iGEM 2019 — Team TU_Dresden

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# Team:TU Dresden/Notebook

Team TU-Dresden | the DipGene project

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[## ![TU Dresden 2019](https://static.igem.org/mediawiki/2019/c/cb/T--TU_Dresden--logo3.png)](https://2019.igem.org/Team%3ATU_Dresden)

* Project
  + [Motivation](https://2019.igem.org/Team%3ATU_Dresden/Motivation)
  + [Description](https://2019.igem.org/Team%3ATU_Dresden/Description)
  + [Design](https://2019.igem.org/Team%3ATU_Dresden/Design)
  + [Results](https://2019.igem.org/Team%3ATU_Dresden/Results)
  + [Demonstrate](https://2019.igem.org/Team%3ATU_Dresden/Demonstrate)
  + [Sweet Spirulina](https://2019.igem.org/Team%3ATU_Dresden/Spirulina)
* Lab
  + [Notebook](https://2019.igem.org/Team%3ATU_Dresden/Notebook)
  + [Parts](https://2019.igem.org/Team%3ATU_Dresden/Parts)
  + [Characterization](https://2019.igem.org/Team%3ATU_Dresden/Characterization)
  + [Protocols](https://2019.igem.org/Team%3ATU_Dresden/Experiments)
  + [Safety](https://2019.igem.org/Team%3ATU_Dresden/Safety)
* Human Practices
  + [Integrated Human Practices](https://2019.igem.org/Team%3ATU_Dresden/Human_Practices)
  + [Public Engagement](https://2019.igem.org/Team%3ATU_Dresden/Public_Engagement)
* [Collaborations](https://2019.igem.org/Team%3ATU_Dresden/Collaborations)
* Team
  + [Our Team](https://2019.igem.org/Team%3ATU_Dresden/Team)
  + [Attributions](https://2019.igem.org/Team%3ATU_Dresden/Attributions)
  + [Sponsors](https://2019.igem.org/Team%3ATU_Dresden/Sponsors)
  + [Crowdfunding](https://www.indiegogo.com/projects/sweet-spirulina-dipgene?fbclid=IwAR3LKhhbd6dBNbMVXAZZ6Uh9-k_P-o5r7Lz0Ba6JeimMBrkAQ_H4mlPYBZo#/)
* [Judging](https://2019.igem.org/Team%3ATU_Dresden/Judging)

## Notebook

#### **Our Idea - in a nutshell**

We aim to provide a fast and easy tool for detecting any nucleic acid sequence of interest from microbial samples and human cells.
To bring this idea to live we had three different subproblems to tackle. Learn more about how we solved each one by clicking on the buttons below.

#### **Disclaimer!**

In the following pages the development of a genetic testing Kit will be described. As a proof-of-concept, the presence of the *sry* gene was tested in several adults. We want to clearly state that this is not intended to be used for the means of discrimination. The detection of the *sry*-gene shall not imply any cultural gender characteristics, therefore we will always refer to people as “*sry*-positive” or “*sry*-negative”, never as “male” or “female”, since this is a matter of personal identification that is not related with the test result. For this reason we request of anybody using this test to do the same.

#### **Designing a novel fusion protein**

![](https://static.igem.org/mediawiki/2019/f/f9/T--TU_Dresden--notebook1.jpg)

[Idea & Planning](#fusionIdea)
[Labwork & Protocols](#fusionLabwork)
[Results & Discussion](#fusionResults)

#### **Establishing a cellulose based DNA extraction method**

![](https://static.igem.org/mediawiki/2019/c/cc/T--TU_Dresden--notebook2.jpg)

[Idea & Planning](#extractIdea)
[Labwork & Protocols](#extractLabwork)
[Results & Discussion](#extractResults)

#### **Characterizing dCas9-cellulose interaction**

![](https://static.igem.org/mediawiki/2019/4/45/T--TU_Dresden--notebook3.jpg)

[Idea & Planning](#charactIdea)
[Labwork & Protocols](#charactLabwork)
[Results & Discussion](#charactResults)

## Idea & Planning

### Experimental Aims:

In this section of the labbook we describe how we designed, cloned expressed and purified our fusion protein. We designed every single step of the cloning process in silico, as can be seen in the [cloning history](https://static.igem.org/mediawiki/2019/b/be/T--TU_Dresden--FullConstruct_Cloning_History.pdf). Then we performed every step in the lab and verified each intermediate result by sequencing. Once the full consruct was complete, we characterized the expression and purification conditions that were optimal for this new protein. Additionally to the fusion protein we were working with the new BioBrick [Bba\_K3037005](http://parts.igem.org/Part%3ABba_K3037005), which is a fusion of MBP-eGFP-dCas9. This was used to do our first experiments with the binding of dCas9 to cellulose and to characterize the guideRNAs we designed and to see in which combinations (called guideRNa-pools) they show best binding to the target.

