iGEM 2017 — Team Baltimore_Bio-Crew

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# Team:Baltimore Bio-Crew/Notebook

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[Home](https://2017.igem.org/Team%3ABaltimore_Bio-Crew)
[Team](https://2017.igem.org/Team%3ABaltimore_Bio-Crew/Team)
[Project](https://2017.igem.org/Team%3ABaltimore_Bio-Crew/Description)
[Results](https://2017.igem.org/Team%3ABaltimore_Bio-Crew/Results)
[Design](https://2017.igem.org/Team%3ABaltimore_Bio-Crew/Design)
[Notebook](https://2017.igem.org/Team%3ABaltimore_Bio-Crew/Notebook)

[Composite Part](https://2017.igem.org/Team%3ABaltimore_Bio-Crew/Composite_Part)
[Safety](https://2017.igem.org/Team%3ABaltimore_Bio-Crew/Safety)
[HP Silver](https://2017.igem.org/Team%3ABaltimore_Bio-Crew/HP/Silver)
 [Experiment](https://2017.igem.org/Team%3ABaltimore_Bio-Crew/Experiments)
 [Attribution](https://2017.igem.org/Team%3ABaltimore_Bio-Crew/Attributions)
[Collaborations](https://2017.igem.org/Team%3ABaltimore_Bio-Crew/Collaborations)
[Interlab](https://2017.igem.org/Team%3ABaltimore_Bio-Crew/InterLab)
[Contributions](https://2017.igem.org/Team%3ABaltimore_Bio-Crew/Contribution)

* [July](#july)
* [August](#august)
* [September](#september)
* [October](#october)

### NoteBook

#### July

7/01/17

Math:

* Worked on getting set up to start math modeling of plastic degradation. Ready to start next week
* Found possible alternatives to our plastic degrading enzymes
* Collaboration: College Park mentoring for plastic degrading simulation

Wiki:

* Created HTML for website from template (not implemented into wiki yet)
* Had difficulties getting the template code to work with the pre-set wiki code
* Emailed UMaryland team to ask for help with wiki programming.

Gene Design:

* Designed genes with Secretion Tag and Periplasmic Tag
* Optimized codons
* Waiting for genes to be shipped

Lab:

* Ran 30 colony PCR reactions
* Ready to run a gel on the PCR and send the successful transformations in for gene sequencing

Outreach and Fundraising:

* Created template for emailing companies
* Reached out to aquarium, Henry Hall program
* Contacted local high school for class time discussion

7/06/17

Lab:

* Prepared all 30 colony PCR samples to run on a gel
* Ran 16 samples, one possible success found
* Other 14 samples ready to be run on Saturday

7/08/17

Interlab:

* signed up
* made 8 chloramphenicol plates

Linearized Backbone Protocol:

* learned the anatomy of the different vectors
* performed restriction digest on vectors

Fundraising:

* found more companies to reach out to
* emailed enzyme assay companies about giving us a discount on assaying materials

Our Constructs:

* ran PCR samples 17-30 on gel. Currently waiting for results

Wiki:

* learned how to use iGEM wiki templates to link to more code files
* added navigation bar
* experimented with the code to learn more about how the iGEM wikis function

7/13/17

Interlab:

* Decided to use Kit Plate 7
* Resuspended 8 DNA samples from plate
* Samples used:
  + Positive Control (BBa\_I20270): well 21B
  + Negative Control (BBa\_R0040): well 21D
  + Test Device 1 (BBa\_J364000): well 21F
  + Test Device 2 (BBa\_J364001): well 21H
  + Test Device 3 (BBa\_J364002): well 21J
  + Test Device 4 (BBa\_J364003): well 21L
  + Test Device 5 (BBa\_J364004): well 21N
  + Test Device 6 (BBa\_J364005): well 21P
* Set up transformations for the 8 samples (transformations later found to be mostly unsuccessful. Did not produce many colonies)

7/15/17

Math:

* learned basics of enzyme kinetics
* started downloading MATLab

Lab:

* ligated new gBlocks with both vectors
* transformed gBlocks
* made more media for plates

Interlab:

* re-transformed all 8 samples

Wiki:

* Uploaded home page
* worked on color theme and logo ideas
* still need to get the rest of the pages up

Waxworms:

* students came in and measured waxworms

#### August

8/03/17

Lab

* Restriction digest of esterase, lipase, C3, and A3 were lost. SO, we started all over again (hopefully for the last time) and did a restriction digest of esterase and lipase again. Then, found C3 and A3 digested from a random previous restriction digestion.
* ran this restriction digest on a gel, and it looked good
* did a PCR of the esterase and lipase stock solutions to produce more.

