iGEM 2020 — Team AshesiGhana

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# Team:AshesiGhana/Notebook

AshesiGhana

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  [Accomplishments](https://2020.igem.org/Team%3AAshesiGhana/Highlights)
  [Contribution](https://2020.igem.org/Team%3AAshesiGhana/Contribution)
* [Project](https://2020.igem.org/Team%3AAshesiGhana/Description)

  [Description](https://2020.igem.org/Team%3AAshesiGhana/Description)
  [Design](https://2020.igem.org/Team%3AAshesiGhana/Design)
  [Engineering](https://2020.igem.org/Team%3AAshesiGhana/Engineering)
  [Results](https://2020.igem.org/Team%3AAshesiGhana/Results)
* [Modelling](https://2020.igem.org/Team%3AAshesiGhana/Model)
* [Notebook](https://2020.igem.org/Team%3AAshesiGhana/Notebook)
* [Human Practices](https://2020.igem.org/Team%3AAshesiGhana/Human_Practices)

  [Communication](https://2020.igem.org/Team%3AAshesiGhana/Education)
* [Team (current)](https://2020.igem.org/Team%3AAshesiGhana/Team)

  [Members](https://2020.igem.org/Team%3AAshesiGhana/Team)
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  [Collaborations](https://2020.igem.org/Team%3AAshesiGhana/Collaborations)
  [Partnership](https://2020.igem.org/Team%3AAshesiGhana/Partnership)

Team Notebook

This is a summary of our iGEM season, a rare glimpse into the minds of the members of this iGEM Team.

Note: **Please click on the month and the corresponding week to see the summary.** We weren’t in the lab this summer and so you’re unlikely to find any lab work here. Happy reading :)!

MAY
JUN
JUL
AUG
SEP
OCT

W1: May 03 - May 09
W2: May 10 - May 16
W3: May 17 - May 23
W4: May 24 - May 30

Summary: Week of May 03 to May 09

* Entered into our last week of online classes
* Preparation for our exams (for the next two weeks)
* Settled on our final project from a group of projects that were presented by each member to the team

© 2020 Ashesi iGEM

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Email: igem@ashesi.edu.gh

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# Team:AshesiGhana/Engineering

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* [Home](https://2020.igem.org/Team%3AAshesiGhana)

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  [Contribution](https://2020.igem.org/Team%3AAshesiGhana/Contribution)
* [Project](https://2020.igem.org/Team%3AAshesiGhana/Description)

  [Description](https://2020.igem.org/Team%3AAshesiGhana/Description)
  [Design](https://2020.igem.org/Team%3AAshesiGhana/Design)
  [Engineering](https://2020.igem.org/Team%3AAshesiGhana/Engineering)
  [Results](https://2020.igem.org/Team%3AAshesiGhana/Results)
* [Modelling](https://2020.igem.org/Team%3AAshesiGhana/Model)
* [Notebook](https://2020.igem.org/Team%3AAshesiGhana/Notebook)
* [Human Practices](https://2020.igem.org/Team%3AAshesiGhana/Human_Practices)

  [Communication](https://2020.igem.org/Team%3AAshesiGhana/Education)
* [Team (current)](https://2020.igem.org/Team%3AAshesiGhana/Team)

  [Members](https://2020.igem.org/Team%3AAshesiGhana/Team)
  [Attributions](https://2020.igem.org/Team%3AAshesiGhana/Attributions)
  [Collaborations](https://2020.igem.org/Team%3AAshesiGhana/Collaborations)
  [Partnership](https://2020.igem.org/Team%3AAshesiGhana/Partnership)

# ENGINEERING SUCCESS AND PREDICTED LAB WORK

Our team is considering a two-phase project, the design phase for this year,
and the implementation phase for next year. For the second phase of our
project which would be implemented in the lab, we envision to use the lab
protocol designed below:

1. Obtaining the (pSB1C3) plasmids and synthesize the other genes of
   interest with their respective promoters and the other necessary parts
2. Digestion of plasmid and respective genetic or DNA sequences
   using the chosen restriction enzymes (EcoRI and XbaI to cut the vector
   and EcoRI and SpeI to cut the insert).
3. Ligation of the compatible ends of the plasmids and DNA sequences using the
   appropriate ligation buffer under the right conditions.
4. Transformation of the cloned plasmid into the V. natriegens bacteria.
5. Using a Chloramphenicol antibiotic LB agar plate, the transformation
   is pipetted onto the agar plate and with a sterilized spreader or glass
   bead, is spread onto the plate
6. Picking single colonies from transformations and growing cell cultures for further studies.
7. Mini prepping to extract plasmid DNA from the bacterial cell suspension.
8. Further digestion is performed, and gel electrophoresis is done for each
   of the five recombinant plasmids to verify the plasmid and check if the DNA
   is present in the plasmid.