In this case our gene of interest is *sry*, in order to quantify, we used Electrophoretic mobility shift assay (EMSA). It is one of the most sensitive methods used for studying DNA binding properties of a protein. Principle behind mobility shift assay is that the free DNA migrates faster on the denaturing polyacrylamide gel compared to DNA bound to the protein, hindering or slowing down its mobility. TBA-Gel image reveals the position of free and bound DNA.

At the heart of our method stands the design of a novel fusion protein (Figure 1).

![](https://static.igem.org/mediawiki/2019/0/02/T--TU_Dresden--notebook-1.png)

Figure 1 - The final construct made up of five different BioBricks, translationally fused by using [RCF25](https://parts.igem.org/Help%3AStandards/Assembly/RFC25), introduced into the expression plasmid ([BBa\_K3037000](http://parts.igem.org/Part%3ABBa_K3037000)) by using RCF10; MBP - Maltose Binding Protein, HRP - Horseradish Peroxidase, dCas9\* - deadCas9 enzyme with a mutation to exclude forbidden EcoRI site; newly designed BioBricks from Team TU Dresden in bold, old BioBricks used from other Teams in a regular font.

---

**Why [RCF25](https://parts.igem.org/Help%3AStandards/Assembly/RFC25) is used?
Why two purification-tags are used?**

The single BioBricks will be joined together using the [RCF25](https://parts.igem.org/Help%3AStandards/Assembly/RFC25) This way a translational fusion is possible and the whole construct will be read as one fusion protein, without the risk of a frameshift at the restriction enzyme scars. Maltose binding protein (MBP) is added for purification via amylose resin and because it by experience enhances the expression of dCas9-fusion proteins. A Strep-tag was added for purification via Strep-column. Both purification methods are needed because of the big size of the complete construct (6 kb) which often poses a problem for . During expression the microorganism often shortens the protein. By having an N-terminal and a C-terminal purification-tag it can be ensured that only the complete fusion protein is purified.

---

**Why does it need a linker and
 what is the expression plasmid?**

Dr. Aliona Bodganova from the protein facility of the local Max Planck Institute (MPI-CBG) taught us how to express dCas9-GFP protein and also sent us the plasmid with her construct of the recombinant protein. Because her construct works very well and robust for many years already and dCas9 is by experience a very difficult to handle protein, we decided to stay as close as possible to the design of her construct and to introduce as little changes as possible, that could cause problems for unknown reasons later on during expression. First of all, we decided to express in the same strain, that she is using  pRARE T7 and took the same linker and same expression plasmid (POCC97). The linker is creating distance between dCas9 and the fused protein, so that there is as little interaction and influencing between the two as possible. The expression plasmid is carrying a lacO promoter and a lacI repressor sequence under a constitutive promoter. By addition of IPTG expression can be induced and before induction there is minimal background expression. Additionally, it is carrying Kanamycin resistance as a selection marker.

---

**Which BioBricks of
old teams did we use?**

The Strep-tag is a BioBrick from the LMU Munich 2012, with the identifier  [Bba\_K823038](http://parts.igem.org/Part%3ABba_K823038). The second old BioBrick used is the [HRP (Bba\_K1800002)](http://parts.igem.org/Part%3ABBa_K1800002).

---

### OUR BIOBRICKS

### **dCas9\***

There are several Cas9-BioBricks in the database of iGEM and even in the distribution Kit of 2019 available, but all of them are optimized for expression in mammalian cells and none for expression in . Since we needed a Cas9, that is codon optimized for  and does not have the endonuclease activity (deadCas9 or dCas9) we had to design our own BioBrick. We based the design on the plasmid we received from Aliona, who kindly approved its use in the frame of the iGEM community. However, a problem arose when we noticed that there was an EcoRI site right in the middle of the coding region of dCas9. Since this is a forbidden restriction site, we used site directed mutagenesis PCR to change the sequence and remove the illegal restriction site. We designed the PCR-primers in a way that they added the prefix and suffix in RCF25 to the sequence as well. The asterix in the name is indicating the mutated site. This dCas9 was obtained from a plasmid named pOCC97.

For general Information on the biology and function of dCas9 in general and our dCas9\*- BioBrick specifically please visit the corresponding registry page: Link to ([Registry](http://parts.igem.org/Part%3ABBa_K3037005)).
BBa\_K3037000

---

### **HRP**

We used an old HRP from the registry, [HRP (Bba\_K1800002)](http://parts.igem.org/Part%3ABBa_K1800002), but to make it possible to use it for translational fusion, we had to transfer it to RCF25. Therefore we ordered it from IDT. To make sure that this changes did not influence the function of HRP we characterized it in comparison to the orginal sequence (which was in RFC10), no changes in the protein activity.

