iGEM 2017 — Team NortheasternU-Boston

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# Team:NortheasternU-Boston/Notebook

[![](https://static.igem.org/mediawiki/2017/6/6c/T--NortheasternU-Boston--NEULogo2.png)](https://northeastern.edu)

NEU IGEM 2017
Expansion of Cell-Free Manufacturing with Post-Translational Modification

#### [Home](/Team%3ANortheasternU-Boston)

#### [Team](/Team%3ANortheasternU-Boston/Team)

#### [Collaborations](/Team%3ANortheasternU-Boston/Collaborations)

#### [Attributions](/Team%3ANortheasternU-Boston/Attributions)

#### [+ Project](#project)

* [Description](/Team%3ANortheasternU-Boston/Description)
* [Design](/Team%3ANortheasternU-Boston/Design)
* [Experiments](/Team%3ANortheasternU-Boston/Experiments)
* [Notebook](/Team%3ANortheasternU-Boston/Notebook)
* [Results](/Team%3ANortheasternU-Boston/Results)

#### [Parts](/Team%3ANortheasternU-Boston/Parts)

#### [Safety](/Team%3ANortheasternU-Boston/Safety)

#### [+ Human Practices](#human-practices)

* [Human Practices](/Team%3ANortheasternU-Boston/Human_Practices)
* [Education and Public Engagement](/Team%3ANortheasternU-Boston/Engagement)
* [Human Practices Silver](/Team%3ANortheasternU-Boston/HP/Silver)

#### [Judging Form](https://igem.org/2017_Judging_Form?team=NortheasternU-Boston)

## Notebook

Our notebook was written and maintained in Benchling, as we planned and performed experiments

Our Generous Sponsors

---

* [![Compass Therapeutics](https://static.igem.org/mediawiki/2017/3/33/T--NortheasternU-Boston--SponsorCompass.png)](http://compasstherapeutics.com "Compass Therapeutics")
* [![IDT Integrated DNA Technologies](https://static.igem.org/mediawiki/2017/8/84/T--NortheasternU-Boston--SponsorIDT.png)](https://www.idtdna.com/site "IDT Integrated DNA Technologies")
* [![NEB New England Biolabs](https://static.igem.org/mediawiki/2017/d/d7/T--NortheasternU-Boston--SponsorNEB.png)](https://www.neb.com "NEB New England Biolabs")
* [![Northeastern University College of Science](https://static.igem.org/mediawiki/2017/5/5e/T--NortheasternU-Boston--SponsorNEUCoS.png)](http://www.northeastern.edu/cos/ "Northeastern University College of Science")
* [![Northeastern University Department of Bioengineering](https://static.igem.org/mediawiki/2017/7/7d/T--NortheasternU-Boston--SponsorNEUBioE.png)](http://www.bioe.neu.edu "Northeastern Univeristy Department of Bioengineering")
* [![Northeastern University Department of Chemical Engineering](https://static.igem.org/mediawiki/2017/3/3a/T--NortheasternU-Boston--SponsorNEUChemE.png)](http://www.che.neu.edu "Northeastern University Department of Chemical Engineering")

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# Team:NortheasternU-Boston/Experiments

[![](https://static.igem.org/mediawiki/2017/6/6c/T--NortheasternU-Boston--NEULogo2.png)](https://northeastern.edu)

NEU IGEM 2017
Expansion of Cell-Free Manufacturing with Post-Translational Modification

#### [Home](/Team%3ANortheasternU-Boston)

#### [Team](/Team%3ANortheasternU-Boston/Team)

#### [Collaborations](/Team%3ANortheasternU-Boston/Collaborations)

#### [Attributions](/Team%3ANortheasternU-Boston/Attributions)

#### [+ Project](#project)

* [Description](/Team%3ANortheasternU-Boston/Description)
* [Design](/Team%3ANortheasternU-Boston/Design)
* [Experiments](/Team%3ANortheasternU-Boston/Experiments)
* [Notebook](/Team%3ANortheasternU-Boston/Notebook)
* [Results](/Team%3ANortheasternU-Boston/Results)

#### [Parts](/Team%3ANortheasternU-Boston/Parts)

#### [Safety](/Team%3ANortheasternU-Boston/Safety)

#### [+ Human Practices](#human-practices)

* [Human Practices](/Team%3ANortheasternU-Boston/Human_Practices)
* [Education and Public Engagement](/Team%3ANortheasternU-Boston/Engagement)
* [Human Practices Silver](/Team%3ANortheasternU-Boston/HP/Silver)