The plasmid (pSB1C3) and other two recombinant plasmids which are cloned with inserts
for plastic degradation (one with a constitutive promoter and the other a high-pH inducible
promoter) are cut with EcoRI and SpeI. The other three recombinant plasmids for the biocementation
process (one with a constitutive promoter another with a high-pH inducible promoter and the last
one with a low pH-inducible promoter) are cut with the restriction enzymes EcoRI and NruI.
Two bands are obtained for each lane however the lane containing the non-cloned plasmid
would only have one band visible since the second band is very small to be visualized.

For the cloned plasmids, two visible bands are obtained; one representing the size (number of base pairs)
of the backbone and one the size (number of base pairs) of the new insert.

For *V. natriegens* with pH inducible promoters (PET degradation & biocementation) and light-inducible
promoters, the culture would be prepared at the pH and wavelength that will trigger the translation
process for PET degradation and biocementation respectively. The experimental procedures described
earlier will be applied in this situation.

Currently, we have not established an experimental technique that will aid the evaluation of the parts under an inducible promoter, but it is something we will work on in the coming year.

In silico Genetic Design

Due to the inability to be in the lab this year, we performed experiments in silico
using the SnapGene Software. After obtaining our genetic parts and coding sequences
from the UniProt database or peer-reviewed articles, we used Snap Gene to clone the
vector (pSB1C3) to obtain five (5) recombinant plasmids which had different inserts
pertaining to the role they performed in our project. We used the Restriction Enzyme
Cloning assembly method while adhering to the BioBrick Standards using the restriction
enzymes, EcoRI, XbaI, and SpeI. The vector was cut with EcoRI and XbaI and the inserts
with EcoRI and SpeI.
These five recombinant plasmids included the following inserts:

* A plastic biodegradation insert with a constitutive promoter

  ![](https://static.igem.org/mediawiki/2020/f/fe/T--AshesiGhana--silico1.jpg)
* A plastic biodegradation insert with a high pH inducible promoter

  [![T--AshesiGhana--silico2.png](/wiki/images/2/24/T--AshesiGhana--silico2.png)](/File%3AT--AshesiGhana--silico2.png)
* A biocementation insert with a constitutive promoter

  [![T--AshesiGhana--silico3.png](/wiki/images/2/2d/T--AshesiGhana--silico3.png)](/File%3AT--AshesiGhana--silico3.png)
* A biocementation insert with a low pH inducible promoter

  [![T--AshesiGhana--silico4.png](/wiki/images/2/2d/T--AshesiGhana--silico4.png)](/File%3AT--AshesiGhana--silico4.png)
* A biocementation insert with a light-inducible promoter promoter

  [![T--AshesiGhana--silico5.png](/wiki/images/8/8f/T--AshesiGhana--silico5.png)](/File%3AT--AshesiGhana--silico5.png)

These inserts were used to clone the vector (pSB1C3). The vector was cut with
restriction enzymes EcoRI and XbaI and all the inserts were cut with the restriction enzymes
EcoRI and SpeI to produce sticky and compatible ends. Since we cloned the inserts differently
into five plasmids, we used a single fragment insert method.