---

### **MBP**

Maltose binding protein (MBP) is commonly used to increase the solubility and cytoplasmic yield of protein expression and to purify proteins via amylose resin.
For general Information on the biology and function of this BioBrick see [Bba\_K3037001](http://parts.igem.org/Part%3ABba_K3037001).

---

### **Linker**

This BioBrick is a well established linker for (d)Cas9 fusion proteins that has been used in the protein facility of the MPI for a long time and was kindly approved for us to share with the iGEM community. We hope additional information on its functionality in different fusion proteins can be added by future teams.

---

### **strep**

The Strep-tag is an old part made by the  [iGEM12\_LMU-Munich](http://parts.igem.org/Part%3ABBa_K823038).

### References:

[1] https://en.wikipedia.org/wiki/Horseradish\_peroxidase, accessed 27th of August

## Labwork & Protocols

##### [Learning to Express and Purify a dCas9-eGFP fusion Protein ![](https://static.igem.org/mediawiki/2019/e/ef/T--TU_Dresden--flechaPau.png)](#answerOne)

#### 5th – 6th July – Nikitha and Mara

![](https://static.igem.org/mediawiki/2019/1/1b/T--TU_Dresden--notebook-2.png)

Figure 2 – SDS-PAGE of the purification process of the dCas9-eGFP fusion protein. During the purification process we took several samples, which were loaded in the end on an SDS-page to monitor the purification process. The fusion protein is very big. dCas9 alone is 160 kDa [1] and eGFP as well as MBP add another 30-40 kDa each [2]

Aliona Bodganova, working at the MPI protein facility kindly agreed to support our project by teaching us how to express, purify and enrich a dCas9-eGFP fusion protein. Furthermore we could keep the purified protein, which was very helpful to do first experiments with the interaction with cellulose and binding to the target DNA, before our own construct was ready. The SDS-PAGE of the protein purification is shown in Figure 2.

The size of the complete product lies therefore around 230 kDa and it can be seen in already in the raw cell lysate as a band above the last band of the protein marker. The protein was once purified via amylose resin column with the a N-terminal-MBP-tag. The MBP-elution shows why this is necessary.
The very big fusion protein is often truncated by the bacteria, and purification via only MBP shows that many truncated versions are present. Only after additional purification on the SP column is the only visible band that of the full transcript.

The complete protocol on how to purify a dCas9-fusion protein, which we wrote after the Days with Aliona can be found
[here](https://2019.igem.org/Team%3ATU_Dresden/Experiments)

### References:

[1] https://www.antibodies-online.com/protein/412983/Enhanced+Green+Fluorescent+Protein+eGFP+protein/
[2] https://www.genscript.com/bacterial-soluble-protein-expression-MBP-tag.html

##### [Getting our BioBricks ready ![](https://static.igem.org/mediawiki/2019/e/ef/T--TU_Dresden--flechaPau.png)](#answerTwo)

![](https://static.igem.org/mediawiki/2019/9/9e/T--TU_Dresden--notebook-3.png)

Figure 3 – Planning of site-directed mutagenesis PCR

Four different new BioBricks were needed to get our planned construct ready: dCas9\*, MBP, HRP and the linker (Figure 3). The Strep-tag that we used was an old BioBrick.

Because of a forbidden restriction enzyme site (EcorRI) in the middle of the dCas9 sequence, it was necessary to do a site-directed mutagenesis. Therefore, we designed four primers, two of which overlap with 20 basepairs around the EcoRI-site and carry the mutated base A->T. The part upstream and downstream of the mutation will be amplified in two separate PCRs (PCR 1 to 2 and 3 to 4). The product of this PCRs will be the substrate for the last PCR (1 to 4), in which the whole sequence is finally amplified.

#### 30th July – Paula and Mara

![](https://static.igem.org/mediawiki/2019/1/1e/T--TU_Dresden--notebook-4.png)
Figure 4 – 1 % agarose gel results
(for the fragment of interest)

The first two PCRs had the difficulty that the additional base pairs added by the prefix and suffix required a change in annealing temperature after the first few cycles. We solved this by designing the PCR in a way, that the first cycles were a three-step and the last cycles a two-step PCR. The number of cycles was generally kept low to minimize the probability of accumulating mutations in the coding sequence.

The PCR was performed in the following way:

   10 µL NEB MasterMix Q5
   7 µl H2O
   1 µL sample
   1 µl each primer
   [20 µl total reaction volume]

\*All later mentioned PCRs were done with this protocol
The PCR was done with the following protocol:

1 % agarose gel showed PCR 1 to 2 and 3 to 4 amplified the fragment of interest successfully, but PCR 1 to 4 only showed a faint band of amplification of the fragment from the 1 to 2 PCR (Figure 4). The PCR programs used are given in the tables below.

