iGEM 2017 — Team Austin_UTexas_LASA

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# Team:Austin UTexas LASA/Notebook

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* **Other Aspects**
  + [Contributions](https://2017.igem.org/Team%3AAustin_UTexas_LASA/Contribution)
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* **Team & iGEM**
  + [Team](https://2017.igem.org/Team%3AAustin_UTexas_LASA/Team)
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  + [Medal Criteria](https://2017.igem.org/Team%3AAustin_UTexas_LASA/Medals)

# Notebook

* [Sep](#tab1default)
* [Oct](#tab2default)
* [Nov](#tab3default)
* [Dec](#tab4default)
* [Jan](#tab5default)
* [Feb](#tab6default)
* [March](#tab7default)
* [April](#tab8default)
* [May](#tab9default)
* [June](#tab10default)
* [July](#tab11default)
* [Aug](#tab12default)
* [Sep](#tab13default)
* [Oct](#tab14default)

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### Sept 1 - Sept 9, 2016

Week 1: Got aliquots of HpaBC from lab, grew up parts (Hpab, HpaC, pp2551) and digested them to check lengths.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### Oct 3- Oct 7, 2016

Week 1: Did PCR to get HpaB out of the HpaBC lab plasmid, sequence verified ppddc in BTK001 backbone, sequence verified pp2551 and HpaC, made glycerol stocks of sequence verified parts

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### Oct 10 - Oct 14, 2016

Week 2: Ordered HpaBC operon from IDT, grew up pEM7 [BTK156], did Gibson assembly of HpaBC in YTK001 entry vector and grew it up

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### Oct 17 - Oct 21, 2016

Week 3: Split parts into different teams, each team working on assembling and sequence verifying individual parts into entry vecotrs[ppddc, HpaBC pEM7, pp2551]

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### Oct 24 - Oct 28, 2016

Week 4: Grew up and miniprepped HpaBC, and pp2551, but transformation of HpaBC did not work.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### Nov 1- Nov 3, 2016

Week 1: Did Gibson assembly, transformed and miniprepped ppddc, pEM7, and HpaBC, found that Gibson assembly for HpaBC did not work

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### Nov 6 - Nov 10, 2016

Week 2: Redid Gibson assembly for HpaBC, realized we were cloning our parts into YTK001 [plasmid vector]

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### Nov 13 - Nov 17, 2016

Week 3: Transformed, miniprepped, made glycerol stocks of HpaBC. Did a gibson assembly of ppddc.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### Nov 20 - Nov 24, 2016

Week 4: Transformed pp2551, transformed Gibson product of pEM7, transformed and miniprepped ppddc gibson assembly.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### Dec 1- Dec 8, 2016

Week 1: pp2551 plates had no colonies, streaked with glycerol stocks of HpaBC, redid transformation of pp2551

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### Dec 11 - Dec 15, 2016

Week 2: Transformation of HpaBC, ppddc plates had no colonies, redid Gibson assembly for ppddc

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### Dec 18 - Dec 22, 2016

Week 3: miniprepped and made glycerol stocks of HpaBC, got very low concentrations for the minipreps, looked into origin of the backbone (BTK001). Transformed gibson reaction of ppddc, plate had no colonies.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### Dec 25 - Dec 29, 2016

Week 4: Digested ppddc and backbone (BTK001) to see if there was a problem in the ori, ran on a gel, showed no insert.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### Jan 1- Jan 5, 2017

Week 1: Used Zymo DNA clean up kit to get more concentrated values to send in for sequencing. Established plan for how to proceed with each part.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### Jan 8 - Jan 12, 2017

Week 2: Miniprepped pp2551. Designed primers for ppddc pcr to add golden gate overhangs.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### Jan 15 - Jan 19, 2017

Week 3: Sent in HpaBC and pp2551 to sequencing. Started ‘lab trainings’ with highschool students new to the club.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### Jan 22 - Jan 26, 2017

Week 4: Received primers for ppddc, and diluted them.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### Feb 1- Feb 9, 2017

Week 1: Ran a PCR reaction of ppddc to add golden gate overhangs.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### Feb 12 - Feb 16, 2017

Week 2: Received and grew up an aliquot of new backbone (YTK001) with ColE origin because the original backbone was giving us trouble with getting high enough concentrations to sequence verify it.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### Feb 19 - Feb 23, 2017

Week 3: Miniprepped new backbone (YTK001). Did a PCR clean-up of ppddc.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### Feb 26 - Feb 28, 2017

Week 4: Re-ran PCR reaction and purified ppddc because we were running low.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### March 1- March 10, 2017

Week 1: Ran a gel electrophoresis on ppddc and HpaBC. Golden gate reaction of ppddc and ytk001, then purified it. Transformed ppddc + ytk001, both green and white colonies (transformation successful).

