iGEM 2018 — Team Baltimore_BioCrew

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# Team:Baltimore BioCrew/Notebook

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    + [SUMMARY](https://2018.igem.org/Team%3ABaltimore_BioCrew/Entrepreneurship)

Accordion Menu

# Notebook

[ ]
June 23

### Clot Makers

We found that we may not need to order any BioBricks, as Lethbridge only used the sequences of the parts, and then proceeded synthesized them. We decided that we want

some more time to determine what we need to send for synthesis, as we want to be exact with what we need. We also decided some different ways to continue the second construct, which Lethbridge never got to test. We want to add purification tags to both construct one (clotting blood) and construct two (localizing the clot). We could use Histags, and in construct two we need Factor 13, an antimicrobial peptide.

### Clot Breakers

* Talked about specific direction for project
* Requested TPA part from IGEM
* Talked about goal to reduce the side effects of TPA by making it more targeted (make TPA only function in low oxygen tension next to blood clot)

### Interlab

* Signed up for interlab (Baltimore Bio-Crew)
* Creating contact list to purchase absorbance filter D for plate reader
* Interviewed clot breakers and clot makers for potential assays

### Wiki

* Wrote a first draft of the project description
* Filled out as much as possible of safety form
* Discussed wiki design

### Human Practices

Came up with ideas and people to contact

[ ]
June 30

### Clot Makers

We began looking at places/people we could contact who we can ask for more insight into potential applications for our project. We sent an email to Dr. Lavik, who is

knowledgeable about using nanoparticles to increase the rate of coagulation and lessen the possibility of infection and inflammation. We also talked a little bit about a potential way to test the effectiveness of the enzymes we produce with our constructs. We also introduced the idea of potentially adding a numbing agent into the construct.

### Clot Breakers

* Designed and ordered primers
* Researched more about redox switches

### Interlab

* We looked at the interlab protocol and verified that our machine has all of the settings and requirements to participate EXCEPT for the Filter D for absorbance
* We successfully poured 20 plates with Chlor antibiotic for next Saturday transformations
* Understanding the interlab this year and why its important

### Wiki

* Came up with some logo designs
* Discussed colors for wiki

[ ]
July 7

### Clot Makers

Did research to figure out how to best design constructs

### Clot Breakers

* Primers arrived
* Transformed tPA biobrick into E.coli

### Interlab

* Transformed our cells w/ different parts
* Ensured that we can use a different filter to complete interlab for plate reading protocol
* Part of the team got to understand the plate reader and the way that we are going to obtain data
* Made a couple Clor plates for transformations

### Wiki

* Short wiki meeting today
* Looked at other websites for inspiration

### Human Practices

* Open house at BUGSS today
* Sent follow-up emails

[ ]
July 14

### Clot Makers

* Designed a potential assay to test the effectiveness of the clotting agent in a simulated injury scenario
* Discussed other assays that could potentially be used through all stages of testing
* Ordered our constructs

### Clot Breakers

* Discussed gene design and looked for bacterial promoters to use
* Need to find bacterial promoters to replace the mammalian promoter on the the TPA gene
* Miniprepped TPA plasmids from last time using Monarch Plasmid Miniprep Kit
* Measured concentration of DNA with nanodrop
Nanodrop Results:
E: 57.0 ng/uL Purity: 1.74
J: 39.8 ng/uL Purity: 1.78
Z 75.8 ng/uL Purity: 1.71* Digested DNA in 1 spot in order to open the plasmids so that we would be able to see the length of them on a gel later
* Used Typical RD Protocol from NEB
* Ran a gel electrophoresis on a 1% gel of the three digests and one lane of uncut plasmid

### Interlab

Finished calibration protocol for absorbance and fluorescence to obtain needed measurement parameters for our experiment

Absorbance settings:- Room temperature
- ABS 550 on plate reader (excitation 535)

Fluorescence settings:

- Room temperature
- Reading from top
- Optimal reading

Complete protocol for absorbance/fluorescence for parts 1-6 (not controls) and recorded data