#### [Judging Form](https://igem.org/2017_Judging_Form?team=NortheasternU-Boston)

# Experiments

### GFP Expression Check

To verify the effectivity of the general schema for our DNA
[ Promoters Long ([BBa\_I712074](http://parts.igem.org/wiki/index.php?title=Part:BBa_I712074))
and Short ([BBa\_I719005](http://parts.igem.org/wiki/index.php?title=Part:BBa_I719005)),
5’ UTR ([BBa\_K1758100](http://parts.igem.org/wiki/index.php?title=Part:BBa_K1758100)),
Terminator ([BBa\_B0012](http://parts.igem.org/wiki/index.php?title=Part:BBa_B0012)) ]
we decided to test our system in a bacterial plasmid under T7 control via the IPTG inducible lac operon.

This experiment was conducted by first cloning plasmid components together.
A G-block was synthesized by IDT containing 5’UTR, Superfolder GFP
([BBa\_I746916](http://parts.igem.org/wiki/index.php?title=Part:BBa_I746916)),
and Terminator ([BBa\_B0012](http://parts.igem.org/wiki/index.php?title=Part:BBa_B0012)).
These were then amplified with overhang PCR via primers containing promoter sequences
for T7 polymerase, two separate promoters were tested: Long
([BBa\_I712074](http://parts.igem.org/wiki/index.php?title=Part:BBa_I712074))
and Short ([BBa\_I719005](http://parts.igem.org/wiki/index.php?title=Part:BBa_I719005)).
The now full translational units were amplified again with gibson primers for
insertion into pSB1C3 amplified with complimentary gibson primers.
These linear amplicons were verified via gel electrophoresis, purified,
and finally gibsoned together and transformed into NEB T7 express
competent E. coli. The transformed colonies were exposed to
broad spectrum UV radiation to excite GFP and phenotypically select for colonies
with the correct construct. Colonies with correct expression were taken into
liquid culture for larger scale verification of fluorescence and miniprep.

### AMP Bacterial Killing by OD600 Broad Investigation

Functional killing of AMPs was tested in a broad format.
Each of our Antimicrobial peptide candidates was constructed as a
G-block which contained the 5'UTR, the AMP gene, and the Terminator.
These were then amplified with tailed primers containing the promoter
sequences for the short and long promoter respectively.
These amplified constructs were verified via gel electrophoresis and
purified and quantified via Nanodrop.

This experiment was designed to broadly test the bacteriostatic effect of each
AMP under the control of each promoter and with or without the presence of the
methionine aminopeptidase enzyme. Antimicrobial peptides were and no template
controls were expressed over 4 hours at 37C using our in-house prepared cell-free
reaction mixture. A culture of BL21 E. coli was raised overnight in LB and then
diluted to apx 1\*10^8 CFU/ml. 90ul of the dilute culture was combined with 10ul
of each AMP reaction mixture and then placed in a plate reader shaking at 37C
for several hours. Cell-free reaction mixtures that had no template DNA were
also added to several wells as a growth control. OD600 was measured
every 15 minutes.

### AMP Bacterial Killing by OD600 Specific Investigation

The first bacterial killing assay had shown several AMP candidates which
potentially had a bacteriostatic effect. To verify this effect in a more
rigorously controlled experiment with replicates was conducted.
The AMPs most consistent across promoter type and presence or absence of
methionine aminopeptidase were: ISGCock\_Contig05\_0593
([BBa\_K2431011](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2431011)),
BP100 ([BBa\_K2431016](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2431016)),
and Micrococcin C7 ([BBa\_K2431010](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2431010)).
These AMPs and additional Methionine aminopeptidase
([BBa\_K2431018](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2431018))
were re-amplified from their respective G-blocks and subsequently purified using
the same process detailed above. Antimicrobial peptides and no template controls
were expressed in triplicate over 4 hours at 37C using our in-house prepared
cell-free reaction mixture. A culture of BL21 E. coli was raised overnight
in LB and then diluted to apx 1\*10^8 CFU/ml. 90ul of the dilute culture was
combined with 10ul of each AMP reaction mixture and then placed in a plate
reader shaking at 37C for 18 hours. Cell-free reaction mixtures that had no
template DNA were also added to several wells as a growth control.
OD600 was measured every 15 minutes.

