iGEM 2019 — Team Tec-Chihuahua

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# Team:Tec-Chihuahua/Notebook

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  [Demonstrate](/Team%3ATec-Chihuahua/Demonstrate)
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* Human Practices

  [Human Practices](/Team%3ATec-Chihuahua/Human_Practices)
  [Public Engagement](/Team%3ATec-Chihuahua/Public_Engagement)
* [Model](/Team%3ATec-Chihuahua/Model)
* [Entrepreneurship](/Team%3ATec-Chihuahua/Entrepreneurship)
* Judging

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* [Wiki](/Team%3ATec-Chihuahua/Wiki)

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# Notebook

# Notebook

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# Tec-Chihuahua © 2019

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# Team:Tec-Chihuahua/Experiments

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* Project

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  [Brainstorm](/Team%3ATec-Chihuahua/Brainstorming)
* Lab

  [Notebook](/Team%3ATec-Chihuahua/Notebook)
  [Experiments](/Team%3ATec-Chihuahua/Experiments)
  [Characterization](/Team%3ATec-Chihuahua/Contribution)
  [Lab Results](/Team%3ATec-Chihuahua/Results)
  [Demonstrate](/Team%3ATec-Chihuahua/Demonstrate)
  [Safety](/Team%3ATec-Chihuahua/Safety)
* Human Practices

  [Human Practices](/Team%3ATec-Chihuahua/Human_Practices)
  [Public Engagement](/Team%3ATec-Chihuahua/Public_Engagement)
* [Model](/Team%3ATec-Chihuahua/Model)
* [Entrepreneurship](/Team%3ATec-Chihuahua/Entrepreneurship)
* Judging

  [Awards](/Team%3ATec-Chihuahua/Requirements)
* [Wiki](/Team%3ATec-Chihuahua/Wiki)

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# experiments

# Protocols

[### Cell Transformation](#collapse1)

[### Chemically Competent Cells](#collapse2)

[### Colony PCR](#collapse3)

[### IPTG Protein Induction](#collapse4)

[### Ligation](#collapse5)

[### Restriction Digest](#collapse6)

[### SDS Page](#collapse7)

[### Soluble Protein Extraction](#collapse8)

[### PureLink® Quick Plasmid Miniprep Kits](#collapse9)

[### IC50 antimicrobial assay Benzimidazole](#collapse10)

[### Growth Kinetics *V. dahliae*](#collapse11)

[### AMPs Inhibition Assay against Bacteria](#collapse12)

[### AMPs Inhibition Assay against Fungus](#collapse13)

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# Tec-Chihuahua © 2019

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# Team:Tec-Chihuahua/Results

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* Project

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* Lab

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  [Lab Results](/Team%3ATec-Chihuahua/Results)
  [Demonstrate](/Team%3ATec-Chihuahua/Demonstrate)
  [Safety](/Team%3ATec-Chihuahua/Safety)
* Human Practices

  [Human Practices](/Team%3ATec-Chihuahua/Human_Practices)
  [Public Engagement](/Team%3ATec-Chihuahua/Public_Engagement)
* [Model](/Team%3ATec-Chihuahua/Model)
* [Entrepreneurship](/Team%3ATec-Chihuahua/Entrepreneurship)
* Judging

  [Awards](/Team%3ATec-Chihuahua/Requirements)
* [Wiki](/Team%3ATec-Chihuahua/Wiki)

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# Results

# Achievements

* The construction of five new BioBricks™ with sequences that are reported to code for our
  proteins of
  interest:

+ [BBa\_K2959004](http://parts.igem.org/Part%3ABBa_K2959004)
+ [BBa\_K2959005](http://parts.igem.org/Part%3ABBa_K2959005)
+ [BBa\_K2959007](http://parts.igem.org/Part%3ABBa_K2959007)
+ [BBa\_K2959008](http://parts.igem.org/Part%3ABBa_K2959008)
+ [BBa\_K2959010](http://parts.igem.org/Part%3ABBa_K2959010)