- (Incubated ½ samples for 6 hours in 37\* incubator, and on ice for as 0 hour time point)
- Made ~40 Chlor plates for next week interlab and CFU protocol

[ ]
July 21

### Clot Makers

* Completed task list for weeks coming up
* Edited clot making ppt for presentation on August 11

### Clot Breakers

* Tissue Plasminogen Activator (TPA) genes were designed

### Interlab

We finished all calculations for LB+Chlor media

Finished taking absorbance and fluorescence of time point 0 hours data

We made 70% alcohol

Labeled and prepped plates and tubes for CFU protocol

- Example of label -1a/ - = Negative control 1= (1st set), a= first replicate

Completing dilutions for protocol and then needs to plate

.1 dilution was not precise, here is the absorbance data from that (measuring 550ABS)

[ ]
July 28

### Clot Breakers

LWe checked the sequence of the TPA we amplified by aligning it to the biobrick parts sequence.

We used Clustal Omega to do this.

We checked from around 580 bp to the end and everything was great (almost perfect matches).

We also checked the quality of the sequence using a chromatography viewer.
(Quality was very good)

Next Step: Will request that VF2 sample be resequenced

#### Elena’s guest lecture

Areas to look more into:

Learned that tPA is very potent and only used as a treatment in high-risk/severe stroke cases. What makes it so potent? Is there a way to modify that aspect?

Methods of delivery for tPA? Literature shows some use for targeted treatment of blood clots. (Reference: https://www.ncbi.nlm.nih.gov/pubmed/28796540)

[ ]
August 4

### Clot Breakers

Plasmid prep with bacterial biobrick plasmid backbone BBa-I746909 (906 bp) using Monarch Miniprep kit. We obtained plasmid concentration and purity using the Nanodrop.

- 1: 105.7 ng/microliter, 260/280: 1.67, 260/230: .91; trial 2; 59.7, 1.79, 1.54
- 2: 72.2 ng/microliter 260/280: 1.56, 260/230: .67; trial 2: 19.8, 1.66, 1.15
- 3: 74.2 ng/microliter 260/280: 1.75, 260/230: 1.36
- 4: 73.2 ng/microliter 260/280: 1.73, 260/230: 1.15

Ran it through gel
Gene designing (shorter TPA: reteplase) for optimized expression in E. Coli
Changing codons from mammalian preference to codons based on E.Coli codon preference.
Lisa ordered three constructs for us:- 1) reteplase with T7 promoter
- 2) TPA with inducible T7 promoter
- 3) TPA with weak constitutively active promoter

[ ]
August 11

### Clot Makers

* Recut, ligated and purified gblocks and vectors
* Ran gel electrophoresis to verify
![Electro1](https://static.igem.org/mediawiki/2018/b/b0/T--Baltimore_BioCrew--Electropheresis_CM.png)
![Electro2](https://static.igem.org/mediawiki/2018/9/91/T--Baltimore_BioCrew--Electropheresis1_CM.png)

#### Wednesday August 15

* Transformed the ligated genes and plated them

### Clot Breakers

* PCR amplified plasmid backbone samples
* Ran a gel of amplified samples

[ ]
August 18

## Interlab

Started to work with assays we created one that had the following materials DPBS,Trypsin and Acetate.
With these materials we did the following- 1.Add 27 ug fluorescein to 1.5 ml acetate.
- 2.5 ml DPBS and 1.5 ml acetate and trypsin mix
- 3. Did 3 of 10 señal dilution
  100 ml trypsin (not in well 12)
  100 ml DPBS, acetate and fluorescein
When it comes to setting the plates :- Put in plate reader
- shake for 5 seconds
- Room temperature
- Read from top
Recorded 5 times every five minutes
Result:- Dilution 3 is most (5/9mg/ml)

## Clot Breakers

- ran digestions
- Performed PCR screening
- purification(The final elutions were not displayed in tubes so purification will be repeated)

[ ]
August 25

### Clot Makers

Performed colony PCR screening, with a couple potential positives.