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### March 13 - March 17, 2017

Week 2: Miniprepped ppddc + ytk001. Transformed pp2551 from January. Grew up pp2551 for glycerol stocks and Hpabc for miniprep to do a golden gate reaction with ytk001.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### March 20 - March 24, 2017

Week 3: Miniprepped HpaBC and nanodropped, but concentrations too low to do a golden gate assembly. Re-did liquid cultures then miniprep of HpaBC.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### March 27 - March 31, 2017

Week 4: Figured out/planned how to combine the two separate sensing plasmids (venus cassette and pp2551 cassette). Decided to do multigene assembly using a new backbone. Looked into addition of lac operon in the sensing assembly.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### April 1- April 7, 2017

Week 1: n/a, people out for school.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### April 10 - April 14, 2017

Week 2: Did a BsaI golden gate reaction for venus cassette plasmid (VCP). Used a backbone (BTK402) with a pMMB67EH origin, ppddc promoter, rpoc terminator, and ConL1 and ConRE connectors.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### April 17 - April 21, 2017

Week 3: Dilutions of each part of the venus cassette plasmid. Grew them up to have enough of our own stock.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### April 24 - April 31, 2017

Week 4: Did a BsaI golden gate reaction for production assembly (HCP) with a CP25 promoter, HpaBC, rpoc terminator, conLS and conRE connectors.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### May 1- May 5, 2017

Week 1: Miniprepped each of the cassette plasmid parts. Transformed VCP golden gate reaction. Did a \_\_\_golden gate reaction for pp2551 cassette plamisd (PCP).

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### May 8 - May 12, 2017

Week 2: Made glycerol stocks of VCP. Transformed HCP, PCP, and VCP. No growth on plates. Looked into why golden gate didn’t work. Found out the low-copy origin is not efficient, and z-comp cells we used for transformation are less efficient.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### May 22 - May 26, 2017

Week 3: Redid golden gate reaction of HCP, PCP, and VCP using EcoRI enzyme. When we spun down liquid cultures, the cells were all red. Re-picked colonies (white) from the HCP and VCP plate because PCP plate only had red colonies. Did a miniprep of HCP and VCP.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### May 29 - May 31, 2017

Week 4: Digested and ran gel electrophoresis of HCP and VCP. No insert seen.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### June 1- June 9, 2017

Week 1: Out for Vacation

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### June 12 - June 16, 2017

Week 2: Out for Vacation

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### June 19 - June 23, 2017

Week 3: Tried golden gate reaction of pp2551, negative results. Ran production assembly, gel looked good. Decided to focus on sensing assembly.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### June 26 - June 30, 2017

Week 4: Out for Vacation

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### July 1- July 7, 2017

Week 1: Tried digesting all parts necessary for golden gate reaction of pp2551’s cassette plasmid, digest did not run properly. Received evpp2551 from Simon (backup), PCRed to add golden gate overhangs.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### July 10 - July 14, 2017

Week 2: Went back to retrying golden gate reaction of pp2551 after eliminating several possible places of error, didn’t work. Put evpp2551 into a golden gate vector.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### July 17 - July 21, 2017

Week 3: Digested all parts necessary for golden gate reaction of pp2551, several oddities with smaller pieces. Decided to move on to possibilities of gibson assembly of sensing assembly. Started working on Human Practices and Wiki in earnest.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### July 24 - July 31, 2017

Week 4: Designed primers for overhangs of CP25, pp2551, and Venus for gibson assembly. Retried a golden gate reaction to assemble the pp2551 cassette plasmid, grew up, but did not seem to have any colonies with the correct insert (all colonies were red instead of white from the rfp dropout). Looked into a new backbone to clone the cassette assemblies into.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### July 1- July 7, 2017

Week 1: Tried digesting all parts necessary for golden gate reaction of pp2551’s cassette plasmid, digest did not run properly. Received evpp2551 from Simon (backup), PCRed to add golden gate overhangs.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### July 10 - July 14, 2017

Week 2: Went back to retrying golden gate reaction of pp2551 after eliminating several possible places of error, didn’t work. Put evpp2551 into a golden gate vector.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### July 17 - July 21, 2017

Week 3: Digested all parts necessary for golden gate reaction of pp2551, several oddities with smaller pieces. Decided to move on to possibilities of gibson assembly of sensing assembly. Started working on Human Practices and Wiki in earnest.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### July 24 - July 31, 2017

Week 4: Designed primers for overhangs of CP25, pp2551, and Venus for gibson assembly. Retried a golden gate reaction to assemble the pp2551 cassette plasmid, grew up, but did not seem to have any colonies with the correct insert (all colonies were red instead of white from the rfp dropout). Looked into a new backbone to clone the cassette assemblies into.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### July 1- July 7, 2017

Week 1: Tried digesting all parts necessary for golden gate reaction of pp2551’s cassette plasmid, digest did not run properly. Received evpp2551 from Simon (backup), PCRed to add golden gate overhangs.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### July 10 - July 14, 2017