8/05/17

Lab

* Used nanodrop to determine the DNA concentrations of our restricted digest of esterase, lipase, A3, and C3. Data is in Shatera’s notebook. We did this to determine the ratio of microliters of the vector and DNA insert to use for ligation.
* Worked out the ratio for ligations using stoichiometry
* Ligated using ratios.
* Transformed bacteria by heat shocking. We have two groups of the ligation and two groups of the transformed bacteria. After incubating the ligations in room temp for 30 min, the ligations were incubated with some of the bacteria (heat shocked for transformation). Then, after an hour, the bacteria were plated and grown. Then, with the other half of the ligations, we continued to incubate the ligations overnight at 44 degrees after sitting for 30 minutes in room temp. Then, we would transform the bacteria the next day. Then, after an hour, they should be plated to grow.

Presentation

* worked on putting together an outline of the presentation

8/10/17

Lab

* Bacteria Colony PCR- we finally got positives!
* Isolated plasmid DNA from colonies to screen as well (in case the PCRs aren’t working).

8/12/17

* Made DNA stocks of positive clones which means that we did plasmid isolation with the positive colonies and used nanodrop to determine concentrations a purity of plasmid. Then, ethanol and salts were added to the isolated plasmids to increase concentration. The plasmids were sent to sequencing to Johns Hopkins.
* Worked on presentation

8/17/17

* Took the tour of the inner harbor and the trash wheel, and learned about the efforts to clean the harbor

8/19/17

* worked on completing the presentation slides and script

8/26/17

* worked on completing the presentation slides and script

#### September

9/02/17

Lab:

* Recloning the Lipase:
  + PCR Amplified: lipase g-block, previously digested lipase, C3 vector from J04450 plasmid, and A3 vector from J04450 plasmid
  + Miniprepped A3 + esterase plasmids to be used as a control for the PCR gel
  + Ran PCR gel (1 ul loading dye in each sample). Gel Key:
    1. Ladder (1 ul)
    2. C3 vector (5 ul)
    3. A3 vector (5 ul)
    4. lipase from previous digest (5 ul)
    5. lipase from g-block (5 ul)
  + PCR purified all samples
  + Ran Nanodrop on PCR purification results. Nanodrop results:
    1. C3: 16.4ng/ul
    2. A3: 19.1ng/ul
    3. lipase digested: 48.1ng/ul
    4. lipase g-block: 25.0 ng/ul
    5. 1st esterase A3 plasmid miniprep: 30.4ng/ul
    6. 2nd esterase A3 plasmid miniprep: 52.6ng/ul
  + Diluted all samples to 10ng/ul
  + Ran restriction digest on all samples
* Purified esterase enzymes
* Ran protein gel of esterase enzymes. Gel key:
  1. standard 20ul
  2. un-induced 20ul
  3. induced 20ul
  4. un-induced 40ul
  5. induced 40ul
* Working Interlab- Must start over the initial protocol with the spectrometer and incubation for 2, 4, 6 hours.

Presentation:

* rehersed

Wiki:

* worked on design and color scheme

9/06/17

Purification of Protein and Interlab:

