iGEM 2022 — Team uma-malaga

![](unset)

# Notebook

![](https://static.igem.wiki/teams/4368/wiki/10.png)
![](https://static.igem.wiki/teams/4368/wiki/march.png)

![](https://static.igem.wiki/teams/4368/wiki/4.png)
![](https://static.igem.wiki/teams/4368/wiki/april.png)

![](https://static.igem.wiki/teams/4368/wiki/11.png)
![](https://static.igem.wiki/teams/4368/wiki/17.png)
![](https://static.igem.wiki/teams/4368/wiki/19.png)
![](https://static.igem.wiki/teams/4368/wiki/20.png)
![](https://static.igem.wiki/teams/4368/wiki/27.png)
![](https://static.igem.wiki/teams/4368/wiki/may.png)

![](https://static.igem.wiki/teams/4368/wiki/2.png)
![](https://static.igem.wiki/teams/4368/wiki/8.png)
![](https://static.igem.wiki/teams/4368/wiki/9.png)
![](https://static.igem.wiki/teams/4368/wiki/16.png)
![](https://static.igem.wiki/teams/4368/wiki/18.png)
![](https://static.igem.wiki/teams/4368/wiki/22.png)
![](https://static.igem.wiki/teams/4368/wiki/30.png)
![](https://static.igem.wiki/teams/4368/wiki/june.png)

![](https://static.igem.wiki/teams/4368/wiki/10.png)
![](https://static.igem.wiki/teams/4368/wiki/11.png)
![](https://static.igem.wiki/teams/4368/wiki/12.png)
![](https://static.igem.wiki/teams/4368/wiki/14.png)
![](https://static.igem.wiki/teams/4368/wiki/15.png)
![](https://static.igem.wiki/teams/4368/wiki/19.png)
![](https://static.igem.wiki/teams/4368/wiki/20.png)
![](https://static.igem.wiki/teams/4368/wiki/26.png)
![](https://static.igem.wiki/teams/4368/wiki/28.png)
![](https://static.igem.wiki/teams/4368/wiki/july.png)

![](https://static.igem.wiki/teams/4368/wiki/august.png)

![](https://static.igem.wiki/teams/4368/wiki/8.png)
![](https://static.igem.wiki/teams/4368/wiki/13.png)
![](https://static.igem.wiki/teams/4368/wiki/14.png)
![](https://static.igem.wiki/teams/4368/wiki/20.png)
![](https://static.igem.wiki/teams/4368/wiki/22.png)
![](https://static.igem.wiki/teams/4368/wiki/26.png)
![](https://static.igem.wiki/teams/4368/wiki/30.png)
![](https://static.igem.wiki/teams/4368/wiki/september.png)

![](https://static.igem.wiki/teams/4368/wiki/10.png)
![](https://static.igem.wiki/teams/4368/wiki/26.png)
![](https://static.igem.wiki/teams/4368/wiki/27.png)
![](https://static.igem.wiki/teams/4368/wiki/october.png)

❮
❯

![](unset)

# Engineering

# Introduction

Due to the need to find a solution to a local problem such as contamination by vegetal wastes, a gene
construct
was designed as an alternative to transform these products into a value-added substance, starch. For this
purpose, we used optimized synthetic gene constructs from Escherichia coli and Cellulomonas fimi, which were
subsequently transformed into E.coli bacteria.

Once the final construct based on two plasmids, pSBC1Cel1 and pSBA1Sta1, was obtained, a search was carried
out
to determine the existing detection methods that would allow verification of this construct. Through a
literature search, it was possible to determine that simple tests such as Red Congo Assay, as well as
staining
with lugol and glucose oxidase test (GOD) allowed verifying the correct functioning of the composition
created.
They were accompanied by a final fluorescence revealed due to emission at the wavelengths corresponding to
RFP
and GFP.

[![](https://static.igem.wiki/teams/4368/wiki/text-protocol.png)](#p1)
[![](https://static.igem.wiki/teams/4368/wiki/text-silico.png)](#p2)
[![](https://static.igem.wiki/teams/4368/wiki/text-tests.png)](#p3)
![](https://static.igem.wiki/teams/4368/wiki/engranaje.png)

# In silico design

For this purpose, the different gene constructs that make up our glucose-sensing-based regulatory mechanism
(Parts) were designed computationally in a software named Benchling.

Firstly, a sequence of an extracellular export motor protein was introduced into the cenA and cex constructs
that in conjunction with the bglX gene allows extracellular cellulose to be degraded into glucose.

Secondly, codon optimization was performed on the gene encoding glgC16 by removing an EcoRI restriction
target. In addition, the basic part of bglX (BBa\_K523002) has been modified by removing an overlapping
native RBS.