![](https://static.igem.org/mediawiki/2019/a/ae/T--TU_Dresden--notebook-5.png)

![](https://static.igem.org/mediawiki/2019/3/3a/T--TU_Dresden--notebook-6.png)
Figure 5 – 1 % agarose gel results (for the linker)

In an additional PCR we tried the amplify the linker-sequence, from a plasmid provided by Aliona Bodganova from the MPI protein facility. The plasmid carries a dCas9-GFP fusion protein with a linker sequence in between. Since this product has proven to work, we decided to use the same linker fragment, getting it out of the plasmid by PCR and adding the prefix and suffix via primer overhangs. The linker is very short and with the prefix and suffix would be around 100 bp of length. As can be seen in the gel this very short amplicon is not visible (Figure 5).

Since also the bands of the ladder of smaller size are not visible we decided to repeat the PCR and run on a higher percentage agarose gel.

#### 31st July – Paula and Mara

![](https://static.igem.org/mediawiki/2019/4/49/T--TU_Dresden--notebook-7.png)
Figure 6 – TAE gel results (for the fragment of interest)

To get the final PCR with primers 1 and 4 to work, we changed the annealing temperature to 55 °C and increased the extension time to 2 minutes.

The first two PCRs were repeated to have more material to work with while establishing the final PCR. With the adjusted parameters PCR 1 to 4 showed three different amplicons. The amplicon of interest at 4.2 kb, but also the shorter amplicons of the first two PCRs. Eventhough, only 1 µL of PCR 1 to 2 and of PCR 3 to 4 were taken as sample for the final PCR it might not have been enough of a dilution factor and the wrong amplicons are caused by leftover primers from the first PCRs. Therefore the PCR product was loaded on a TAE gel and the band at 4.2 kb (shown in Figure 6) was cut out from the gel and purified via gel purification Kit (Test-samples received from Jena Bioscience).

![](https://static.igem.org/mediawiki/2019/4/4a/T--TU_Dresden--notebook-8.png)
Figure 7 – 2% agarose gel results (for the linker)

The PCR for amplifying the linker fragement was repeated with the same PCR program but then run on a 2% agarose gel and successfull amplification of the small fragment could be seen (Figure 7).

The product of the PCR was as well loaded on a TAE gel and purified with the same Kit.

Overnight cultures were set from pSB1C3 - [RFP] and pSB1C3 - [strep] plasmids. Both of which were kindly provided by an old iGEM member of TU Dresden, Philipp Popp.

#### 1st July – Paula and Mara

The overnight cultures were minipreped and the pSB1C3 - [RFP] digested with EcoRI (E) and PstI (P). The PCR products for the linker and the complete dCas9 were as well digested with the same enzymes ligated and transformed as given in the NEB BioBrick instructions. The bands seen on the gel were thought to have a high enough concentration to make a transformation possible.

The pSB1C3 - [strep] was sent for sequencing with our generously sponsored Eurofins overnight sequencing barcodes (with primer TM2889). The sequence was successfully verified: Link to sequencing result. 100 µL of ligation were plated on chloramphenicol containing plates.

#### 20th July – Sebastian and Nikitha

From the plates with the ligated linker and dCas9 fragments, clones were picked and grown over night at 37°C, 950 rpm in a thermoshaker.

#### 21st July – Sebastian and Nikitha

![](https://static.igem.org/mediawiki/2019/4/41/T--TU_Dresden--notebook-9.png)
Figure 8 – in silica results in SnapGene
![](https://static.igem.org/mediawiki/2019/e/eb/T--TU_Dresden--notebook-10.png)
Figure 9 – Obtained gel results

MW: 1 kb DNA Ladder
1: pSB1C3-Linker
EcoRI + PstI
1. 2029 bp
2. 102 bp

2: pCas9-19\_pOCC97-cas9-dead
EcoRI + PstI
1. 7828 bp
2. 2809 bp

3: pSB1C3-dCas9\_RFC25
EcoRI + PstI
1. 4173 bp
2. 2029 bp

From the in silica gel created with SnapGene (Figure 8), it is obvious that none of the clones was positive (Figure 9).

#### 30th July – Sebastian and Nikitha

The gBlocks ordered for the two BioBricks of MBP and HRP arrived from IDT, were resuspended in water and digested the following way:

HRP-isoform C and MBP:
gBlock 43 µL
10x 2.1 NEB Buffer 5 µL
EcoRI-HF 1 µL
PstI 1 µL

500 ng of pSB1C3 were digested as given by NEB, the linearizd plasmid was run on an agarose TAE gel, the lane of interest was cut from the gel and purified. The yield of purified linearized plasmid was at 15 ng/µl.