* Grew up transformed bacteria that we know have our esterase gene! We grew the bacteria in 5mL overnight cultures. We used PSB1A3 + number 4 and PSB1A3 + number 6. PSB1C3 + number 5 did not grow.
* Then, we added 2mL of overnight culture into 50 mL of LB AMP and we let it grow. Then, we checked the OD of our cultures so it could get to .6:
  + A4
    1. .140 - 10:15
    2. .402 - 11:15
    3. .67 - 12:15
  + A6
    1. .144- 10:15
    2. .385 - 11:15
    3. .740 - 12:15
* We induced our four flasks. 2 were induced with 1 mM IPTG 62.5 microliter of .8 M stock. Then, 2 were not induced
* After each time point of 0, 1, 2, 3, 4 in a shaker, we obtained 6 mL from all four flasks. We put one mL of bacteria in 4 microcentrifuge tubes from each flask. Then, we placed 5mL from all four flasks into four tubes. We centrifuges all eight tubes in centrifuge for 10 min at 400 speed to form pellets of cells. Then, the microcentrifuge tubes will be used in a gel. Then, the tubes will be frozen and later lysed and purified using a nickel column to verify the length of proteins; this will happen on another day. For now, the pellets are frozen. The group of samples that will be used for the gel will be ran on a gel another day as well.

9/16/17

Interlab:

* Started on cell measurement protocol
* Protocol was followed incorrectly and interlab would need to be re-done later

Lab:

* Ran protein gel of clones:
  + A3+Esterase #4
  + A3+Esterase #6

9/21/17

Lab:

* Ran colony PCR of Lipase+C3 plasmid

9/22/17

Learned more about micro plastics at a seminar at the Baltimore Underground Science Space

9/23/17

Started on interlab again:

* Took several timepoints:
  + t0 = 10:30
  + t1 = 12:30
  + t2 = 2:30
  + t3 = 4:30
* measured florescence at each timepoint and recorded it in the provided spreadsheet

Lab:

* ran protein gel for Esterase+C3 #5. Loaded 10ul of each sample, 5ul ladder. Gel Key:
  1. protein ladder (NEB Broadspectrum)
  2. psb1C3+Esterase #5 uninduced timepoint 4hr (sample 2 test)
  3. psb1C3+Esterase #5 uninduced timepoint 4hr (sample 3 test)
  4. psb1C3+Esterase #5 uninduced timepoint 4hr (Elute 1)
  5. psb1C3+Esterase #5 uninduced timepoint 4hr (Elute 2)
  6. psb1C3+Esterase #5 induced timepoint 4hr (test sample 1)
  7. psb1C3+Esterase #5 induced timepoint 4hr (test sample 2)
  8. psb1C3+Esterase #5 induced timepoint 4hr (test sample 3)
  9. psb1C3+Esterase #5 induced timepoint 4hr (Elute 1)
  10. psb1C3+Esterase #5 induced timepoint 4hr (Elute2)

9/28/17

Interlab:

* OD600 reference point data lost. Went through protocol again.
* Submitted interlab data. not accepted because we didn't have time to do enough replicates.

9/30/17

Due to interlab being no longer an option to complete our requirements, we considered other options to meet these requirements.

* for our contribution requirement, we decided to write a protocol for testing for allergenicity in different proteins. This would make it easy for other teams to see if their biobricks would produce proteins that could contain allergens, and allow us to characterize other parts by testing for allergens in them.

Lab:

* Because our protein gels were consistently negative for esterase, we hypothesized that the enzyme was toxic to E.coli. To test this, we decided to try transcribing and translating our esterase constructs using a cell free kit. We also wanted to sequence our biobricks by themselves, so that we could be sure that there were no errors in them before we started. Because of this, we needed to PCR amplify our biobricks to get more DNA to work with. PCR amplified:
  1. esterase biobrick (2017)
  2. lipase biobrick (2017)
  3. esterase biobrick (2016)
  4. lipase biobrick (2016)
* Ran gel of PCR

#### October

10/05/17

Lab:

* gel showed PCR to be unsuccessful. The wrong primers were used to amplify.
* Re-did PCR with correct primers
* Ran gel of PCR. Used 1ul of each ladder and 10ul of each sample. Gel key:
  1. 1kb ladder
  2. X
  3. Lipase 2017 1
  4. Lipase 2017 2
  5. X
  6. Esterase 2017 1
  7. Esterase 2017 2
  8. X
  9. Lipase 2016 1
  10. Lipase 2016 2
  11. X
  12. Esterase 2016 1
  13. Esterase 2016 2
  14. X
  15. 100bp ladder

10/07/17

Presented to the public and the UMaryland iGEM team for Baltimore Innovation Week. Received feedback on project and presentaion

Lab:

* Gel purified samples L1, L2, E1, and E2
* Ran a gel with gel purified samples. Ran 6ul of each sample. Gel key:
  1. ladder
  2. X
  3. L1
  4. L2
  5. X
  6. E1
  7. E2
* Took nanodrop concentrations of the gel purified samples.
* Despite unreliable readings from the nano drop, all of the results for the gel were positive.