# Establishment of a protocol for the final construct

Our construct is based on the existence of only two plasmids, pSBC1Cel1 and pSBA1Sta1, which combine all the
genes required for starch production. For this purpose, a paired ligation protocol based on the BioBrick®
Assembly Kit has been designed. For more information on how this procedure or every other one of our
experimentation was performed, please click on the button below to get a copy of our protocols.

[PROTOCOLS](https://static.igem.wiki/teams/4368/wiki/protocols.pdf)

# Screening tests

For final testing that our verified construct is working optimally, colorimetric (congo red and lugol) and
enzymatic (glucose oxidase) assays are performed. If you require more information on how these procedures
have been approached, please visit the Verification section of Experiments.

![](unset)

Close

# Experiments

![](https://static.igem.wiki/teams/4368/wiki/experiments.png)

![](https://static.igem.wiki/teams/4368/wiki/introductionposit.png)

![](https://static.igem.wiki/teams/4368/wiki/cen.png)

![](https://static.igem.wiki/teams/4368/wiki/cex.png)

![](https://static.igem.wiki/teams/4368/wiki/p-glx.png)

![](https://static.igem.wiki/teams/4368/wiki/glgc.png)

![](https://static.igem.wiki/teams/4368/wiki/rfp.png)

![](https://static.igem.wiki/teams/4368/wiki/gfp.png)

![](https://static.igem.wiki/teams/4368/wiki/ci.png)

![](https://static.igem.wiki/teams/4368/wiki/ligation.png)

![](https://static.igem.wiki/teams/4368/wiki/verification.png)

# Results

* [![](https://static.igem.wiki/teams/4368/wiki/results-1.png)

  Introduction](#p1)
* [![](https://static.igem.wiki/teams/4368/wiki/results-2.png)

  Results](#p2)
* [![](https://static.igem.wiki/teams/4368/wiki/results3.png)

  Future perspectives](#p3)

# Introduction

The aim of our project was to obtain a transformed bacterial population containing both
overexpressed glycogenosynthetic and cellulolytic genes for the final starch-like production.

# Results

For this, in the first instance, gene constructs based on the Part Registry were developed
computationally, with subsequent modification and optimization to avoid restriction targets to
be used within the insert.

The first step of the experimental design is the digestion of the inserts and their ligation
into individual plasmids for characterization. As can be seen in the agarose electrophoresis
gels, the digestion of the seven inserts have been successful and individualized plasmids of
each gene have been obtained.

![](https://static.igem.wiki/teams/4368/wiki/fig1.png)

![](https://static.igem.wiki/teams/4368/wiki/fig2.png)

![](https://static.igem.wiki/teams/4368/wiki/fig3.png)

![](https://static.igem.wiki/teams/4368/wiki/fig4.png)

![](https://static.igem.wiki/teams/4368/wiki/fig5.png)

![](https://static.igem.wiki/teams/4368/wiki/fig6.png)

![](https://static.igem.wiki/teams/4368/wiki/fig7.png)

Subsequently, pairwise ligation of the inserts was performed as previously described. Correct
inclusion of the gene pairs into a plasmid with a different antibiotic resistance was observed.
Although the results were conclusive in that the digests were performed correctly, the ligation
of the two inserts had very low efficiency, resulting in a low number of colonies or even empty
plates. Even so, the ligation of the plasmid containing the cellulolytic genes was performed
correctly.

Finally, the colorimetric test for the functionality of the psBC1Cel1 plasmid was performed. The
congo red test was positive in two of the eight samples tested, showing a hydrolysis halo around
the bacterial growth. However, the resulting halo is similar to that observed in the literature,
indicating a good degradation efficiency.

![](https://static.igem.wiki/teams/4368/wiki/fig8.png)

# Future perspectives

Due to time constraints, it was only possible to verify the assay to determine the hydrolysis
capacity of cen and cex cellulases, obtaining cellobiose as a product. For this purpose, the
commercial reagent m-methyl cellulose was used, which made it possible to make the culture
plates on which the presence of this reaction was revealed with congo red (for more information
on the procedure, see the section Verification of Experiments).

Once the correct performance using such a substrate has been determined, future alternatives are
proposed to use other cellulose-rich sources, such as plant waste itself, as well as other
products such as porous filter paper. The extraction of the cellulose-rich fraction and its
subsequent development in the same way will allow the determination of the stereospecificity of
this reaction, as well as an optimization of the gene construction for its maximum yield.

However, a more in-depth study is not excluded in which the possibility of the bacteria being
able to work directly on plant residues without the need to homogenize and treat them is
considered.