Engineering
Please follow us to Proposed Implementation and Proof of Concept to learn how we utilized both concepts or back to Design to understand the initial design ideas.
Engineering of our constructs
In order to successfully implement our idea of the biological sensor and the selected design, a lot
of literature research was conducted at the beginning of the project. As we had no experience with
MagAcreated part:
BBa_K3338000 and LPS-dependent gene expression, we used different papers to get a first well-founded
impression. Furthermore, the right software for planning of the cloning had to be found. We decided
to use and learn the software of SnapGene, which was kindly provided to us by SnapGene. After a
short training period the first constructs could be designed. Here we proceeded schematically step
by step.
First the individual constructs were designed. For this purpose, the sequences were searched from Addgene, assembled via SnapGene and prepared for the DNA HIFI Assembly reaction of NEB. In order to obtain the individual sequences, which should be present in the constructs, the plasmids were ordered from Addgene. The individual plasmids we have chosen can be seen in the table below.
| Addgene number of plasmid | Fragment | Material information |
|---|---|---|
| 21751 | MagAcreated part: BBa_K3338000 |
MS-1 MagAcreated part: BBa_K3338000 was a gift from Elliot Meyerowitz (Addgene plasmid # 21751 ; RRID:Addgene_21751) |
| 22522 | Gaussia Luciferasecreated part: BBa_K3338001 |
phGluc was a gift from Christopher Buck (Addgene plasmid # 22522 ; RRID:Addgene_22522 ) |
| 79491 | IL6-promotercreated part: BBa_K3338008 |
pENTR221-IL6.Pro was a gift from Peter Jon Nelson (Addgene plasmid # 79491 ; RRID:Addgene_79491 ) |
| 45350 | mCherryused part: BBa_J04450, P2Acreated part: BBA_K3338003 |
AmCyan-P2Acreated part: BBA_K3338003-mCherry was a gift from Ilpo Huhtaniemi (Addgene plasmid # 45350 ; RRID:Addgene_45350 ) |
| 21041 | IREScreated part: BBA_K3338004 |
pmRFP_a_tubulin_IRES_puro2b was a gift from Daniel Gerlich (Addgene plasmid # 21041 ; RRID:Addgene_21041 ) |
| - | pEGFP-C2created part: BBA_K3338020 |
avaiable in the lab |
The plasmids were multiplied, isolated and cut via restriction digestions. This should create the right backbones for our constructs. The fragments, which should be arranged in different ways within the backbone, were amplified by PCR and suitable primers. In order to perform the reaction with the HIFI DNA Assembly Kit, the compatibility of the fragments to each other and to the backbone must be ensured.
To verify the correct incorporation of the individual fragments and the success of the reaction, test digestions were carried out. Only the correctly cloned plasmid could lead to the correct band length and arrangement. After this was validated, test PCRs were performed. Also, at this point only correct constructs could lead to an amplification of the desired length. Only when these steps were reached, the finished plasmid was sent for sequencing to check the correct base sequence, the correct frame and any mutations.
Unfortunately, this was sometimes not the case. In theses cases, the fragment was not inserted, or not in the correct order. This was due to errors in planning and could then be tracked via SnapGene. As soon as the source of the error was found, the sequence was changed, new primers were ordered and the cloning was performed again. Possible error sources were mutations inserted via SnagGene and mutation primers, wrong restriction sites or errors in the normal primer sequences.
After the plasmids had passed all test sections, they were multiplied by a MaxiPrep to be used for transfection experiments.