[ ]
September 1

## Clot Makers

Miniprepped genes and sent them to Johns Hopkins for screening

## Clot Breakers

set up four ligations:- TPA-T7 (from gblock)
- TPA weak (from gblock)
- Reteplase (from gblock)
- Mammalian TPA (from PCR of biobrick)

[ ]
September 8

## Clot Makers

Redigested and ligated g-blocks

## Clot Breakers

Observations of plates:- about 3 colonies on
- about 5 colonies on
- no colonies on Reteplase
- dozens of colonies on mammalian TPA plates

Colony PCR screen to verify that insert is present

[ ]
September 15

## Clot Makers

Redigested and ligated g-blocks

## Clot Breakers

Colony PCR screen
https://www.neb.com/protocols/2012/09/06/protocol-for-onetaq-2x-master-mix-with-standard-buffer-m0482
There were many colonies on Reteplase today.
Observations:- 2 colonies on TPA weak
- 4 colonies on T7
- Dozens of colonies on Reteplase 1:2
- Around a dozen from Reteplase 1:1

Colony PCR screen to check for insert

[ ]
September 22

## Clot Makers

Screened colonies with colony PCR

Various positive samples that need to be sequenced to determine whether they’re our samples or not

## Clot Breakers

looked at sequencing results- The Clot Makers also had issues when using the linearized vector. Following their lead, we’ll try ligating our 3 inserts into the BBxxxx vector (contains RFP)

3 reactions -- T7-TPA, weak-TPA, reteplase

[ ]
September 29

## Clot Makers

Conducted PCR Screening on our next set of potential positives and sent the rear third of construct to be sequenced

## Clot Breakers

No colonies found on reteplase
16 colonies on weakpromoter-TPA
10 colonies on T7-TPA
check for insert using colony PCR screen- select 5 colonies from each of two plates (10 colonies total)
- primers VF2 and VR; NEB oneTaq

[ ]
October 6

### Clot Makers

Prepped for a His Tag Screening. Decided to use samples 4-3 and 1-5 (and some of 1-4). Didn’t finish.

## Clot Breakers

Performed PCR cleanup of 9 colonies

Concentrated samples using NanoSep

[ ]
October 10

### Clot Makers

Finished His Tag Screening and ran a protein gel.


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# Team:Baltimore BioCrew/Experiments

* [HOME](https://2018.igem.org/Team%3ABaltimore_BioCrew)
* TEAM
  + [THE CREW](https://2018.igem.org/Team%3ABaltimore_BioCrew/Team)
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  + [Thank you Ed](https://2018.igem.org/Team%3ABaltimore_BioCrew/Part_Collection)* PROJECT
    + [DESCRIPTION](https://2018.igem.org/Team%3ABaltimore_BioCrew/Description)
    + [DESIGN](https://2018.igem.org/Team%3ABaltimore_BioCrew/Design)+ [EXPERIMENTS](https://2018.igem.org/Team%3ABaltimore_BioCrew/Experiments)
      + [NOTEBOOK](https://2018.igem.org/Team%3ABaltimore_BioCrew/Notebook)
      + [INTERLAB](https://2018.igem.org/Team%3ABaltimore_BioCrew/InterLab)
      + [MODEL](https://2018.igem.org/Team%3ABaltimore_BioCrew/Model)
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  * [SAFETY](https://2018.igem.org/Team%3ABaltimore_BioCrew/Safety)
  * HUMAN PRACTICES
    + [HUMAN PRACTICES](https://2018.igem.org/Team%3ABaltimore_BioCrew/Human_Practices)
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  * AWARDS
    + [MODEL](https://2018.igem.org/Team%3ABaltimore_BioCrew/Model)
    + [SUMMARY](https://2018.igem.org/Team%3ABaltimore_BioCrew/Entrepreneurship)

# Experiments

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

## Introduction

Our project was split into two sections: Clot Makers, who focused on the quick coagulation of wounds using an enzyme found in the Russel's Viper's and Desert Horned Viper's venom. Conversely, the Clot Breakers focused on using a shortened version of tPA called Reteplase to decoagulate unordinary clots, such as those that could appear in blood vessels, causing strokes and heart diseases.