* The correct transformation of *E. coli* expression strain SHuffle T7 Express®️ for parts
  [BBa\_K2959005](http://parts.igem.org/Part%3ABBa_K2959005) (PsDef1-
  Erv1p) and [BBa\_K2959007](http://parts.igem.org/Part%3ABBa_K2959007)
  (WAMP1b); and the correct transformation of BL21 (DE3) Competent *E.
  coli* for part
  [BBa\_K2959010](http://parts.igem.org/Part%3ABBa_K2959010) (AtPFN1).
* Protein production characterized in part [BBa\_R0010](http://parts.igem.org/Part%3ABBa_R0010), a lacI regulated promoter, in *E. coli* expression
  strain
  SHuffle T7 Express®️, confirming IPTG induction functionality in our parts.
* The amplification of our BioBrick™ sequences through colony PCR performed upon our transformed
  cells
  to
  confirm the presence of our expression plasmids.
* Corroboration of protein expression in *E. coli* BL21 (DE3) transformed with [BBa\_ K2959010](http://parts.igem.org/Part%3ABBa_K2959010)
  through an SDS-
  PAGE at 12%.
* The antifungal activity was proved with the three peptides: AtPFN1 [[BBa\_K2959010]](http://parts.igem.org/Part%3ABBa_K2959010),PsDef1 [[BBa\_K2959005]](http://parts.igem.org/Part%3ABBa_K2959005),WAMP1b [[BBa\_K2959007]](http://parts.igem.org/Part%3ABBa_K2959007) against *V.dahliae*.

# BioBrick™ Assembly

Three of our DNA sequences (AtPFN1, PsDef1-Erv1p, and WAMP1b) were synthesized by IDT®️ with the
BioBrick™ prefix and
suffix flanking the composite part. This made possible the correct digestion with restriction
enzymes
EcoRI-HF and
PstI. After the digestion, ligation was performed with T7 ligase in order to place our constructs
into
the pSB1C3
linearized backbone with chloramphenicol resistance, which was previously digested with the same
restriction enzymes.
Using the SnapGene®️ software, we could model our ligated expression plasmids, and the final parts
resulted in a
sequence of 2,590 bp for WAMP1b, 2,651bp for AtPFN1 and 3,106 bp for PsDef1-Erv1p. Thereupon,
*Escherichia coli*
[BL21(DE3) for AtPFN1 and SHuffle for PsDef1+Erv1p and WAMP1b] cultures were transformed by heat
shock
for following
antibiotic selection of clones.

![](https://static.igem.org/mediawiki/parts/d/d3/T--Tec-Chihuahua--WAMP1bff.png)

## a)

![](https://static.igem.org/mediawiki/parts/2/21/T--Tec-Chihuahua--AtPFN1ff.png)

## b)

![](https://static.igem.org/mediawiki/parts/7/77/T--Tec-Chihuahua--PsDef-Erv1pff.png)

## c)

**Figure 1.** SnapGene®️ maps of a)
BioBrick™ [BBa\_K2959007](http://parts.igem.org/Part%3ABBa_K2959007), b)
BioBrick™ [BBa\_K2959010](http://parts.igem.org/Part%3ABBa_K2959010), and c)
BioBrick™ [BBa\_K2959005](http://parts.igem.org/Part%3ABBa_K2959005) ligated
into backbone
pSB1C3.

The next step was to amplify our BioBrick™ sequences through colony PCR performed upon our
transformed
cells to confirm
the presence of our expression plasmids inside of our chassis. With the help of the specific forward
BioBrick™ prefix
[[BBa\_G1004](http://parts.igem.org/Part%3ABBa_G1004)] and the specific
reverse BioBrick™ suffix [[BBa\_G1005](http://parts.igem.org/Part%3ABBa_G1005)], we were able to amplify our sequences
exclusively.
Through agarose gels we confirmed the correct transformation. The PCR action from SnapGene®️ was
used to
predict the
size of the amplified sequences which resulted in a size of 579 bp for WAMP1b, 640 bp for AtPFN1,
and
1,095 bp for
PsDef1+Erv1p.

![](https://static.igem.org/mediawiki/parts/c/c5/T--Tec-Chihuahua--wamp1b.pcr.ff.png)

![](https://static.igem.org/mediawiki/parts/3/3f/T--Tec-Chihuahua--ELECTROFORESISWAMP1b.jpeg)

**Figure 2.** (On top)
SnapGene®️ amplification through PCR of [BBa\_K2959007](http://parts.igem.org/Part%3ABBa_K2959007). (Left) Agarose gel electrophoresis of
[BBa\_K2959007](http://parts.igem.org/Part%3ABBa_K2959007) compared with
NEB Quick-Load Purple 1Kb Plus DNA Ladder, where the highlighted band corresponds to approximately
579
bp.