Week 2: Went back to retrying golden gate reaction of pp2551 after eliminating several possible places of error, didn’t work. Put evpp2551 into a golden gate vector.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### July 17 - July 21, 2017

Week 3: Digested all parts necessary for golden gate reaction of pp2551, several oddities with smaller pieces. Decided to move on to possibilities of gibson assembly of sensing assembly. Started working on Human Practices and Wiki in earnest.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### July 24 - July 31, 2017

Week 4: Designed primers for overhangs of CP25, pp2551, and Venus for gibson assembly. Retried a golden gate reaction to assemble the pp2551 cassette plasmid, grew up, but did not seem to have any colonies with the correct insert (all colonies were red instead of white from the rfp dropout). Looked into a new backbone to clone the cassette assemblies into.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### August 1- August 4, 2017

Week 1: Ordered primers for sequence verification and gibson assembly of cassette plasmid parts. Redid PCR reactions of parts (CP25, evpp2551, venus, backbone) to add gibson overhangs). Decided to try using a new backbone with lacI gene. Digested and ran gels to check lengths of PCRed parts. All parts looked fine. Got in contact with Power for Parkinson’s

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### August 7 - August 11, 2017

Week 2: Did golden gate reactions for backbone (ColE + lacI). Ran a gibson reaction of parts (CP25, evpp2551, venus, backbone). Started looking into testing the promoter pddc background. Visited and helped out at Power for Parkinson’s (Public Engagement)

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### August 21 - August 18, 2017

Week 3: Sent in the gibsoned multigene assembly and backbone (btk524) for sequencing. Transformed Venus cassette plasmid into BL21 DE3s. Sequencing did not work.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### August 28 - August 31, 2017

Week 4: Digested the sensing multigene assembly.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### September 1- September 8, 2017

Week 1: Ran the digests on a gel. Restreaked and grew up plates of Venus cassette plasmid. Ran a pre-experiment with Venus Casette plasmid with a plate reader.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### September 11 - September 15, 2017

Week 2: Sent sensing multigene assembly for sequencing again using PCR primers.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### September 18 - September 22, 2017

Week 3: Could not assemble the backbone with a ColE origin. Decided to stick with a p15A origin and LacI.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### September 25 - September 29, 2017

Week 4: Digested and ran Venus cassette plasmid and evpp2551 cassette plasmid. Extracted and purified gel. Looked into testing plasmids of another sensing plasmid from the lab that has a similar construct.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### October 1- October 6, 2017

Week 1: Conducted experiment on the already made sensing mulitigene assembly from the lab. Did a golden gate assembly of the evpp2551 cassette plasmid.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### October 9 - October 13, 2017

Week 2: Sent evpp2551 cassette plasmid for sequencing. After sequence verifying, moved onto a multigene assembly.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### October 16 - October 20, 2017

Week 3: Reached out to igem mentor for help. Ordered designed primers for Venus cassette plasmid and evpp2551 cassette plasmid. Did two step digest for backbone (pET28), venus cassette, evpp2551 cassette. Gel extracted them and ligated. Grew up and prepared liquid cultures for part characterization. Acquired several different promoters along with the biobrick promoter to characterize.

![](https://static.igem.org/mediawiki/2017/d/d6/T--Austin_UTexas_LASA--Month.jpg)

#### October 23 - October 31, 2017

Week 4: Did experiments for part characterization and sensing plasmid.

## Task Division

Lab: Ashley, Anna, Eli, Anusha, Anika, Shreya, Sumin, Shruti, David, Christian

Wiki: Anika, Shreya, Ashley, Anna

Human Practices: David, Shruti, Anna

Public Relations: Sumin, Christian

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# Team:Austin UTexas LASA/Experiments

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* **Other Aspects**
  + [Contributions](https://2017.igem.org/Team%3AAustin_UTexas_LASA/Contribution)
  + [Collaborations](https://2017.igem.org/Team%3AAustin_UTexas_LASA/Collaborations)
  + [Public Engagement](https://2017.igem.org/Team%3AAustin_UTexas_LASA/Engagement)
* **Team & iGEM**
  + [Team](https://2017.igem.org/Team%3AAustin_UTexas_LASA/Team)
  + [Attributions](https://2017.igem.org/Team%3AAustin_UTexas_LASA/Attributions)
  + [Medal Criteria](https://2017.igem.org/Team%3AAustin_UTexas_LASA/Medals)

# Experiments

## **Protocols**

#### [PCR (Polymerase Chain Reaction)](#collapseOne)

## Reactants

| 10x Reaction Mix | 5 uL |
| --- | --- |
| For primer (10 uM) | 1 uL |
| Rev primer (10 uM) | 1 uL |
| Template | 1 uL |
| Taq Polymerase | 1 uL |
| MgCl2 (25 mM) | 1 uL |
| DMSO | 1 uL |
| NF H2o | 41 uL |
| **Total** | **50 uL** |