### AMP Bacterial Killing by Plate using PURExpress

The goal of this experiment was to use the PURExpress system to produce the
AMPs of potential effect in the initial broad OD600 investigation.
Evaluate microbial killing effectivity of a droplet of the expression rxn
on a BL21 bacterial lawn in order to check whether or not the in-house
preparation of the cell free reaction mixture was effective at expressing AMPs.
PURExpress reactions were prepared for BP100
([BBa\_K2431016](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2431016)),
and Micrococcin C7
([BBa\_K2431010](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2431010))
with both promoter arrangements, a no DNA added reaction was also prepared as a control.
These reactions proceeded at 37C for 3 hours for expression to occur.
Reactions were then added to plates containing a lawn of BL21 E. coli.
For each reaction 1 20ul droplet of undiluted reaction mixture and 1 20ul
droplet of reaction mixture diluted 1:4 were added to separate plates.

Our Generous Sponsors

---

* [![Compass Therapeutics](https://static.igem.org/mediawiki/2017/3/33/T--NortheasternU-Boston--SponsorCompass.png)](http://compasstherapeutics.com "Compass Therapeutics")
* [![IDT Integrated DNA Technologies](https://static.igem.org/mediawiki/2017/8/84/T--NortheasternU-Boston--SponsorIDT.png)](https://www.idtdna.com/site "IDT Integrated DNA Technologies")
* [![NEB New England Biolabs](https://static.igem.org/mediawiki/2017/d/d7/T--NortheasternU-Boston--SponsorNEB.png)](https://www.neb.com "NEB New England Biolabs")
* [![Northeastern University College of Science](https://static.igem.org/mediawiki/2017/5/5e/T--NortheasternU-Boston--SponsorNEUCoS.png)](http://www.northeastern.edu/cos/ "Northeastern University College of Science")
* [![Northeastern University Department of Bioengineering](https://static.igem.org/mediawiki/2017/7/7d/T--NortheasternU-Boston--SponsorNEUBioE.png)](http://www.bioe.neu.edu "Northeastern Univeristy Department of Bioengineering")
* [![Northeastern University Department of Chemical Engineering](https://static.igem.org/mediawiki/2017/3/3a/T--NortheasternU-Boston--SponsorNEUChemE.png)](http://www.che.neu.edu "Northeastern University Department of Chemical Engineering")

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

[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:NortheasternU-Boston/Results

[![](https://static.igem.org/mediawiki/2017/6/6c/T--NortheasternU-Boston--NEULogo2.png)](https://northeastern.edu)

NEU IGEM 2017
Expansion of Cell-Free Manufacturing with Post-Translational Modification

#### [Home](/Team%3ANortheasternU-Boston)

#### [Team](/Team%3ANortheasternU-Boston/Team)

#### [Collaborations](/Team%3ANortheasternU-Boston/Collaborations)

#### [Attributions](/Team%3ANortheasternU-Boston/Attributions)

#### [+ Project](#project)

* [Description](/Team%3ANortheasternU-Boston/Description)
* [Design](/Team%3ANortheasternU-Boston/Design)
* [Experiments](/Team%3ANortheasternU-Boston/Experiments)
* [Notebook](/Team%3ANortheasternU-Boston/Notebook)
* [Results](/Team%3ANortheasternU-Boston/Results)

#### [Parts](/Team%3ANortheasternU-Boston/Parts)

#### [Safety](/Team%3ANortheasternU-Boston/Safety)

#### [+ Human Practices](#human-practices)

* [Human Practices](/Team%3ANortheasternU-Boston/Human_Practices)
* [Education and Public Engagement](/Team%3ANortheasternU-Boston/Engagement)
* [Human Practices Silver](/Team%3ANortheasternU-Boston/HP/Silver)

#### [Judging Form](https://igem.org/2017_Judging_Form?team=NortheasternU-Boston)

# Results

### GFP Expression in Cells

To verify that our DNA schema was appropriate for expression with the T7 promoter
we decided to validate it with an easy phenotypic readout: GFP in T7 express competent *E. coli.*

![](https://static.igem.org/mediawiki/2017/6/61/T--NortheasternU-Boston--GFPKit1.png)