The digested gBlocks were ligated into the linearized plasmid following the NEB protocol and electroporated at 1350 V into electrocompetent  prepared with the following protocol: Link to protocol. After electroporation the bacteria were given time to recover in media without LB for one hour in a thermoshaker at 37 °C, 950rpm.

Afterwards a 1:100 dilution was prepared and 100 µL of the dilution plated on Chloramphenicol containing plates.

#### 3rd August – Paula and Mara

To verify the successful insertion of the gBlocks in the plasmid, 1.5 mL liquid cultures containing 30 ng/µl chloramphenicol were set up with colonies picked from the plates plated on the 30th July. Ten colonies were picked for each BioBrick.

#### 4th August – Paula and Mara

The colonies from the day before were grown overnight and minipreped the next morning with a kit provided by our sponsor (Zymo Research). The obtained plasmids were digested with E and P and loaded on an agarose gel as follows.

![](https://static.igem.org/mediawiki/2019/2/27/T--TU_Dresden--notebook-11.png)
Figure 10 – Results of in silica gel (left) and agarose gel (right) of pSB1C3 – [MBP]

Comparing with the in silica gel made with SnapGene it is obvious that the colony number 8 (marked with an asterix) was positive (Figure 10). The bands match the expected sizes (given to the right) very well, eventhough the third band of the complete plasmid is also visible, which shows that the digest was not complete.

![](https://static.igem.org/mediawiki/2019/1/16/T--TU_Dresden--notebook-12.png)
Figure 11 – Results of in silica gel (left) and agarose gel (right) for pSB1C3 – [HRP]

Comparing to the in silica gel and the expected sizes calculated in SnapGene it can be seen that three colonies carry the insertion (clones 3, 7 and 8) (Figure 11). The clones of interest were again grown overnight in a liquid culture, because it was not enough plasmid DNA left to send for sequencing.

#### 5th August – Paula and Mara

The cultures from the positive clones identified the day before were minipreped with the Zymo-Kit. The protocol was changed by omitting the binding column and precipitating the DNA with isopropanol instead, which increased the yield. The plasmid DNA was sent for sequencing and the sequences were successfully verified: Link to sequencing result.

To elucidate the problem of why the PCR for mutating the dCas9 sequence did not work, three PCRs were performed. We wrote the following program presented in Table 4.

![](https://static.igem.org/mediawiki/2019/3/37/T--TU_Dresden--notebook-13.png)
Table 4 – PCR program

![](https://static.igem.org/mediawiki/2019/e/ea/T--TU_Dresden--notebook-14.png)
Figure 12 – Results of PCR on agarose gel

Three PCRs were performed:

A - Y = 6; X = 11
B - Y = 12, X = 22
C - the program was split into two PCRs in which the 3-step part had no additional primers added
(only the overhangs from primers 2 and 3) and before starting the 2-step part primers 1 and 4 were added.

The amplicon of interest was only amplified in PCR B. It might be that because of the increased cycle number the lane is visible in B but not in A and amplification was also successful in A. In PCR C only the two smaller fragments were amplified but not the big one (Figure 12).

#### 7th August – Paula and Mara

The BioBricks that were successfully verified by sequencing, MBP, HRP and strep were midipreped to have a large amount for later exeriments. At the same time the pSB1C3-[RFP] and the pSB1A3-[RFP] plasmids were midipreped as well. All midipreps were done with a Zymo-Kit with 50 mL cultures.

From each midiprep 200 ng were digested with E+P and separated on an agarose gel (Figure 13).

![](https://static.igem.org/mediawiki/2019/7/77/T--TU_Dresden--notebook-15.png)
Figure 13 – Separation results on agarose gel

The plasmids with RFP insert and the BioBricks were successfully verified. PCR 1 to 2 was redone successfully, as well as PCR 3-4 (gel not shown) to have a sufficient amount of DNA for the purification (with a PCR purification Kit from Jena Bioscience) and subsequent repeat of the proven to work PCR B as done on the 5th August. The unspecific amplicons of the smaller fragments seen on that day, were probably taking activity and ressources from the polymerase, so the amount of DNA we got was too small for the transformation we tried. For this reason purifying the new PCR 1 to 2 and 3 to 4, removing all the old primers, would hopefully lead to a single amplicon of sufficient concentration.

#### 9th August – Paula and Mara

![](https://static.igem.org/mediawiki/2019/d/df/T--TU_Dresden--notebook-94.png)
Figure 14 – Agarose gel electrophoresis

The PCR 1 to 4 was repeated with the purified samples from the 7th of August with the PCR protocol as given on the 30th July.