###### Protocol

1. On ice, add all reagents to PCR tube, Taq polymerase last.
2. Run thermocycler program:

* 95 C for 5 min (2 min if NOT using Hotstart)

> 10 cycles:
95 C for 20 s
0.3 C/s to 50 C
72 C for # s (extension = 1 min/kb)

> 15 cycles:

95 C for 20 s

55 C for 20 s

72 C for 1 min

72 C for 10 min

4 C forever

#### [PCR Clean Up](#collapseTwo)

Reagents and protocol from Zymo DNA Clean & Concentrator Kit

**Reagents**

| DNA Binding Buffer | Refer to table below. |
| --- | --- |
| DNA Wash Buffer | Refer to table below. |
| NF H2O | Volume dependent on objective. |

**Protocol:**all centrifugation steps should be performed between 10,000 - 16,000g.

1. On ice, add DNA Binding Buffer (2-7 volumes) and the sample (see table below) in a microcentrifuge tube and mix briefly by vortexing.

**Application** | **DNA Binding Buffer: Sample** | **Example** || Genomic DNA (> 2 kb) | 2 : 1 | 200 uL : 100 uL |
| PCR Product, DNA Fragment | 5 : 1 | 500 uL : 100 uL |

2. Transfer mixture to a minicolumn inside of a collection tube and centrifuge for 30 seconds, discard flow-through.
3. Add 200 uL DNA Wash Buffer to the column and centrifuge for 30 seconds. Repeat 1x.
4. Add water (volume dependent on objective) directly to the column, and incubate at room temperature for 1 min.
5. Transfer the column to a clean microcentrifuge tube and centrifuge for 1 min to elute the DNA.

#### [Gels & Gel Electrophoresis](#collapseThree)

Reagents:

| TAE Buffer | ~ 50 mL per gel, more necessary for electrophoresis chamber. |
| --- | --- |
| NF Agarose (generally Seakem LE) | Grams of agarose : mL buffer = percentage of gel |
| Ethidium Bromide | ~ 1 drop |

\*\*Don’t forget ladder.

Protocol (gel):

1. Mix the TAE buffer and agarose once measured in microwavable flask. Stir with stir bar in flask and magnetic stirrer.
2. Afterwards, microwave for 1-3 mins until agarose is completely dissolved.
   Pause the microwave if the solution starts bubbling over, then stir the solution by slightly shaking the flask, and put the flask in the microwave again.
3. Let the flask cool until it is cool enough to carry with the orange heat “gloves.”
4. Add ethidium bromide and let gel solidify (10 - 20 min). Remember combs.
5. Usually, use the gel for gel electrophoresis right away, but if needed, the gel can be stored in a container filled with TAE buffer and 1-2 drops of ethidium bromide.

Protocol (gel electrophoresis):

1. Add reagents together (always at least ladder and sample).

* This can occur in PCR tubes or parafilm.

# bp ladder - 2 uL

Loading dye - 4 uL (1:3.5 ratio)

Water - 8 uL

Sample - volume depends on objective. Remember: try to have at least 50 ng to visualize on gel.

Loading dye - 1:3 ratio with sample.

2. Place the gel in the gel tray in the gel electrophoresis chamber and fill with TAE buffer to max line.
   * Since DNA is negatively charged, the wells should be near the positive end (black) and DNA will “run to red.”
3. Put the ladder and sample(s) in the wells with the ladder in Well 1, etc. Close the chamber properly, and run the gel electrophoresis chamber at 100 - 130 V.
4. Wait for about 30 minutes before stopping the gel electrophoresis chamber and removing the gel tray.
5. Place the gel in the Gel Imager (UV Light) next to the computer that will show the image. Run the program “Ethidium Bromide” to see DNA bands.
   * Make sure to position the gel in the center.
   * Remember: smaller fragments are farther away from the wells.
6. Clean up area, making sure to throw the gel away in the gel waste bin.

#### [Gel Purification](#collapseFive)

Reagents

| Membrane Binding Solution | 1 mL per each g of gel |
| --- | --- |
| Membrane Wash Solution | 1200 uL |
| NF water | 15 uL |