10/14/17

helped run a table at the FamFest event at the Y, and talked about synthetic biology with the public

10/19/17

Lab:

* The concentrations of three samples, were taken on a nano drop. The two unmodified biobricks from this year, L1 and E1, did not have a reliable measurement on the nano drop. However, the successful plasmid from this year, CE 5, did have a good nano drop concentration reading. Because of this, we decided to only run the cell free kit on the CE 5 plasmid.
* Sample CE 5 was transcribed and translated in-vitro using a cell free kit.

10/21/17

Lab:

* Ran protein gel of the cell free protein expression samples and the time course samples for the positive control bacteria and induced engineered E.coli, some induced with lactose and some induced with IPTG.
* Gel 1 Key:
  1. ladder
  2. control 2
  3. control 3
  4. control4
  5. CE5-1 IPTG 2hr
  6. CE5-2 IPTG 2hr
  7. CE5-3 IPTG 2hr
  8. CE5-1 lac 2hr
  9. CE5-2 lac 2hr
  10. CE5-3 lac 2hr
* Gel 2 Key:
  1. CE5-1 IPTG 5hr
  2. CE5-2 IPTG 5hr
  3. CE5-3 IPTG 5hr
  4. ladder
  5. CE5-1 lac 5hr
  6. CE5-2 lac 5hr
  7. CE5-3 lac 5hr
  8. X
  9. X
  10. X
* Gel 3 Key:
  1. CE5-1 IPTG
  2. CE5-2 IPTG
  3. CE5-3 IPTG
  4. CE5-1 lac
  5. CE5-2 lac
  6. CE5-3 lac
  7. ladder
  8. X
  9. CE5 (in-vitro)
  10. positive control

10/26/17

Wiki:

* compiled a list of what still needed to be done

Completed Judging form and worked on finishing up poster

10/28/17

Lab:

* Completed flouricine diacetate enzyme assay on the products of cell free expression by the Esterase and Lipase biobricks. Showed positive for enzymes!

Wiki:

* worked on getting finished up
* put together protocols page content, project description content, and notebook page

## Sponsors

#### The Baltimore Bio-Crew thanks our sponsors for their generous support of our team that made our project and travel to the Jamboree possible. Thank you!

[![BD Medical Technology, Advancing the World of Health - BD](https://upload.wikimedia.org/wikipedia/en/f/f8/Update_Color_BD_PNG_Logo.png)](http://www.bd.com/en-us)
[![Family League of Baltimore](http://baltimoreattendance.org/wp-content/uploads/2015/08/flbcinc-360x230.png)](http://familyleague.org/)
![Fabian Kolker Foundation](https://static.igem.org/mediawiki/2017/6/6c/T--Baltimore_Bio-Crew--fabian_kolker_small_icon.png)
[![VWR Charitable Foundation](https://static.igem.org/mediawiki/2016/1/1a/T--Baltimore_Biocrew--VWR_Foundation_LOGO.jpeg)](http://vwrfoundation.org/)
[![Maryland Recycling Network](https://media.licdn.com/mpr/mpr/shrink_200_200/AAEAAQAAAAAAAAI8AAAAJDY0ZDg0ZjlkLWVlMTItNGI1Mi1iNWEwLWYzMDVlYWMwMTZhZg.png)](http://www.marylandrecyclingnetwork.org/)
[![The Robert W. Deutsch Foundation](https://static.igem.org/mediawiki/2016/6/65/T--Baltimore_BioCrew--DeutschFoundation.png)](https://www.rwdfoundation.org/)

[![](https://static.igem.org/mediawiki/2017/b/bf/HQ_menu_logo.jpg)](https://2017.igem.org)


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# Team:Baltimore Bio-Crew/Experiments