[![T--AshesiGhana--silico6.png](/wiki/images/5/5b/T--AshesiGhana--silico6.png)](/File%3AT--AshesiGhana--silico6.png)

Fig. In silico Cloning in SnapGene of a vector (pSB1C3) with biocementation
insert with a light-inducible promoter promoter

[![T--AshesiGhana--silico7.png](/wiki/images/5/5f/T--AshesiGhana--silico7.png)](/File%3AT--AshesiGhana--silico7.png)

Fig. Cloning History of the vector (pSB1C3) with biocementation insert with
a low pH inducible promoter promoter

After the cloning was done, we ran an agarose gel simulation using SnapGene to check if
inserts have been properly inserted into the vector. We used 1.0% Agarose and used GeneRulerTM
1kb DNA Ladder as the MW marker. We used a total of six lanes that had the first lane containing
the DNA for the vector and the second to sixth lanes contained the DNA of the five recombinant
plasmids. The description of each lane in this simulation are:

Lane 1:
Uncloned Vector (pSB1C3)

Lane 2:
Cloned vector with plastic biodegradation insert with a constitutive promoter

Lane 3:
Cloned vector with plastic biodegradation insert with a high pH inducible promoter

Lane 4:
Cloned vector with biocementation insert with a constitutive promoter

Lane 5:
Cloned vector with biocementation insert with a low pH inducible promoter

Lane 6:
Cloned vector with biocementation insert with a light-inducible promoter promoter

The results from the Gel electrophoresis can be found in the results section.

[![T--AshesiGhana--silico8.png](/wiki/images/1/1d/T--AshesiGhana--silico8.png)](/File%3AT--AshesiGhana--silico8.png)

Fig. Agarose gel simulation of cloned Vector with the five different inserts

 [See Results ->](https://2020.igem.org/Team%3AAshesiGhana/Results)

© 2020 Ashesi iGEM

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Email: igem@ashesi.edu.gh

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# Team:AshesiGhana/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:AshesiGhana/Results

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* [Home](https://2020.igem.org/Team%3AAshesiGhana)

  [Medal Checklist](https://2020.igem.org/Team%3AAshesiGhana/Medal_Checklist)
  [Accomplishments](https://2020.igem.org/Team%3AAshesiGhana/Highlights)
  [Contribution](https://2020.igem.org/Team%3AAshesiGhana/Contribution)
* [Project](https://2020.igem.org/Team%3AAshesiGhana/Description)

  [Description](https://2020.igem.org/Team%3AAshesiGhana/Description)
  [Design](https://2020.igem.org/Team%3AAshesiGhana/Design)
  [Engineering](https://2020.igem.org/Team%3AAshesiGhana/Engineering)
  [Results](https://2020.igem.org/Team%3AAshesiGhana/Results)
* [Modelling](https://2020.igem.org/Team%3AAshesiGhana/Model)
* [Notebook](https://2020.igem.org/Team%3AAshesiGhana/Notebook)
* [Human Practices](https://2020.igem.org/Team%3AAshesiGhana/Human_Practices)

  [Communication](https://2020.igem.org/Team%3AAshesiGhana/Education)
* [Team (current)](https://2020.igem.org/Team%3AAshesiGhana/Team)

  [Members](https://2020.igem.org/Team%3AAshesiGhana/Team)
  [Attributions](https://2020.igem.org/Team%3AAshesiGhana/Attributions)
  [Collaborations](https://2020.igem.org/Team%3AAshesiGhana/Collaborations)
  [Partnership](https://2020.igem.org/Team%3AAshesiGhana/Partnership)

# RESULTS

Since we did not get into the lab this year,
we designed our project in silico using the SnapGene software.
The genetic check has results from our design in SnapGene after
running an agarose simulation in SnapGene and the functional check
which are predicted results from some predicted experiments to evaluate
the performance of our gen constructs.

Genetic Check

[![T--AshesiGhana--results1.png](/wiki/images/3/38/T--AshesiGhana--results1.png)](/File%3AT--AshesiGhana--results1.png)

Fig.1

The results obtained from Agarose gel simulation
in SnapGene can be found in the figure above.
Using 1.0% Agarose and the GeneRulerTM 1kb DNA Ladder as the MW marker,
we obtained the following bands. The first lane (1) shows the band of the
vector yet to be cloned which has a total of 2070 base pairs (bp). This band is used
to draw inferences when analyzing the bands of the cloned vector with inserts in the
other lanes. Using the GeneRulerTM 1kb DNA Ladder, we can tell the size of each band

From the gel image, we confirmed that all inserts were properly inserted into the vector by comparing
the size of the bands with that of the initial size or number of base pairs of the inserts.