As can be seen from the gel, the PCR was finally successful, with a strong amplification of the amplicon of interest and no unspecific amplification (Figure 14). To test if the EcoRI site was successfully mutated and not recognized by the restriction enzyme anymore, 200 ng of DNA were digested with EcoRI and run on a gel. As can be seen on the right the fragment is not digested. The former forbidden restriction enzyme site is therefore proven to be removed.

#### 10th August – Paula and Mara

The PCR amplified dCas9\* sequence was digested with E and P, ligated into pSB1C3, electroporated into  GB05 and plated as described earlier.

#### 11th August – Paula and Mara

From the plates 10 clones were picked and set up to grow in 2 mL liquid media with Cm overnight.

#### 12th August – Paula and Mara

![](https://static.igem.org/mediawiki/2019/0/0d/T--TU_Dresden--notebook-16.png)
Figure 15 – Agarose gel electrophoresis

The overnight cultures were minipreped, digested with E and P and separated on an agarose gel.
Five clones were positive and two of then were sent for sequencing, to check for the accumulated mutations during the PCR cycles.

##### [Adding BioBricks together to the final construct ![](https://static.igem.org/mediawiki/2019/e/ef/T--TU_Dresden--flechaPau.png)](#answerThree)

![](https://static.igem.org/mediawiki/2019/b/b2/T--TU_Dresden--notebook-20.png)

Figure 16 – The scheme shows systematically how we were adding our different BioBricks together to the final construct, All basic BioBricks were cloned in pSB1C3, for the first fusions transferred to pSB1A3 and for the final construct cloned in pOCC97 (Kn resistance), bold and darker shaded BioBricks are newly designed by our team and lighter are BioBricks used from old teams. On each arrow the enzymes for this specific digest are indicated with their first letter.

#### 12th August – Paula and Mara

As first two BioBricks the HRP and the Strep-tag were added together with the RCF25 enzymes. Therefore the following digest was set up:

pSB1C3 - [HRP] with E and AgeI (A) in CutSmart
pSB1C3 - [Strep] with P and NgoMIV (N) in NEB buffer 1.1
pSB1A3 - [RFP] with E and P in NEB buffer 2.1

Ligation, transformation and plating was performed as described before.

#### 13th August – Paula and Mara

Seven clones were picked from the plates plated the day before and grown for 5 hours in liquid media. The cultures were then minipreped with the Zymo-Kit and isopropanol precipitation.

#### 15th August – Paula and Mara

To be able to submit the pOCC97 expression plasmid, in which we will clone our final construct, we designed primers flanking both sides of the plasmid around the insertion site and amplified the backbone in a PCR with the following program shown in Table 5.

![](https://static.igem.org/mediawiki/2019/c/c8/T--TU_Dresden--notebook-21.png)

Table 5– PCR pOCC97 backbone program

pOCC97\_fw: tactagtagcggccgctgcagCCGTTATAGAAGCTTGAGTATT
pOCC97\_rev: gaattcgcggccgcttctagagGCCCATGGATATATCTCCTTCT

#### 17th August – Paula and Mara

The linker BioBrick which could not be amplified by PCR was ordered from IDT in RCF25 standard as a gBlock. The dried gBlock was resuspended, digested with E and P and ligated into pSB1C3-[RFP].

#### 20th August – Paula and Mara

![](https://static.igem.org/mediawiki/2019/d/d2/T--TU_Dresden--notebook-22.png)

Figure 17 – Agarose gel electrophoresis (linker)

The cultures from the day before were and minipreped and restriction digested with EcoRV, PstI the different enzyme was chosen to make it possible to see the very tiny fragment (100 bp) of the linker in comparison to the pSB1C3[RFP], which was digested with EcoRI, EcoRV, PstI.

The expected bands for the linker are at 1133 bp and 998 bp (Figure 17). For the pSB1C3[RFP] they are at 1133 bp, 1110 bp, 896 bp. The difference of the band at 896 bp and of 998 bp can clearly be seen. All clones successfully took up the linker fragment.

#### 23rd Septermber – Paula and Sebastian

![](https://static.igem.org/mediawiki/2019/4/4d/T--TU_Dresden--notebook-23.png)

Figure 18 – Agarose gel eletrophoresis

The dCas9 sequences were sequenced of several positive clones from the PCR of the 12th of August. The following cloning was done. MBP digested with E and N, Cas9 with A and P, HRP-strep with A and P, pSB1A3 with E and P, Linker with E and N. They were ligated in the following way:

MBP with dCas9 in pSB1A3
Linker with HRP-strep in pSB1A3

As described before the samples were transformed, plated, grown, picked, minipreped, E and P digested and analyzed on an agarose gel.

As can be seen on the gel, only the plasmid backbone is visible in all samples and not the fragment of interest (Figure 18). Therefore, the cloning was not successfull.