![](https://static.igem.org/mediawiki/parts/4/40/T--Tec-Chihuahua--atpfn.pcr.png)

![](https://static.igem.org/mediawiki/parts/d/d2/T--Tec-Chihuahua--ELECTROFORESISAtPFN.jpeg)

**Figure 3.** (On top) SnapGene®️ amplification through PCR of [BBa\_K2959010](http://parts.igem.org/Part%3ABBa_K2959010). (Left) Agarose
gel
electrophoresis of
[BBa\_K2959010](http://parts.igem.org/Part%3ABBa_K2959010) compared with NEB
Quick-Load Purple 1Kb Plus DNA Ladder, where the highlighted band
corresponds
to
approximately 640 bp.

![](https://static.igem.org/mediawiki/parts/5/5c/T--Tec-Chihuahua--psdef1.pcr..png)

![](https://static.igem.org/mediawiki/parts/e/ec/T--Tec-Chihuahua--ELECTROFORESISPsDefE.jpeg)

**Figure 4.** (On top) SnapGene®️ amplification through PCR of [BBa\_K2959005](http://parts.igem.org/Part%3ABBa_K2959005). (Left) Agarose
gel
electrophoresis of
[BBa\_K2959005](http://parts.igem.org/Part%3ABBa_K2959005) compared with NEB
Quick-Load Purple 1Kb Plus DNA Ladder, where the highlighted band
corresponds to
approximately 1,095 bp.

# Protein Production

### IPTG Induction and Extraction

Following the construction of each BioBrick™, it was necessary to induce protein production. Since
T7
promoter for
AtPFN1 is used due its high levels of transcription in *E. coli* BL21 (DE3), isopropyl β-D-1
thiogalactopyranoside (IPTG)
is employed as an inducer for T7 RNA polymerase production. The concentration of IPTG utilized was
0.2
mM, followed by
incubating the cultures at 37°C and 225 rpm for 5 hours. Production of PsDef1 and WAMP1b was induced
under the lacI
regulated promoter in *E. coli* SHuffle cells with IPTG 0.2 mM and incubated at 30°C and 225
rpm
for 5 hours. This was
followed by protein extraction by lysis solution to which lysozyme was added, in order to obtain our
soluble peptides.

### SDS-PAGE

Electrophoresis in a 12% polyacrylamide gel was performed (**Figure 5**) in order to corroborate
that the protein of
interest was
indeed expressed. Each well was loaded with 50 μg of total protein from the soluble extracts of the
cell
lysates.

![](https://static.igem.org/mediawiki/parts/b/b4/T--Tec-Chihuahua--Gel.jpeg)

**Figure 5.** Expression of AtPFN1 visualized in a SDS-PAGE (12%) of *E. coli* BL21
(DE3) transformed with [BBa\_K2959010](http://parts.igem.org/Part%3ABBa_K2959010) (**Negative control:**  *E.coli* BL21 (DE3) untransformed cells, **1.** ATPFN1 transformed cells without induction, **2.** ATFPN1 transformed cells with 0.2mM of IPTG, **3.** ATFPN1 transformed cells with 0.4mM of IPTG, **4.** ATFPN1 transformed cells with 1mM of IPTG, **5.** ATFPN1 transformed cells with 3mM of IPTG, **6.** ATFPN1 transformed cells with 3mM of IPTG).

As shown, bands are present at the approximate weight of 14.3 kDa which corresponds to
AtPFN1 as
corroborated by UniProt. Visible bands of AtPFN1 correspond to protein extracts of transformed *E.
coli* BL21 (DE3)
induced with different concentrations of IPTG. No band can be appreciated in the first column
following
the protein
ladder Precision Plus ProteinTM Dual Xtra Prestrained Protein Standars, which belongs to a control of untransformed BL21 (DE3) cells, confirming that the bands are
expressed
exclusively in transformed cells and are indeed AtPFN1. No basal level expression was observed on
the
uninduced control
(transformed cells with no IPTG) as seen in column 1. Therefore, functionality of part [BBa\_K2959010](http://parts.igem.org/Part%3ABBa_K2959010) as a
generator of
AtPFN1 under induction by IPTG was confirmed.