Protocol

1. Weigh a microcentrifuge tube for each DNA fragment to be isolated and record weight
2. Use a UV lamp to excise the DNA fragment of interest in a minimal volume of agarose using a clean scalpel or razor blade
3. Transfer the gel slice to the weighed microcentrifuge tube and record the weight
4. Subtract the weight of the empty tube from the total weight to obtain the weight of the gel slice
   * The gel slice may be stored at 4°C or at –20°C for up to one week in a tightly closed tube under nuclease-free conditions before continuing purification
5. Add Membrane Binding Solution to the gel slice
6. Vortex the mixture and incubate at 50–65°C for 10 minutes or until the gel slice is completely dissolved
7. Vortex the tube every few minutes to increase the rate of agarose gel melting
8. Centrifuge the tube briefly at room temperature to ensure the contents are at the bottom of the tube
   * Once the agarose gel is melted, the gel will not resolidify at room temperature
9. Transfer the dissolved gel mixture or prepared PCR product to the SV Minicolumn assembly
10. Incubate for 1 minute at room temperature before centrifugation for 1 minute and discarding supernatant
11. Wash with 700µL of Membrane Wash Solution, centrifuge for 1 minute, and discard supernatant
12. Wash with 500µL of Membrane Wash Solution, centrifuge for 5 minutes, and discard supernatant
13. Centrifuge 1 minute with the microcentrifuge lid open to allow evaporation of any residual ethanol
14. Transfer SV Minicolumn to a new microcentrifuge tube
15. Add Nuclease Free water directly to the center of the column
    * Membrane should be completely covered with nuclease-free water
16. Incubate at room temperature for 1 minute, centrifuge for 1 minute at 16,000 × g, and repeat
17. Discard the SV Minicolumn
18. Store the tube containing the eluted DNA at 4°C or –20°C
    * Check the purity of DNA in the nanodrop
    * If concentration not high enough, speed vac and nanodrop again

#### [Gibson Cloning](#collapseSix)

Reagents:

| DNA insert | 3:1 insert to vector ratio. Should have 500 ng. |
| --- | --- |
| DNA vector | 3:1 insert to vector ratio. |
|| Gibson Master Mix | 15 uL |
| --- | --- |
| NF H2O | Remaining volume necessary. |
| Total | 20 uL |

Procedure

1. Calculate the amount of insert, vector, and water needed. Use calculations on spreadsheets in drive.
   * 3:1 insert to vector ratio
   * Should have 500 ng of template DNA

| Reagents | Concentration (ng/uL) | Concentration (g/uL) | # bp | Mol weight (amu) | M (Concentration/MW) | uL necessary for # fmol |
| --- | --- | --- | --- | --- | --- | --- |
| Insert | given | Conc (ng/uL) \* 10^(-9) | given | # bp \* 650 | Conc (g/uL) / mol weight | # fmol / M |
| Vector | given | Conc (ng/uL) \* 10^(-9) | given | # bp \* 650 | Conc (g/uL) / mol weight | # fmol / M |
| Gibson Master Mix | n/a | n/a | n/a | n/a | n/a | 15 |
| NF H2O | n/a | n/a | n/a | n/a | n/a | 20 - (sum) |
|  |  |  |  | Total |  | 20 uL |

2. On ice, put the insert, vector, and water in a PCR tube with 15 uL of the gibson master mix.
   * There should already be aliquots of 15 uL of gibson master mix (GA) in the fridge. Restock if not.
3. Run thermocycler program.

50 C for 1 hr

4 C forever

#### [Golden Gate Cloning](#collapseSeven)

Reagents

| DNA inserts/parts | 20 fmol DNA (per each insert/part) per 10 uL rxn. Calculations can be done with spreadsheet from Gibson calculations. Modify fmol calculations at end. |
| --- | --- |
| T4 Ligase Buffer 10X | 1 uL buffer per 10 uL rxn |
| T7 Ligase | 0.5 uL enzyme per 10 uL rxn |
| Type II Restriction Enzyme (BsaI or BsmbI) | 0.5 uL enzyme per 10 uL rxn |
| NF H2O | Remaining volume necessary. |

Protocol:

1. Calculate amount of reagents and DNA needed based on wanted volume of reaction and add all reagents to a PCR tube on ice, restriction enzyme last.
   * 20 fmol DNA (for each insert/part) per 10 uL rxn
2. Run thermocycler program.

> 25 cycles
(Incubation temperature) for 2 min
16 C for 5 min

(Incubation temperature) for 60 min
(Inactivation temperature) for 10 - 20 min
4 C forever

#### [Transformation (electrotransformation w E. Coli)](#collapseEight)

Reagents:

| Soc Medium | 1 mL per 1 uL cells |
| --- | --- |
| DNA | ~ 250 ng |

Protocol:

1. Using electrocomp cells, let cells thaw on ice (a few minutes, do not leave out for longer than necessary). Keep cuvette and cuvette holder on ice as well.
2. Add DNA to cells once thawed.
   Need ~ 250 ng for transformation.
   Do not incubate.
3. Add cells to slit in cuvette, put cuvette in holder and push back into clasp of electroporator.
   Remember to press down on pulse until beep.
   Check time constant to make sure transformation successful.
4. Using sterile technique, add SOC medium to cuvette then immediately pipette draw SOC up with cells.
   Place in pre-prepared 1.5 mL tube.
5. Incubate cells in shaking incubator for 1 hr.
6. Afterwards, spin down at 3000 rpm for 5 min to pellet bacteria.
7. Take out SOC, but leave ~ 60 - 80 uLs. Resuspend pellet and plate.