#### 28th August – Sebastian and Paula

Restriction digestion was performed again to check if one of these clones have the construct with them. Restriction digestion was done with following enzymes: pSB1A3A with E and P, MBP with E + A, dCas9 with N + P HRP-strep with N +P. Appropriate fragments digested was cut out the gel and gel clean was performed (Figure 19). Two ligation was performed first, between MBP, dCas9 and pSB1A3 and second- pSB1A3, linker and HRP-strep. Ligated products were transformed and plated.

![](https://static.igem.org/mediawiki/2019/8/8d/T--TU_Dresden--notebook-24.png)

Figure 19 – Results for digested fragments

#### 29th August – Sebastian and Paula

Transformed colonies were grown and miniprepped and loaded onto 0.8 % agarose gel to check if the ligation reaction worked (Figure 20). For MBP and dCas9 ligation, clone 2 worked with ligated product seen at 500 bp. In the second ligation reaction between Linker, HRP and Strep, all the clones had ligated product at 1000 bp and ligation was successful.

![](https://static.igem.org/mediawiki/2019/5/59/T--TU_Dresden--notebook-25.png)

Figure 20 – Results for the ligation of MBP-dCas9 and Linker-HRP-Strep

#### 1st September – Sebastian and Paula

Ligation reaction between MBP-dCas9 (clone 3) and Linker-HRP-strep (clone 5) from 29.09.19. MBP-dCas9 was digested with E+A, linker-HRP-Strep with N + P and pSB1C3 with E +P. Triplicates of mini prepped clone 3 was digested and mini prep of 5 clones was performed and after restriction digest respective fragments (marked in red) was cut out and gel purification was carried out for final ligation (Figure 21).

![](https://static.igem.org/mediawiki/2019/2/28/T--TU_Dresden--notebook-26.png)

Figure 21 – Results for the ligation between MBP-dCas9-Linker-HRP-Strep

#### 7th September – Mara and Paula

Ligation reaction between MBP-dCas9 (clone 3) and Linker-HRP-strep (clone 5) from 29.09.19. MBP-dCas9 was digested with E+A, linker-HRP-Strep with N + P and pSB1C3 with E +P. Triplicates of mini prepped clone 3 was digested and mini prep of 5 clones was performed and after restriction digest respective fragments (marked in red) was cut out and gel purification was carried out for final ligation (Figure 21).

![](https://static.igem.org/mediawiki/2019/4/4f/T--TU_Dresden--notebook-27.png)

Figure 22 – Results for ligation and cloning of full construct: MBP-dCas9- Linker- HRP-Strep

#### 8th September – Mara and Paula

1. Ligating HRP-Strep in pOCC97 (expression vector) using E+P and optimizing CutSmart buffer for improved digestion by enzymes.

2. Cloning of Full construct in pOCC97 expression vector.

![](https://static.igem.org/mediawiki/2019/9/98/T--TU_Dresden--notebook-28.png)

Figure 23 – Results for ligation of full construct: HRP-Strep

Expected fragment size of HRP-Strep (insert) 1 kb and the pOCC97 backbone is 5 kb. We do not see band of HRP-strep at 1 kb, most likely that this fragment ran out of gel and we only see the pOCC97 backbone (Figure 23).

![](https://static.igem.org/mediawiki/2019/c/cc/T--TU_Dresden--notebook-90.png)

Figure 24 – Results for ligation and cloning of full construct

Full construct was cloned into pOCC97 and digested with X +P to get two fragments at 5kb and 6 kb (Figure 24). However, these fragments were too close and could be resolved and visualized as individual bands.

![](https://static.igem.org/mediawiki/2019/b/b9/T--TU_Dresden--notebook-91.png)

Figure 25 – Agarose gel image simulation in SnapGene

In Figure 25 the agarose gel image stimulation of X +P digestion and resolution of the two fragments are presented.

It can be seen in the first lane (digested with X and P), that the difference in size between the backbone (5 kb) and the full construct (6 kb) is not sufficient to be properly separated on an agarose gel. Therefore a new restriction digest had to be prepared in which a triple digest with X, P and PmlI additionally was set up. The fragments of different size makes it easy to distinguish a successful insertion of the final construct in the expression plasmid.

#### 9th September – Mara and Paula

1.Triple digestion of ligated full construct into pOCC97
2.Cloning of K1800002 into pOCC97 and digestion
3.Cloning of HRP-strep into pOCC97 and digestion

![](https://static.igem.org/mediawiki/2019/1/13/T--TU_Dresden--notebook-92.png)

Figure 26 – Results for ligation and cloning of full construct (two replicates), K1800002 and HRP

From the ligation replicate 1 and 2 we see three fragments in the 3rd lane and 8th lane, respectively (suggesting successful ligation into the pOCC97). However, HRP-Strep cloning into pOCC97 was not successful, since we could not see the third fragment.