Functional Check

The following experiments have been predicted to evaluate the processes involved in this
project. Below are the experiments and graphs predicting their performance as described in
the project design. The graphs however do not represent the modeling results of our project.

[![T--AshesiGhana--results2.jpg](/wiki/images/a/ae/T--AshesiGhana--results2.jpg)](/File%3AT--AshesiGhana--results2.jpg)

Fig.2: Experiment for evaluation of the plastic degradation parts.

[![T--AshesiGhana--result3.jpg](/wiki/images/b/b7/T--AshesiGhana--result3.jpg)](/File%3AT--AshesiGhana--result3.jpg)

Fig.3: A graph showing the change in the mass of PET film with time.

We expect a decline in the mass of PET with an increase in time.

[![T--AshesiGhana--result4.jpg](/wiki/images/8/85/T--AshesiGhana--result4.jpg)](/File%3AT--AshesiGhana--result4.jpg)

Fig.4: Experiment for evaluation of the biocementation parts

To test the mechanical strength of the bio-brick and also to monitor the progress
of the biocementation process we plan to put a weighty object, for example, a metal
ball into the tank at regular time intervals. As shown in the diagram we expect that
the ease with which the object will sink to the bottom of the tank will decrease as time goes by.

[![T--AshesiGhana--result5.jpg](/wiki/images/5/52/T--AshesiGhana--result5.jpg)](/File%3AT--AshesiGhana--result5.jpg)

Fig.5: A pictorial representation of the biocementation experiment.

We expect a proportional relationship between the mechanical strength and the number
of days. We also expect a linear increase in the mechanical strength of the slurry as
time goes by until it reaches its maximum strength and plateaus.

[![T--AshesiGhana--result6.jpg](/wiki/images/f/f4/T--AshesiGhana--result6.jpg)](/File%3AT--AshesiGhana--result6.jpg)

Fig.6

For V.natriegens with pH inducible promoters (PET degradation & biocementation) and
light-inducible promoters, the culture would be prepared at the pH and wavelength
that will trigger the translation process for PET degradation and biocementation
respectively. The experimental procedures described earlier will be applied in this
situation.

Currently, we have not established an experimental technique that will aid
the evaluation of the parts under an inducible promoter, but it is something we will
look into.

References

[1]"ISF6\_4831 - Poly(ethylene terephthalate) hydrolase precursor - Ideonella sakaiensis
(strain NBRC 110686 / TISTR 2288 / 201-F6) - ISF6\_4831 gene & protein", Uniprot.org.
Available: https://www.uniprot.org/uniprot/A0A0K8P6T7.

[2]"lipase [Ideonella sakaiensis] - Protein - NCBI", Ncbi.nlm.nih.gov.
Available: https://www.ncbi.nlm.nih.gov/protein/GAP38373.1.

[3]"B.pasteurii ureA, ureB and ureC genes - Nucleotide - NCBI", Ncbi.nlm.nih.gov.
Available: https://www.ncbi.nlm.nih.gov/nuccore/X78411.

[4]"ureC - Urease subunit alpha - Sporosarcina pasteurii - ureC gene & protein",
Uniprot.org. Available: https://www.uniprot.org/uniprot/P41020.

[5]"ureB - Urease subunit beta - Sporosarcina pasteurii - ureB gene & protein",
Uniprot.org. Available: https://www.uniprot.org/uniprot/P41021.

[6]J. Hoff, B. Daniel, D. Stukenberg, B. Thuronyi, T. Waldminghaus, and G. Fritz,
"Vibrio natriegens: an ultrafast‐growing marine bacterium as emerging synthetic biology
chassis", Environmental Microbiology, 2020.

[7]"Team:Harvard BioDesign/Basic Part - 2016.igem.org", 2016.igem.org, 2016.
Available: https://2016.igem.org/Team:Harvard\_BioDesign/Basic\_Part#.

[8]"ISF6\_0224 - Mono(2-hydroxyethyl) terephthalate hydrolase precursor -
Ideonella sakaiensis (strain NBRC 110686 / TISTR 2288 / 201-F6) - ISF6\_0224 gene
& protein", Uniprot.org. Available: https://www.uniprot.org/uniprot/A0A0K8P8E7.

© 2020 Ashesi iGEM

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Email: igem@ashesi.edu.gh