For parts [BBa\_K2959005](http://parts.igem.org/Part%3ABBa_K2959005) and [BBa\_K2959007](http://parts.igem.org/Part%3ABBa_K2959007), visualization
of PsDef1 and WAMP1b in a polyacrylamide gel
could not be
carried out given the small size of the peptides, 9 and 4.5 kDa respectively. Due to a lack of
reagents
and equipment,
we were unable to adapt our SDS-PAGE protocol and optimize the visualization of small proteins.
However,
to confirm
that our peptides were being expressed, we characterized the lacI regulated promoter ([BBa\_R0010](http://parts.igem.org/Part%3ABBa_R0010)), which
controls
expression of these two parts, in *E. coli* SHuffle. The functionality of this promoter as well
as
ideal IPTG
concentrations and incubation temperatures were confirmed. Thus, [BBa\_K2959005](http://parts.igem.org/Part%3ABBa_K2959005) and [BBa\_K2959007](http://parts.igem.org/Part%3ABBa_K2959007) work as
generators of
PsDef1 and WAMP1b when induced with 0.2 or 0.4 mM IPTG at 30°C. Read more about it in our
[Characterization](https://2019.igem.org/Team%3ATec-Chihuahua/Contribution)page.

# Demonstrate

In addition to the molecular characterization, we decided to characterize our parts in a more realistic
environment by confronting them against the fungus. For this reason, we decided to set up a test on a
microplate where the confrontation of our peptides adjacent to *V.dahliae* took place. Here,
peptides’ inhibition capability was detected through a growth curve of the fungus.
Derived from this, we could achieve positive results where two or more concentrations of our peptides (raw
extraction) showed some kind of inhibition: for more info click the button below.

[Demonstrate](https://2019.igem.org/Team%3ATec-Chihuahua/Demonstrate)

# Successes & Failures

During our iGEM experience, we learned a lot of unique things that led to significant
achievements after facing unexpected obstacles. After months of hard work, we
believe the effort was worthwhile.

Successes

* The antifungal activity was proved with the three peptides: AtPFN1 [[BBa\_K2959010]](http://parts.igem.org/Part%3ABBa_K2959010), PsDef1 [[BBa\_K2959005]](http://parts.igem.org/Part%3ABBa_K2959005), WAMP1b [[BBa\_K2959007]](http://parts.igem.org/Part%3ABBa_K2959007) against *V.dahliae.*
* Characterization of LacI regulated promoter ([BBa\_R0010](http://parts.igem.org/Part%3ABBa_R0010)) and RFP Coding Device ([BBa\_J04450](http://parts.igem.org/Part%3ABBa_J04450)).
* Protein production characterized with a LacI promoter ([BBa\_R0010](http://parts.igem.org/Part%3ABBa_R0010)) in *E.
  coli* SHuffle T7 Express®️, confirming IPTG induction functionality in our parts.
* We achieved the assembly of three new BioBricks. [BBa\_K2959005](http://parts.igem.org/Part%3ABBa_K2959005) that
  codes for PsDef1-Erv1p, [BBa\_K2959007](http://parts.igem.org/Part%3ABBa_K2959007) that codes for WAMP1b. [BBa\_K2959010](http://parts.igem.org/Part%3ABBa_K2959010) that
  codes for AtPFN1. The three of them are expression systems for antimicrobial peptides.

Unsuccessful

* We first started to work with *E. coli* DH5α but later had to change to Mach1 due to
  the small colonies we achieved during transformation.
* We also discovered that one of the strains we thought was PsDef1 didn’t actually have the
  protein. After transformation, we saw colonies and assumed that it had been correctly
  transformed. Electrophoresis was performed and it showed the band of the whole construct.
  After working with that construct for a long time, we performed electrophoresis again only
  to see that the band of the size of pSB1C3 was observed but not the rest, it just vanished,
  and we are not sure where it vanished while we were working. Up to this day, we are not sure
  how it disappeared.
* SHuffle T7 Express®️ was the most important chassis of two of our parts and they were
  correctly transformed for the first half of summer. The transformation and competent cells
  protocol were placed aside, and we didn’t use it for a while since we were working on other
  parts of the project. When we used SHuffle in order to transform other parts, the strain
  didn’t work properly. The LB - CAM plates disseminated with control competent cells, showed
  colony growth and this was odd for us. Our LB + CAM plates didn’t show any growth, which
  meant we didn’t have any contamination and our RFP had grown but the colonies weren’t red.
  We got to the conclusion that our competent cells weren’t completely competent anymore, that
  they had lost this characteristic due to the amount of time it was stored at -70ºC.

# Future Plans

For future experimentation we have the following plans to impulse our project:

[### Experimentation Plan & Considerations](#collapse1)

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# Tec-Chihuahua © 2019

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