#### [Liquid Cultures](#collapseNine)

Reagents:

| Nutrient Broth | 4 mL |
| --- | --- |
| Antibiotic | 1:1000 ratio between uL antibiotic and uL broth |

Equipment:

* Plate of Transformed Colonies
* Large Tubes in Tube Rack
* Bunsen Burner (sterile technique throughout)
* Shaking Incubator

Protocol:

1. In front of the flame from the bunsen burner, put the nutrient broth and antibiotic in the large tube
   * Superior broth has more nutrients than LB and therefore has a higher yield
   * Antibiotic depends on antibiotic resistance of transformed colonies
2. Pick up a colony from the plate of colonies by touching the colony with a tip (on pipette)
3. When transferring the colonies to the large tubes, tilt the large tube so the nutrient broth is closer to the opening
4. Put the very end of the tip (which touched the plate and has the colony) into the broth
   * Do not dip the entire tip in the nutrient broth because the tip is not that sterile
5. Shake the tip a little to transfer the colony into the nutrient broth
6. Take the very end of the tip out of the tube, discard the tube, and cap the tube
7. Put the tubes in a shaking incubator overnight
   * Can either miniprep immediately or freeze culture tubes to miniprep later

Freezing Liquid Culture Tubes

1. Take liquid culture tubes out of the shaking incubator and centrifuge at 3000 rpm for 5 minutes
2. Dump the supernatant (liquid) into a beaker
3. Add 10% bleach to the beaker
4. Place the large tubes in the freezer covered with plastic wrap
   * Take out the large tubes only when going to do the miniprep immediately

#### [Miniprep](#collapseTen)

Purpose:
To purify plasmid from cells, usually with the liquid cultures created from a transformation

Reagents

| Plasmid Resuspension Buffer (B1) | 400 µL |
| --- | --- |
| Plasmid Lysis Buffer (B2) | 400 µL |
| Plasmid Neutralisation Buffer (B3) | 800 µL |
| Plasmid Wash Buffer 1 | 200 µL |
| Plasmid Wash Buffer 2 | 400 µL |
| DNA Elution Buffer | 40 µL |

Equipment:

Protocol:

1. Pellet liquid culture in 1.5 mL microcentrifuge tube by centrifugation at 13000 rpm for 5-10 minutes, and discard the supernatant
   * Pipette the liquid culture into a labelled microcentrifuge tube 1 mL at a time
   * Centrifuge the 1 mL-filled tubes and discard the supernatant
   * Repeat this process, putting 1 more mL of the liquid culture into the same microcentrifuge tube, until the liquid culture tube is empty
   * Pellet should form inside the microcentrifuge tube
2. Resuspend the plasmid in 400 µL Plasmid Resuspension Buffer (B1)
   * Vortex or pipette until there are no clumps present
3. Add 400 µL Plasmid Lysis Buffer (B2), and gently invert the tube 5-6 times
4. Incubate at room temperature for 1 minute
5. Add 800 µL Plasmid Neutralisation Buffer (B3), and invert the tube until the entire solution is completely yellow
   * Plasmid Neutralisation Buffer (B3) should be stored in the 4 C freezer
6. Incubate at room temperature for 2 minutes
7. Centrifuge at 13000 rpm for 10 minutes
8. Transfer the supernatant into the spin column (with the collection tube)
9. Centrifuge at 13000 rpm for 1 minute, and discard the flow-through (in the collection tube)
10. Add 200 µL Plasmid Wash Buffer 1 to the spin column
11. Centrifuge at 13000 rpm for 1 minute, and discard the flow-through (in the collection tube)
12. Add 400 µL Plasmid Wash Buffer 2 to the spin column
13. Centrifuge at 13000 rpm for 1 minute, and discard the collection tube with the flow-through
    * If there is any doubt, centrifuge at 13000 rpm for 1 more minute before discarding the collection tube with the flow-through
    * Do not touch the spin column to the flow-through in the collection tube when removing the spin column
14. Transfer the spin column to a clean microcentrifuge tube
15. Add >= 30 µL Elution Buffer
    * Note: Using more elution buffer will increase overall yield of DNA but will result in final solution of eluted DNA being less concentrated.
    * Tip: To increase the yield, use 20 uL of Michelle’s Zymo Elution Buffer instead
    * Tip: To decrease chemical contamination from ethanol, after step 13 spin the tubes with the filter for 1 min at 13,000 rpm without the lid in order to evaporate ethanol from the filters
    * Tip: To increase yield heat elution buffer using a heating block to 60℃
16. Incubate at room temperature for 1 minute
17. Centrifuge at 13000 rpm for 1 minute
    * Resulting flow-through in the microcentrifuge tube is the purified DNA

(Based on Monarch Miniprep protocol with revisions from Michelle Byrom)

#### [Digest](#collapseEleven)

Reagents:

| DNA | ~ 250 ng in 2 uL |
| --- | --- |
| NF H2O | Remaining volume necessary to fill total volume. |
| Buffer | 2 uL per enzyme |
| Enzyme | 0.5 per enzyme |
| **Total** | **20 uL** |

Protocol:

1. Add all reagents to a PCR tube on ice. Add enzyme last.
   * Don’t forget to flick tube!!
2. Run thermocycler program.