Successful expression of GFP in our DNA system in T7 express E. coli under the long promoter
([BBa\_I712074](http://parts.igem.org/wiki/index.php?title=Part:BBa_I712074))
with the 5’UTR
([BBa\_K1758100](http://parts.igem.org/wiki/index.php?title=Part:BBa_K1758100))
superfolder GFP
([BBa\_I746916](http://parts.igem.org/wiki/index.php?title=Part:BBa_I746916))
and Terminator
([BBa\_B0012](http://parts.igem.org/wiki/index.php?title=Part:BBa_B0012))
in pSB1C3 vector.
The same DNA sequence but as a linear PCR amplicon was expressed using NEBs PURExpress kit

![](https://static.igem.org/mediawiki/2017/9/9e/T--NortheasternU-Boston--GFPKit2.png)

### Bacterial Killing Assay by OD600

![](https://static.igem.org/mediawiki/2017/0/0a/T--NortheasternU-Boston--ResultsFig1.png)

**Figure 1:** The OD of liquid cultures at 600nm over time in the presence
of cell-free AMP reactions. **L** and **S** indicate the long
([BBa\_I712074](http://parts.igem.org/wiki/index.php?title=Part:BBa_I712074))
and short promoter
([BBa\_I719005](http://parts.igem.org/wiki/index.php?title=Part:BBa_I719005)),
respectively. “MetAP” indicates that methionine aminopeptidase
([BBa\_K2431025](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2431025))
was present in the reaction. The control reaction contained all components of
the cell-free reaction except for input DNA. Error bars shown are 95% confidence intervals, n=4.

![](https://static.igem.org/mediawiki/2017/e/ea/T--NortheasternU-Boston--ResultsFig2.png)

**Figure 2:** The OD of liquid cultures at 600nm over time in the presence of ISGCock
([BBa\_K2431022](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2431022)) ,
BP100 ([BBa\_K2431002](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2431002)) ,
and Micrococcin ([BBa\_K2431000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2431000))
AMPs. **L** and **S** indicate the long
([BBa\_I712074](http://parts.igem.org/wiki/index.php?title=Part:BBa_I712074))
and short promoter ([BBa\_I719005](http://parts.igem.org/wiki/index.php?title=Part:BBa_I719005)),
respectively. “MetAP” indicates that methionine aminopeptidase
([BBa\_K2431025](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2431025))
was present in the reaction. The control reaction “Control average” contained
all components of the cell-free reaction except for input DNA. Error bars
shown are 95% confidence intervals, n=6. The reaction “MetAP Control Average”
contained all components of the cell-free reaction as well as MetAP DNA.
The reaction “Cells Only Control” contained only media and cells.

In the first attempt of the killing assay, we tested all twelve of our AMPs
under both long and short promoters and in the presence of MetAP.
The goal of this broader assay was to identify which AMP’s under which configuration
were effective. Most of the AMPs under all configurations had OD’s within range
of the 95% confidence interval of the control reaction. This control reaction
contained cell-free buffer and water at the same volume as the AMP reaction in
order to account for toxicity. However,
ISGCock ([BBa\_K2431022](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2431022)),
BP100 ([BBa\_K2431002](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2431002)),
and Micrococcin ([BBa\_K2431000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2431000))
showed slightly lower OD’s than the control at most, if not all, of the promoter and
MetAP configurations (Figure 1). However, due to the lack of DNA for replicates
of these AMP reactions, no statistical conclusions could be drawn. In addition,
for all of the AMPs there was a steady decrease in OD after about 5 hours,
leading to widely varied data points that could not be analyzed. This was likely
because of the evaporation of the media, due to a lack of a seal on the plate.
Therefore, the data shown in Figure 1 only includes data points up to that point.
In order to achieve replicates, we proceeded to do a second killing assay using only
ISGCock ([BBa\_K2431022](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2431022)),
BP100 ([BBa\_K2431002](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2431002)),
and Micrococcin ([BBa\_K2431000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2431000)).
Our second assay was run with sealed plates, which helped eliminated cell
death due to evaporation. However, this assay indicated no difference in cell death
between AMP reactions and control reactions (Figure 2). The OD’s for all reactions
were remarkably consistent, indicating that our AMP was most likely not expressed
at a meaningful level in our reactions.

In addition, the control containing only cells had a higher OD in general than
all reactions containing cell-free buffer components, indicating that any toxicity
was most likely due to the presence of this buffer. The discrepancy between the
two killing assays could be due to multiple factors. Firstly, the AMP DNA for
the second assay was prepared separately from the first assay. Any impurities
in the final DNA template, such as ethanol or guanidinium may have contributed
to cell death. Another factor that contribute to this discrepancy was the evaporation
of the liquid cell cultures. This evaporation could have led to cell death in
some reactions early on in the time-course, creating the appearance of a toxic AMP.
In addition, the first assay did not have replicates for the AMP reactions,
furthering the uncertainty in the effectiveness of the AMPs. In the second assay,
all AMP reactions were performed in triplicate and were consistently within 95%
confidence interval of all other reactions, save the cells only control.
Once again, this points to a lack of sufficient expression of the AMP and a small
toxic effect due to cell-free buffer conditions.