From the individual plasmids and fragments, the first step was to start with MagAcreated part:
BBa_K3338000. The transmembrane
transporter [1] cannot be displayed by any simple
visualization methods. To check the cloning and transfection efficiency, a fluorescent protein
(EGFPused part:
BBA_K1123017)
was cloned in fusion to MagAcreated
part:
BBa_K3338000. To be more precise, the sequence of the MagAcreated part:
BBa_K3338000 was placed in the
multiple cloning site of a pEGFP-C2CREATED
PART:
BBA_K3338020
backbone vector. This was assembled in the composite part BBa_K3338012. This should result in a fusion protein that can
be visualized by fluorescence microscopy. Unfortunately, the first attempt was unsuccessful. In the
test restriction digestion described above, the correct bands could not be detected in any of the
eight clones used. Thus, the approach was changed in such a way that the backbone would be cut via a
different restriction enzyme. The basic procedure and the finally successful transfection attempt
are shown in the figure below.
BBa_K3338000 was cloned into the pEGFP-C2CREATED PART:
BBA_K3338020 backbone via a single restriction digest. This resulted in a fusion protein of MagAcreated part:
BBa_K3338000 and EGFPused part:
BBA_K1123017, whose localization and expression could be shown correctly in the cell membrane by fluorescence microscopy.
Since Gaussia Luciferasecreated part:
BBa_K3338001 can be indicated by a simple coelenterazine reaction in medium, there was no
need to clone a fusion protein. For better comparability, the gene of luciferase, hGluccreated part:
BBa_K3338001, was also
cloned into the same backbone of the pEGFP-C2created part:
BBA_K3338020
vector (see BBa_K3338017).
For this purpose, the plasmid was cut at
different sites to remove the fluorescent protein EGFPused part:
BBA_K1123017.
Only the gene of Gaussia Luciferasecreated
part:
BBa_K3338001 was
obtained under the control of the CMV-promoterused
part:
BBa_I712004. The basic procedure and the successful luminescence
measurement are shown in the figure below. The signal proves the correct cloning, which has already
been validated by sequencing. In addition, the figure also shows that the functionality of Gaussia Luciferasecreated part:
BBa_K3338001 is maintained and that it successfully leads to luminescence via the conversion of the
substrate coelenterazine [2].
BBa_K3338001, hGluc, was cloned behind the CMV-promoterused part:
BBa_I712004 in the pEGFP backbone. Successfully transfected cells secreted the luciferase into the medium, whereby luminescence could be successfully detected via the substrate of the luciferase, the coelenterazine.
In order to get not only a visual proof of the expression of the two proteins, both sequences, MagAcreated part:
BBa_K3338000
and hGluc, were labelled with a FLAG tag using appropriate primers (see BBa_K3338016 and BBa_K3338018). This should also allow the
successful expression to be detected by a Western blot. The constructs could be successfully cloned
and sequenced . Due to the time constraints and the closure of the laboratory due to the corona
pandemic, this experiment was postponed and could not be performed successfully so far.
Nevertheless, the basic plan is shown in the figure below using the example of Gaussia Luciferasecreated part:
BBa_K3338001.
BBa_K3338001. In addition to visualization by luminescence or fluorescence microscopy, the expression of both reporter proteins should also be shown by a Western blot.
For the further progress of the project it was necessary to find a suitable combination of the two
reporter proteins. Therefore, an internal ribosome entry site (IREScreated part:
BBA_K3338004)
and a P2Acreated part:
BBA_K3338003
peptide were used.
More details on the biological background can be found in the design chapter.
To validate the two
separation possibilities, two fluorescent proteins, namely EGFPused part:
BBA_K1123017
and mCherryused part:
BBa_J04450 were combined
once via
an IREScreated part:
BBA_K3338004
and once via a P2Acreated part:
BBA_K3338003
sequence in a pEGFP-C2created part:
BBA_K3338020
backbone (see BBa_K3338013 and BBa_K3338014). The extent of expression was reproduced
by fluorescence microscopy. These images as well as sections of both constructs with the pEGFP-C2created part:
BBA_K3338020
backbone are shown in the figure below.
From our previous results we have concluded that the part BBa_K3338013, which contains a P2Acreated part:
BBA_K3338003
sequence leads to a higher simultaneous
expression of both reporter proteins.