(Incubation temperature) for 1 hr/overnight

#### [Ligation](#collapseTwelve)

Reagents:

| DNA | ~ 250 ng in 2 uL |
| --- | --- |
| NF H2O | Remaining volume necessary to fill total volume. |
| T4 Ligase | 2 uL per enzyme |
| Buffer | 0.5 per enzyme |
| **Total** | **20 uL** |

Protocol:

1. Add all reagents to a PCR tube.
2. Run thermocycler program.

60 C for 2 hr/overnight

#### [LS/SB Broth](#collapseThirteen)

Reagents:

| NF H2O | Dependent on objective. |
| --- | --- |
| Pre-mixed LB/SB Powder | Dependent on company. |

Protocol:

1. Measure out water, add to bottle.
2. Using pre-mixed LB powder, add specified amount per liter. Mix with magnetic stir bar.
3. Take out stir bar.
4. Autoclave on liquid cycle for 20 min at 15 psi.

#### [Glycerol Stocks](#collapseForteen)

Reagents:

| Liquid Culture | 500 uL for 1 stock |
| --- | --- |
| 50% Glycerol | 500 uL for 1 stock |

Protocol:

1. Inoculate liquid cultures using the standard protocol for liquid cultures and leave overnight or until there is bacterial growth.
2. Using sterile procedure. add 500 µL of the liquid culture and 500 µL of 50% glycerol to a 1.5 mL microcentrifuge tube. Close lid and gently mix by inverting the tube or vortexing to ensure that the culture and glycerol are not separated.
   * Stock doesn’t always need to consist of 500 uL liquid culture and 500 uL glycerol, but liquid culture and glycerol must be in a 1:1 ratio.
3. Put the glycerol stock in the -80℃ freezer to freeze. Glycerol stock should be stable for years so long as it is left at -80℃. Subsequent thaw and freeze cycles will reduce shelf life.
4. To recover bacteria from a glycerol stock open the top and use a sterile hoop to scrape some frozen bacteria off the top. Streak onto a plate containing the appropriate antibiotic.
5. Grow on plates overnight at 37℃ (or a lower temperature like 30℃ if a slower growth rate is required).

#### [Production of L-DOPA\*](#collapseFifteen)

Reagents:

| Liquid Cultures of Production Assembly and Controls | 100 uL |
| --- | --- |
| NF H2O | Dependent on objective. |
| Pre-mixed Nutrient Powder (LB) | Dependent on objective. |
| Tyrosine | Enough to make 1 mM and 5 mM solution. |

Protocol:

1. Make 3 different batches of LB Broth: plain LB, LB with 1 mM tyrosine, and LB with 5 mM tyrosine.
   * Weigh out enough tyrosine to make 1 mM and 5 mM solution.
   * Use standard LB protocol to make all three, with exception of adding tyrosine.
2. Use different batches of LB to make liquid culture stocks with production assembly as well as a control, another plasmid that doesn’t produce L-DOPA.
   * Use already grown liquid cultures. Take 100 uL grown culture and add to 4 mL of LB/LB w 1mM tyrosine/LB w 5 mM tyrosine.
   * Remember antibiotic.
   * Make liquid cultures in triplicates.
3. Leave new culture tubes in shaking incubator.
4. Let cultures sit until turn dark (brown color).

#### [Sensing and Reporting Dopamine\*](#collapseSixteen)

Reagents:

| LB Broth | Dependent on objective. |
| --- | --- |
| Dopamine | Enough to make 10 mM stock solution. |
| L-Ascorbic Acid | 1 mg/uL solution. |
| Liquid Cultures of Sensing Assembly and Controls | 100 uL |
| IPTG | Enough for 1 mM solution. |

Protocol:

1. First make 10 mM DOPA stock solution in order to make other test conditions.
   * Calculate mLs of 10 mM DOPA stock solution needed. Using sterile technique, add however many mLs of LB needed (for example, if 20 mLs of stock solution needed, add 20 mL LB and base calculations off of that) to a tube.
   * Add enough dopamine to make 10 mM solution (continuing off example of 20 mL solution, add 0.0363 g of dopamine to make 10 mM solution).
   * Add ascorbic acid in concentration of 1 mg/uL solution.
   * Once all added, vortex tube for ~15 - 20 min until dopamine goes into solution.
   * Sterilize solution by using a syringe and filter.
2. After having made 10 mM DOPA stock solution, make a 1:10 dilution of 10 mM stock solution to make a 1 mM solution. (If 20 mL of 10 mM stock solution, dilute 2 mL of 10 mM solution in 18 mL LB to make 20 mL of 1 mM DOPA solution). Remember to add ascorbic acid to new stock solution. Then sterilize like before.
3. Repeat this step until there is 10 mM, 1 mM, 100 uM, 10 uM, and 1 uM DOPA solution.
4. Use 1 mM, 100 uM, 10 uM, and 1 uM DOPA solution to make liquid cultures from already grown liquid cultures of sensing assembly and controls.
   * Make 1:50 dilutions of already grown liquid cultures with different media conditions. For example, for 4 mL culture, add 80 uL of already grown culture to 4 mL of LB or a certain DOPA solution.
   * Do triplicates for each condition for each assembly/control tested.
   * Remember to make two identical sets, one without IPTG and one with.
5. Grow for ~ 6 hrs.
   * When cell OD reaches .6, add 4 uL (1:10 of media) of 1 mM IPTG (after ~3 hours)
   * Put cultures back into the shaking incubator
6. Spin down cultures
   * ~3000 rpm for 5 minutes
   * Pour out supernatant
7. Resuspend in 1x Buffer
   * 4 mL of 1x PBS
   * Pipette or vortex to resuspend cells
8. Plate reading
   * Take 100 uL of the resuspended cells
   * Load into a 96 well transparent plate
   * Measure absorbance and fluorescence with Venus assay (excitation: 515 nm, emission: 528 nm)

#### [Measuring Fluorescence\*](#collapseSeventeen)

Reagents:

| Liquid Cultures | 100 uL |
| --- | --- |
| PBS | 5 mL |

Protocol:

1. Liquid cultures
   * Pick colonies from plate
   * Multiple controls
   * Overnight in the 37 incubator
2. Liquid cultures of already grown liquid cultures
   * 100 uL of grown liquid cultures into 5 mL of media
   * Remember antibiotic
   * Incubate cultures for another 4-6 hours
3. Spin down cultures
   * 3000 rpm for 5 minutes
4. Resuspend in 1x Buffer
   * 4 mL of 1x PBS
   * Pipette or vortex to resuspend cells
5. Plate reading
   * Take 100 uL of the resuspended cells
   * Load into a 96 well transparent plate
   * Measure absorbance and fluorescence with GFP assay (excitation: 495 nm, emission: 509 nm)

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# Team:Austin UTexas LASA/Results

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# Results

**Overview**

   - Assembled and sequence verified Venus cassette plasmid and pp2551 cassette plasmid
    - Assembled the multigene sensing assembly
    - Tested another version of the sensing assembly and demonstrated that it works

**Production Circuit**

Our team did not have an assay to get quantitative measurements concerning the production of L-DOPA in our production assembly. However, by itself, L-DOPA will oxidize to melanin. As a result, to get qualitative results concerning our production assembly, we grew up our production cells in LB media containing 5 mM tyrosine and 0 mM tyrosine and then observed if a color change occurred. The diagram above shows such a color change. Our production assembly did observe a similar color change, but the change in color was not very pronounced.

**Sensing Circuit**

We tested a sensing construct that was similar to our sensing construct in design but built with different methods and parts. Instead of Venus fluorescence protein, this sensing circuit expressed e2-crimson, and was inserted into a pMMB67EH vector, while in our sensing construct, the Venus gene was inserted into a pET28 vector. Both constructs were transformed into Top 10 E. Coli strain and used in the fluorescence detection assay. We found that the fluorescence intensity in the cell increased as the amount of L-DOPA in the media increased for the lab construct. However, we found that our sensing construct, in the presence of L-DOPA, showed increased expression of fluorescence when certain amounts of L-DOPA were present in the media. You can see a slight increase in fluorescence from no L-DOPA to 100 uM of L-DOPA. However, the data shows that a lower level of fluorescence was detected when 1 mM of L-DOPA was present, which is different than the predicted outcome. These data suggest that different combination of vector plasmid and E. coli strain might have a different effect on the inducibility of sensing construct. Therefore, further study is needed to find out the best combination of vector and E.coli strain for this sensing construct.

![](https://static.igem.org/mediawiki/2017/c/cb/T--Austin_UTexas_LASA--result1.png)

Fig. 1. A chart showing the detected level of fluorescence (E2-Crimson) expressed by a sensing cell in different conditions of L-DOPA in the media.

![]( https://static.igem.org/mediawiki/2017/b/bf/T--Austin_UTexas_LASA--result.png)

Fig. 2. A chart showing the detected level of fluorescence (Venus) expressed by a sensing cell in different conditions of L-DOPA in the media.

![](https://static.igem.org/mediawiki/2017/8/88/T--Austin_UTexas_LASA--result2.jpg)