### Protocol: Clot Makers

[ ]
Gel electrophoresis

Adapted from Addgene, (https://www.addgene.org/protocols/gel-electrophoresis/)

Pouring the Gel

* Measure out agarose on scale slowly (for a standard 1% gel, use 1 g of agarose. For other concentrations, calculate accordingly). Stay within ~.05 g of 1 g.
* Mix agarose powder with 100 mL 1xTAE in a microwavable flask.
* Microwave for 1-3 min until the agarose is completely dissolved, stopping every 30 seconds to swirl the flask. Keep an eye on it to make sure it does not overboil. Wear oven mitts when handling the flask.
* Place agarose solution on counter until the flask can be comfortably held
* Set up the gel tray with the well comb in place, as one person slowly pours the agarose solution into the well. Avoid bubbles.
* Let gel sit for 20-30 minutes until completely solidified (the gel will be milky white and opaque).

Loading Samples

* Add equal amount of loading buffer to each of your samples.
* Place the agarose gel into the electrophoresis unit.
* Fill gel box with 1xTAE until the gel is covered and container is filled.
* Carefully load a molecular weight ladder into the first lane of the gel. Place your elbow on the counter to prop yourself up so you can look at the gel from an angle to see the wells. Place the tip of the pipette tip just above the well and slowly push so the sample fills the entire well.
* Load the rest of the samples in this manner.
* Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel for about 1 hour. Note: Black is negative, red is positive. Always Run to Red.
* Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
* Visualize your DNA fragments under UV light.

[ ]
Plasmid Purification

We did these using the QIAprep Spin Mini Prep Kit Protocol:

[ ]
Restriction Digest

Master mix:

* 76 uL H2O
* 20 uL 10x NEB2
* 1 uL EcoR1
* 1 uL Pst1
* 1 uL Dpn1
* 1 uL BSA

Mix together:

* 10 uL Sample DNA
* 10 uL Master Mix

[ ]
Ligation

Mix together:

* 2 uL plasmid
* 3.5 uL insert
* 1 uL T4 Ligase buffer
* 0.5 uL T4 Ligase
* 4.5 uL H2O

[ ]
Transformations

We used the iGEM Parts Registry Transformation Protocol:

[https://static.igem.org/mediawiki/parts/6/67/IGEM\_Registry\_-\_Transformation\_Protocol.pdf>](https://static.igem.org/mediawiki/parts/6/67/IGEM_Registry_-_Transformation_Protocol.pdf)

Changes made: We omitted step 13, and used one petri dish rather than two.

[ ]
Colony PCR

Master Mix:

1. 316.8ul dIH20
2. 44ul Taq Buffer
3. 22ul foreward primer
4. 22ul reverse primer
5. 33ul dNTPs
6. 2.2ul Taq

Add 1ul DNA to 19ul Master Mix

Settings:

1 Cycle

1. 95°C - 1min

30 Cycles

1. 95°C - 30s
2. 55°C - 45s
3. 72°C - 45s

1 Cycle

1. 72°C - 2min

Hold at 4°C

[ ]
Ni-NTA Spin Column Purification of 6xHis-Tagged Proteins (E. coli Lysates)

1. Resuspend a pellet derived from 5ml cell culture volume in 630 ul Lysis buffer (NPl-10). Add 70ul Lysozyme Stock Solution (10mg/ml) and add 3 units/ml culture volume Benzonase Nuclease
2. Incubate for 15-30min
3. Centrifuge lysate at 12000 x g for 15-30 min at 4 degrees C. Collect the supernatant.
4. Save 20 ul of the lysate for SDS-PAGE analysis
5. Equilibrate the Ni-NTA spin column with 600 ul buffer NPl-10. Centrifuge for 2 minutes at 2900 rpm. Spin without lid to ensure it is done in two minutes
6. Load up to 600 ul of the cleared lysate containing the His-Tagged protein onto the (pre-equilibrated) Ni-NTA spin columns. Spin for 5 minutes at about 1600 rpm, and collect the flow-through. To ensure efficient binding, don’t go higher than 1600 rpm, though do it with an open lid
7. Wash the column twice with 600 ul Buffer NPl-20. Centrifuge for 2 min. at about 2900 rpm. Save the flow-through for analysis by SDS-PAGE
8. Elute the protein twice with 300ul Buffer NPl-500. Centrifuge for 2 minutes at about 2900, and collect the eluate. Most of the protein will be in the first eluate. The rest will be in the second. 300ul each time