Our indication of a biofilm was thus represented by the following amplification sequence:
BBa_I712004 with high basal expression. The simultaneous expression of both reporter proteins is mediated by a P2Acreated part:
BBA_K3338003 sequence.
The basal expression of the CMV-promoterused
part:
BBa_I712004 is very high. This promoter was only used to validate the
successful cloning and was replaced by several other promoters in the further course of the project.
These promoters should respond to LPS stimulation, one toxin of a bacterial biofilm. LPS should lead
to an increased expression of the downstream genes via the NFκB pathway. To make this
possible, the IL6-promotercreated part:
BBa_K3338008
was used first. However, because this promoter is not compatible with the
BioBrick system of the iGEM Foundation, an additional mutated promoter (IL6mutcreated part:
BBa_K3338005)
was synthesized.
Therefore the thymine at position 371 was replaced by an adenine.
Furthermore, two promoters (NF-κB-AP1-minCMV1created part:
BBa_K3338007
and
NF-κB-AP1-minCMV2created part:
BBa_K3338002)
were designed and developed by ourselves. The minimal CMV-promoterused
part:
BBa_I712004 was
taken as a basis and extended in two different variants with binding sites for the inflammatory
molecules AP-1 and NFκB. These are induced by the bacterial toxin LPS. This should enable a
differentiation of lipopolysaccharide-induced expression of reporter proteins from basal expression.
These self-designed promoters are shown in the figure below.
The process of engineering was completed with the subsequent successful cloning. The self-designed
promoters and both IL6-promotercreated part:
BBa_K3338008s
were kindly provided by IDT and could be ligated directly into the
pEGFP-C2created part:
BBA_K3338020
backbone by clever selection of the connection points. Now the LPS dependence of the
individual promoters could be traced by the expression of the reporter proteins. The data of the
different experiments can be read in the chapter results.
Engineering of our measuring chamber
The chamber measuring principle is intuitive. We follow the principle of magnetophoresis, which aims to sort the particles inside the chamber in two output channels based on their magnetic properties. For this, a magnet is placed on one side of the chamber, attracting the magnetic particles and thus sorting magnetic and non-magnetic particles into different channels.
As source for an external magnetic field, we use a neodymium-alloy magnet. Two solutions stream through the input channels into the chamber. One being the probe solution with the magnetic particles, the other one being a buffer solution. Because of the low stream velocities and the small size of the particles, a laminar flow is build up inside the chamber. This prevents unwanted mixing of the two solutions.
Evaluation of the experiment is done by counting the number of particles at each output for different strengths of the magnetic field and calculating the ratio of the counts. If the particles in the probe solution exhibit magnetic properties, the count ratio will change significantly, when changing the magnetic field strength.
In the mixing channel, the magnetic field of the magnet interacts with the magnetic dipole moment of the particle. This interaction results in a force exerting on the magnetic particles moving inside the mixing channel. As a result, the trajectory of the particle is altered. If paramagnetic particles are used, the force is attractive, which means that the particles are accelerated towards the magnet [3]. In this case, it is important, that the probe solution is streamed in the input channel facing away from the magnet. Only in this case, a significant increase in the number of paramagnetic particles in the output channel can be observed, when adding an external magnetic field. Comparing the number of particles at each output channel, more particles will be counted at the channel faced towards the magnet. If the ratio of the counts is calculated, it will increase or respectively decrease with a change in the magnetic field strength.
Bibliography and references
- Uebe, R., Henn, V., & Schüler, D. (2012). The MagAcreated part:
BBa_K3338000 protein of Magnetospirilla is not involved in bacterial magnetite biomineralization. Journal of bacteriology, 194(5), 1018–1023. - Tannous B. A. (2009). Gaussia Luciferasecreated
part:
BBa_K3338001 reporter assay for monitoring biological processes in culture and in vivo. Nature protocols, 4(4), 582–591. - Tarn, Mark D., et al. On-chip diamagnetic repulsion in continuous flow. Science and technology of advanced materials, 2009.