![BUGSS Logo](https://static.igem.org/mediawiki/2017/e/e2/Baltimore_biocre_BugssLOGO.png)

[Home](https://2017.igem.org/Team%3ABaltimore_Bio-Crew)
[Team](https://2017.igem.org/Team%3ABaltimore_Bio-Crew/Team)
[Project](https://2017.igem.org/Team%3ABaltimore_Bio-Crew/Description)
[Results](https://2017.igem.org/Team%3ABaltimore_Bio-Crew/Results)
[Design](https://2017.igem.org/Team%3ABaltimore_Bio-Crew/Design)
[Notebook](https://2017.igem.org/Team%3ABaltimore_Bio-Crew/Notebook)

[Composite Part](https://2017.igem.org/Team%3ABaltimore_Bio-Crew/Composite_Part)
[Safety](https://2017.igem.org/Team%3ABaltimore_Bio-Crew/Safety)
[HP Silver](https://2017.igem.org/Team%3ABaltimore_Bio-Crew/HP/Silver)
 [Experiment](https://2017.igem.org/Team%3ABaltimore_Bio-Crew/Experiments)
 [Attribution](https://2017.igem.org/Team%3ABaltimore_Bio-Crew/Attributions)
[Collaborations](https://2017.igem.org/Team%3ABaltimore_Bio-Crew/Collaborations)
[Interlab](https://2017.igem.org/Team%3ABaltimore_Bio-Crew/InterLab)
[Contributions](https://2017.igem.org/Team%3ABaltimore_Bio-Crew/Contribution)

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# Experiment

![](https://static.igem.org/mediawiki/2017/archive/9/9c/20170930173029%21E2.jpeg)

![](https://static.igem.org/mediawiki/2017/e/e8/Baltimore_biocrewexperiment234.jpeg)

![](https://static.igem.org/mediawiki/2017/e/e4/Baltimorebiocrewexperiment7890.jpeg)

### Protocols

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

All plasmid purifications were done using the QIAprep spin mini prep kit protocol.

Restriction Digest

Master mix:

1. 76 uL H2O
2. 20 uL 10x NEB2
3. 1 uL EcoR1
4. 1 uL Pst1
5. 1 uL Dpn1
6. 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

Transformation

We used the iGEM Parts Registry Transformation Protocol: <https://static.igem.org/mediawiki/parts/6/67/IGEM_Registry_-_Transformation_Protocol.pdf>

Changes made: Recovery tubes were made using 500 uL SOC media instead of 200 uL

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 Protocol

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

In Vitro Transcription Translation

Protocol using NEB’s PURExpress kit-

<https://www.neb.com/protocols/0001/01/01/protein-synthesis-reaction-using-purexpress-e6800>

Changes made: RNA Polymerase was added during step two. A control sample was made using no RNA Polymerase.

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.

Allergenicity Testing Protocol

Getting protein Sequence:

1. Use <http://www.fr33.net/translator.php> to convert nucleotide sequence to protein sequences
2. Plug in output sequences into NCBI BLAST to verify which protein sequence is the intended sequence.

Examining Allergenicity

1. Copy nucleotide sequence for the gene that is to be tested.
2. Click on a new tab, and go onto Translator by clicking this link: <http://www.fr33.net/translator.php>. Paste the nucleotide sequence into the box and press translate. This will change your nucleotides into amino acids. Copy this amino acid chain UNTIL the first asterisk (leave out the asterick).Copy this sequence.
3. Then, click on a new tab, and go onto the NCIB Blast by clicking this link: <https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins>. Paste the amino acid chain in the box and press BLAST. This could take a couple seconds for the results to come up. This is used to verify the identity of the protein that is going to be used later in the protocol. The identity of the amino acid chain can be seen by comparing the first result of the NCIB Blast matches the same name as the biobrick name on the IGEM website. If it does match then continue to the next step. If it does not check another translation found in part 2.
4. Click new tab, and go onto AllergenOnline by following this link: <http://www.allergenonline.org/index.shtml>
5. Press Search for full-length alignments by FASTA, and copy and paste your amino acid chain (The same amino acid chain from step 3) into the box. Make sure the bubble that says "Full Fasta" is hit and press submit.
6. This will open up another window. Look at the % identity and % of similarity. If there is a % identity higher than 50% than, the biobrick potentially can be an allergen. If not, then your biobrick is less likely to be an allergen.
7. Now, we will go back to the main page of the AllergenOnline website by clicking this link: <http://www.allergenonline.org/index.shtml>. Then, press Search for 80 amino acid alignments by FASTFA.
8. Paste the amino acid chain (the same chain from steps 3, and 5) into the box and make sure the second bubble is filled in called sliding 80mer window and press submit.
9. The results will open up in a new tab again, and look at and record the % identity and % similarity in the same google document. If the % identity is less than 35%, then the biobrick is less likely to be allergens.