[ ]
Protein Resuspension and Protein Gel Electrophoresis

1. Resuspend protein sample in a 1:1 ratio of sample to cracking buffer
2. Place resuspended sample in boiling water for 5 minutes
3. Load samples into protein gel
4. Run at 100v
5. Run until the gel is about 75-80% of the way down the gel
6. Remove gel from electrodes and plastic casing
7. Wash gel with purified water about 3 times, agitating the vessel the gel and water are in slightly to wash thoroughly, while not tearing the gel
8. Stain the gel with Simplyblue dye

[ ]
PCR Amplification

Mix together:

1. 35.4 uL Water
2. 15 uL 5x Phusion Buffer
3. 1 uL 10 mM dNTPs
4. .8 uL 30 mM Forward Primer
5. .8 uL 30 mM Reverse Primer
6. .5 uL enzyme (Phusion taq)
7. 2 uL Template

Settings

1 Cycle

1. 98°C - 30s

35 Cycles

1. 98°C - 10s
2. 55°C - 30s
3. 72°C - 2min

1 Cycle

* 72°C - 10min

[ ]
Flourocine Diacetate Enzyme Activity Assay

Enzyme activity was assayed using a modification of the fluorocine diacetate assay for hydrolytic enzymes (Gamble and Muriana 2007). 5ul of in vitro transcription/translation product was mixed with 250ul of phospate buffered saline containing 1 mM flourescine diacetate in wells of a Tecan black Tecan micro titer plate. Flouresence generated by cleavage of the acetate groups was measured using a Tecan Genios plate reader with excitation at 485nM and emission at 535 nM.

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Baltimore BioCrew/Results

* [HOME](https://2018.igem.org/Team%3ABaltimore_BioCrew)
* TEAM
  + [THE CREW](https://2018.igem.org/Team%3ABaltimore_BioCrew/Team)
  + [COLLABORATIONS](https://2018.igem.org/Team%3ABaltimore_BioCrew/Collaborations)
  + [Thank you Ed](https://2018.igem.org/Team%3ABaltimore_BioCrew/Part_Collection)* PROJECT
    + [DESCRIPTION](https://2018.igem.org/Team%3ABaltimore_BioCrew/Description)
    + [DESIGN](https://2018.igem.org/Team%3ABaltimore_BioCrew/Design)+ [EXPERIMENTS](https://2018.igem.org/Team%3ABaltimore_BioCrew/Experiments)
      + [NOTEBOOK](https://2018.igem.org/Team%3ABaltimore_BioCrew/Notebook)
      + [INTERLAB](https://2018.igem.org/Team%3ABaltimore_BioCrew/InterLab)
      + [MODEL](https://2018.igem.org/Team%3ABaltimore_BioCrew/Model)
      + [RESULTS](https://2018.igem.org/Team%3ABaltimore_BioCrew/Results)
      + [DEMONSTRATE](https://2018.igem.org/Team%3ABaltimore_BioCrew/Demonstrate)
      + [ATTRIBUTIONS](https://2018.igem.org/Team%3ABaltimore_BioCrew/Attributions)
  * PARTS
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    + [COMPOSITE PARTS](https://2018.igem.org/Team%3ABaltimore_BioCrew/Composite_Part)
    + [IMPROVED PARTS](https://2018.igem.org/Team%3ABaltimore_BioCrew/Improve)
  * [SAFETY](https://2018.igem.org/Team%3ABaltimore_BioCrew/Safety)
  * HUMAN PRACTICES
    + [HUMAN PRACTICES](https://2018.igem.org/Team%3ABaltimore_BioCrew/Human_Practices)
    + [EDUCATION & ENGAGEMENT](https://2018.igem.org/Team%3ABaltimore_BioCrew/Public_Engagement)
  * AWARDS
    + [MODEL](https://2018.igem.org/Team%3ABaltimore_BioCrew/Model)
    + [SUMMARY](https://2018.igem.org/Team%3ABaltimore_BioCrew/Entrepreneurship)