### AMP expressed via PURExpress Plate Based Killing Assay

In order to determine whether or not it was the AMPs or the cell free expression
reaction that was ineffective we expressed BP100
([BBa\_K2431002](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2431002))
and Micrococcin C7
([BBa\_K2431000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2431000))
using NEBs PURExpress cell-free protein expression kit using long and short promoters.
These reactions were then applied to a BL21 E. coli bacterial lawn on LB plates
at reaction concentration and at a 1:4 dilution. Plates were left to grow overnight at 37C.
Pictures below show that under both promoters and typically at both dilutions
both BP100 and Micrococcin C7 are effecitve at killing BL21 E. coli.
11L and 11S indicate BP100 expressed with the long and short promoters respectively.
12L and 12S indicate Micrococcin C7 expressed with the long and short promoters
respectively. “N” indicates the normal undiluted reaction concentration and “D”
indicates the diluted reaction mixture. No Temp plates were plates dosed with
control PURExpress reaction without any DNA template added.The success of the AMPs in killing bacteria in this assay once again indicates that the expression of AMP in the cell free reaction used in the liquid culture killing assay was not sufficient. However, this plate based assay shows the antimicrobial effects of both Micrococcin and BP100.

**Control: D & N**

![](https://static.igem.org/mediawiki/2017/2/28/T--NortheasternU-Boston--PlateControl1.png)

![](https://static.igem.org/mediawiki/2017/f/ff/T--NortheasternU-Boston--PlateControl2.png)

**Part 11: BP100
([BBa\_K2431002](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2431002))**

![](https://static.igem.org/mediawiki/2017/5/51/T--NortheasternU-Boston--11SN.png)

![](https://static.igem.org/mediawiki/2017/e/e9/T--NortheasternU-Boston--11SD.png)

![](https://static.igem.org/mediawiki/2017/6/6f/T--NortheasternU-Boston--11LN.png)

![](https://static.igem.org/mediawiki/2017/9/93/T--NortheasternU-Boston--11LD.png)

**Part 12: Micrococcin C7
([BBa\_K2431000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2431000))**

![](https://static.igem.org/mediawiki/2017/7/75/T--NortheasternU-Boston--12SN.png)

![](https://static.igem.org/mediawiki/2017/5/59/T--NortheasternU-Boston--12SD.png)

![](https://static.igem.org/mediawiki/2017/d/de/T--NortheasternU-Boston--12LN.png)

![](https://static.igem.org/mediawiki/2017/5/51/T--NortheasternU-Boston--12LD.png)

Our Generous Sponsors

---

* [![Compass Therapeutics](https://static.igem.org/mediawiki/2017/3/33/T--NortheasternU-Boston--SponsorCompass.png)](http://compasstherapeutics.com "Compass Therapeutics")
* [![IDT Integrated DNA Technologies](https://static.igem.org/mediawiki/2017/8/84/T--NortheasternU-Boston--SponsorIDT.png)](https://www.idtdna.com/site "IDT Integrated DNA Technologies")
* [![NEB New England Biolabs](https://static.igem.org/mediawiki/2017/d/d7/T--NortheasternU-Boston--SponsorNEB.png)](https://www.neb.com "NEB New England Biolabs")
* [![Northeastern University College of Science](https://static.igem.org/mediawiki/2017/5/5e/T--NortheasternU-Boston--SponsorNEUCoS.png)](http://www.northeastern.edu/cos/ "Northeastern University College of Science")
* [![Northeastern University Department of Bioengineering](https://static.igem.org/mediawiki/2017/7/7d/T--NortheasternU-Boston--SponsorNEUBioE.png)](http://www.bioe.neu.edu "Northeastern Univeristy Department of Bioengineering")
* [![Northeastern University Department of Chemical Engineering](https://static.igem.org/mediawiki/2017/3/3a/T--NortheasternU-Boston--SponsorNEUChemE.png)](http://www.che.neu.edu "Northeastern University Department of Chemical Engineering")

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