---

## Sponsors

#### The Baltimore Bio-Crew thanks our sponsors for their generous support of our team that made our project and travel to the Jamboree possible. Thank you!

[![BD Medical Technology, Advancing the World of Health - BD](https://upload.wikimedia.org/wikipedia/en/f/f8/Update_Color_BD_PNG_Logo.png)](http://www.bd.com/en-us)
[![Family League of Baltimore](http://baltimoreattendance.org/wp-content/uploads/2015/08/flbcinc-360x230.png)](http://familyleague.org/)
![Fabian Kolker Foundation](https://static.igem.org/mediawiki/2017/6/6c/T--Baltimore_Bio-Crew--fabian_kolker_small_icon.png)
[![VWR Charitable Foundation](https://static.igem.org/mediawiki/2016/1/1a/T--Baltimore_Biocrew--VWR_Foundation_LOGO.jpeg)](http://vwrfoundation.org/)
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# Team:Baltimore Bio-Crew/Results

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# BALTIMORE BIO-CREW

#### Bio-Engineering E.Coli To Degrade Plastic and Save The Baltimore Inner Harbor

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### Results

Working hard, we managed to successfully clone the Esterase gene into E.coli. Both colony PCR and sequencing were positive for the CE 5 esterase clone. However, Lipase was not successfully cloned and the sequences came up negative and truncated.

![](https://static.igem.org/mediawiki/2017/e/ee/BaltimoreBiocrewFigure1.png)

Due to the negative results with the lipase, we hypothesized that the lipase enzyme was toxic to the strain of E. coli we were using, causing it to reject the DNA.

We started inducing the positive esterase clone to produce protein, and running the protein produced on a protein gel after purifying on a nickel column. After running multiple protein gels with the esterase, no positive results were found.

Because of this, we hypothesized that the E. coli cells were not producing the esterase because the enzyme was also harmful to the cells.

![](https://static.igem.org/mediawiki/2017/5/5e/T--Baltimore_Bio-Crew--results_fig2.jpg)

To bypass the cell issue and test whether or not our constructs could actually produce the protein, we decided to test the constructs and biobricks on their own by translating them using a cell free kit, and then running the product of that on a protein gel. The protein gel results after the cell free kit were inconclusive, possibly because the cell just wasn’t producing enough protein to show up on the gel.

![](https://static.igem.org/mediawiki/2017/e/ec/T--Baltimore_Bio-Crew--results_fig3.jpg)

We decided to run the cell free kit products through a FDA enzyme test. Fluorescence was seen, indicating that enzyme was being produced! The results of this test are shown on a bar graph below

![](https://static.igem.org/mediawiki/2017/3/38/T--Baltimore_Bio-Crew--results_fig4.jpg)

Additional tests will need to be done, but the FDA test indicated that our constructs can produce protein, even if the enzymes may not be able to be produced in E. coli

---

## Sponsors

#### The Baltimore Bio-Crew thanks our sponsors for their generous support of our team that made our project and travel to the Jamboree possible. Thank you!

[![BD Medical Technology, Advancing the World of Health - BD](https://upload.wikimedia.org/wikipedia/en/f/f8/Update_Color_BD_PNG_Logo.png)](http://www.bd.com/en-us)
[![Family League of Baltimore](http://baltimoreattendance.org/wp-content/uploads/2015/08/flbcinc-360x230.png)](http://familyleague.org/)
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