## Successful Cloning of the Cerastocytin and Russel Viper Snake Venom Genes

We were able to successfully clone five of our six Clot Maker constructs:
1. Cerastocytin with a His tag and the Lac promoter
2. Cerastocytin with a constitutive promoter
3. Russell Viper Snake Venom with a constitutive promoter
4. Russell Viper Snake Venom with a His tag and the Lac promoter
5. Russell Viper Snake Venom with a His tag
We were not able to clone Cerastocytin with a His tag and constitutive promoter

We were able to determine that we successfully cloned the genes by colony-screening PCR using VF2 and VR primers. We also purified plasmids and sent them for sequencing.
![](https://static.igem.org/mediawiki/2018/thumb/4/47/T--Baltimore_BioCrew--Results.png/1200px-T--Baltimore_BioCrew--Results.png.jpeg)

## Successful Expression of the Cerastocytin and Russel Viper Snake Venoms

We were able to successfully express five of our six Clot Maker constructs in E. coli cells. We grew the cells overnight, diluted them in the morning and then diluted and grew them to an OD of 0.6. We lysed the cells and ran them on SDS-PAGE gels.

Lane 1: Cerastocytin with a His tag and the Lac promoter induced with IPTG
Lane 2: Cerastocytin with a His tag and the Lac promoter not induced
Lane 3: Cerastocytin with a constitutive promoter induced with IPTG
Lane 4: Cerastocytin with a constitutive promoter not induced
Lane 5: Cerastocytin with a constitutive promoter induced with IPTG
Lane 6: Cerastocytin with a constitutive promoter not induced
Lane 7: Russell Viper Snake Venom with a constitutive promoter induced with IPTG
Lane 8: Russell Viper Snake Venom with a constitutive promoter not induced
Lane 9: Russell Viper Snake Venom with a constitutive promoter induced with IPTG
Lane 10: Russell Viper Snake Venom with a constitutive promoter not induced
Lane 11: Russell Viper Snake Venom with a His tag and the Lac promote r induced with IPTG
Lane 12: Russell Viper Snake Venom with a His tag and the Lac promoter not induced
Lane 13: Russell Viper Snake Venom with a His tag induced with IPTG
Lane 14: Russell Viper Snake Venom with a His tag not induced
Lane 15: Ladder
![](https://static.igem.org/mediawiki/2018/thumb/c/cb/T--Baltimore_BioCrew--more_results.png/1200px-T--Baltimore_BioCrew--more_results.png.jpeg)

We saw bands on our gel, but we were not sure if they were our protein or just a normal protein in E. coli. So we re-ran our gel with a control from E. coli without a plasmid.
Lane 1: Cerastocytin with a His tag and the Lac promoter induced with IPTG
Lane 2: Cerastocytin with a His tag and the Lac promoter not induced
Lane 3: Cerastocytin with a constitutive promoter induced with IPTG
Lane 4: Cerastocytin with a constitutive promoter not induced
Lane 5: Cerastocytin with a constitutive promoter induced with IPTG
Lane 6: Cerastocytin with a constitutive promoter not induced
Lane 7: Russell Viper Snake Venom with a constitutive promoter induced with IPTG
Lane 8: Russell Viper Snake Venom with a constitutive promoter not induced
Lane 9: Russell Viper Snake Venom with a His tag and the Lac promote r induced with IPTG
Lane 10: Russell Viper Snake Venom with a His tag and the Lac promoter not induced
Lane 11: Russell Viper Snake Venom with a His tag induced with IPTG
Lane 12: Russell Viper Snake Venom with a His tag not induced
Lane 13: Control no plasmid
Lane 15: Ladder
![](https://static.igem.org/mediawiki/2018/4/48/T--Baltimore_BioCrew--Protein47.png)

Since there was no band in our control lane, the band(s) we see in the other lanes are our protein and our gene is working properly and expressing the protein, even if we don't induce it.