest with BamHI and HincII · Benchling

# Restriction Digest with BamHI and HincII

## Introduction

The ligated BBa\_K3277000 + pSB1C3 was cut with BamHI and HincII to insert the annealed siRNA oligos.

## Materials

* Mini-Prepped Plasmid
* BamHI-HF
* HincII
* CutSmart Buffer
* Nuclease Free Water
* PCR tubes
* Thermocycler
* Micropipetter

## Procedure

* Double Digest with BamHI-HF and HincII

1. Add 2μL of CutSmartbuffer to tube

1. Add 1μL of BamHI-HF

1. Add 1μL of HincII

1. Add 5μL of plasmid

1. Fill up to 20μL with nuclease free water

1. Digest at 37℃ for 60 minutes and then heat inactivate at 80℃ for 20 minutes.

Colony PCR (cPCR) with OneTaq 2X Mastermix · Benchling

# Colony PCR (cPCR) with OneTaq 2X Mastermix

## Introduction

Polymerase Chain Reactions on transformed bacterial colonies can be useful to determine if the desired insert DNA is present or absent in plasmid constructs.

## Materials

* OneTaq 2x Mastermix
* 10μM Forward primer
* 10μM Reverse primer
* Resuspended colony (to be used as DNA template)
* Nuclease Free Water
* Thermocycler
* Micropipetter
* PCR tubes

## Procedure

* PCR

1. Select a transformed colony from the plate and resuspend in 20μL of nuclease free water.

1. Add 9μL of nuclease free water to PCR tube.

1. Add 12.5 μL of OneTaq 2x Mastermix.

1. Add 0.5μL of forward primer.

1. Add 0.5μL of reverse primer.

1. Add 1μL of resuspended colony.

1. Place in thermocycler at standard temperatures and adjust accordingly to the primer and primer concentration.

|  |  |  |  |
| --- | --- | --- | --- |
| **Component** | **25μL reaction** | **50μL reaction** | **[Final]** |
| OneTaq 2x Mastermix | 12.5μL | 25μL | 1x |
| Forward primer | 0.5μL | 1μL | 0.2μM |
| Reverse primer | 0.5μL | 1μL | 0.2μM |
| Template DNA | variable | variable | <1000ng |
| Nuclease Free water | up to 25μL | up to 50μL | <1000ng |

|  |
| --- |
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PCR Protocol · Benchling

# PCR Protocol

## Introduction

Polymerase Chain Reaction is used to amplify sections of DNA using specific primers, allowing the production of many copies of that section.

## Materials

* Q5 High-Fidelity 2X Master Mix
* 10 µM Forawrd Primer
* 10 µM Reverse Primer
* Template DNA
* Nuclease Free Water
* PCR Tubes
* Thermocylcer
* Micropipetter

## Procedure

* PCR

1. Add 9 µL of water to PCR Tube

1. Add 12.5 µL of Master Mix to PCR tube

1. Add 2.5 µl of each Primer

1. Add 1 µL of DNA template

1. Place into thermocyler at standard temperatures with adjusted annealing and extension parameters. See table from NEB below.

|  |  |  |  |
| --- | --- | --- | --- |
| **Component** | **25 µl Reaction** | **50 µl Reaction** | **Final Concentration** |
| Q5 High-Fidelity 2X Master Mix | 12.5 µl | 25 µl | 1X |
| 10 µM Forward Primer | 1.25 µl | 2.5 µl | 0.5 µM |
| 10 µM Reverse Primer | 1.25 µl | 2.5 µl | 0.5 µM |
| Template DNA | variable | variable | < 1,000 ng |
| Nuclease-Free Water | to 25 µl | to 50 µl |  |

|  |
| --- |
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PCR Parameters

Annealing Oligos · Benchling

# Annealing Oligos

## Introduction

The siRNAs were ordered as separate oligos, the sense and anti-sense strands. These strands have to be annealed together.

## Materials

* Resuspended sense and anti-sense oligos
* Heatblock
* Eppendorf Tubes

## Procedure

* Annealing

1. Set the temperature on the heatblock to 98℃.

1. Place 10μL of each oligo into an eppendorf tube (total volume should be 20μL).

1. Put the Eppendorf tubes onto the heatblock and turn it off. Allow the temperature to decrease gradually to room temperature (25℃).

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# Team:Bio Without Borders/Results

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TEAM

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PROJECT

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# Results

Unfortunately we were unable to complete the construction of any plasmids for part submission. The only results we have are in our lab notebook page which documents the times we tried to get ligations to work. We have submitted results for characterizing an existing part on the page for [BBa\_K608008](http://parts.igem.org/Part%3ABBa_K608008). We tested this part against three other similar parts. The part that produced the greatest amount of GFP as measured by fluorescence intensity per cell was the construct with a medium promoter and strong RBS.

* [BBa\_K608008](http://parts.igem.org/Part%3ABBa_K608008) has a strong promoter and medium RBS
* [BBa\_K608010](http://parts.igem.org/Part%3ABBa_K608010) has a medium promoter and strong RBS
* [BBa\_K608011](http://parts.igem.org/Part%3ABBa_K608011) has a medium promoter and medium RBS
* [BBa\_K608012](http://parts.igem.org/Part%3ABBa_K608012) has a medium promoter and weak RBS

# Future plans

We will try alternate assembly methods to make our plasmids. We could have the entire BioBrick synthesized. We could use another method to assemble the plasmids such as Golden Gate Assembly. Once we have assembled the plasmids correctly we will test the siRNAs in collaboration with a lab that routinely handles psyllids, as described in our project description.