#### 10th September – Mara and Sebastian

![](https://static.igem.org/mediawiki/2019/9/9c/T--TU_Dresden--notebook-93.png)

Figure 27 – *sry* gene amplification result

We amplified *sry* gene (template buccal swab from Sebastian) using the following forward and reverse primers:

Forward - AGTAAAATAAGTTTCGAACTCTGG

Reverse - AGGCCTTTATTAGCCAGAGAAAA, annealing temperature 55 ℃ (Figure 27).

#### 11th September – Mara and Sebastian

![](https://static.igem.org/mediawiki/2019/1/1f/T--TU_Dresden--notebook-95.png)

Figure 28 – Cloning of K1800002 into pOCC97 followed by digestion and cloning of HRP-strep into pOCC97 and digestion

All the ligated clones were mini prepped and digested with PmlI, EcoRI and Pst1. Positive clones (marked in red) had 1 kb fragment of HRP and they were selected for expression of HRP and HRP- Strep respectively (Figure 28).

#### 12th September – Mara and Sophie

![](https://static.igem.org/mediawiki/2019/e/ef/T--TU_Dresden--notebook-29.png)

Figure 29 – Cloning results for different constructs

Final clone check of pOCC97 expressing dCas9-HRP construct using triple restriction digestion PmlI, X and P.

Final clone check of pOCC97 expressing K1800002 construct using triple restriction digestion PmlI, EcoRI and Pst1.

Final clone check of pOCC97 expressing HRP-strep construct using triple restriction digestion PmlI, EcoRI and Pst1.

Selected clones were used for respective protein expression.

##### [Characterization of expression and purification conditions for full construct![](https://static.igem.org/mediawiki/2019/e/ef/T--TU_Dresden--flechaPau.png)](#answerFour12)

#### 1. Expression

**1st October - Paula and Mara**

The full construct, which is uploaded in a BioBrick as K3037003 was cloned into our expression plasmid K3037000, and was expressed overnight. First expression was done at 37°C for seven hours, induced with 1mM IPTG.

![](https://static.igem.org/mediawiki/2019/3/3c/T--TU_Dresden--notebook101.png)

Figure 32 - Expression of full construct in pOCC97 not optimized (BBa\_K3037000)

The full construct is 230 kDa and can be seen at the expected size on the gel (Figure 32). On the time point before induction, the band of the protein of interest can not be seen. This shows that our induction is specific and the promoter is not leaking.

**2nd October - Sebastian Nikitha and Mara**

To identify the perfect conditions for expression of this novel protein, which did not exist before, we tested different IPTG concentrations and different temperatures (Figure 33). From the fusion protein that we used before (eGFP-dCas9) we knew that a protein of similar size was expressed in the same strain of E.coli pRARE T7 at 18°C, which improved proper folding. Therefore, we wanted to test this condition with our protein. As a comparison we used the natural growth conditions of E.coli, at 37°C, to compare if the amount of protein, we would obtain would reduce with the reduced temperature. At the same time we checked different insertions in the expression plasmid. Which are marked as optimized and not optimized pOCC97.IPTG concentrations were checked from 0.5, 1.0 to 1.5 mM.

![](https://static.igem.org/mediawiki/2019/d/d7/T--TU_Dresden--notebook102.png)

Figure 33 - Different IPTG concentrations and temperature to test the expression of the novel protein

The SDS-PAGEs shown above were analyzed with Fiji (Figure 34). The intensity of each band was normalized against the background of its corresponding lane. Additionally, for the single data points the values were normalized from zero to one. Different temperatures:

![](https://static.igem.org/mediawiki/2019/6/69/T--TU_Dresden--notebook103.png)

Graph 1 - Analysis of novel protein expression performed in Fiji

It is important to note, that the fourth time point is after overnight expression (8 hours after time point 3, the other timepoints are 1 hour apart).

On each graph the Full construct 11 and Clone 7 are the same sequence of our fusion protein. The difference is the way how they are integrated in the expression plasmid. The clone number 11, is using the RBS of the Freiburg standart (Shine-Dalgarno-Sequence), while the clone 7 has additionally a longer space with the native RBS of the POCC97 between the Promoter and the shine-dalgarno sequence. It can not be inferred from the data what exactly is causing differences in expression, but differences can be observed. First of all the total amount of recombinant protein expressed does not differ between both expression constructs, as estimated from the image analysis of the SDS-pages, but the expression with clone 7 seems more robust. Comparing the expression at different temperatures and different IPTG concentrations does not lead to strong differences in the final amount for clone 7, but does show strong differences for clone 11. For clone eleven a strong dependence on the IPTG concentration, which differently influences expression over time, can be seen in Figure 33 and Graph 1.

While the higher amount of IPTG impaired expression at 37°C it improved expression at 18°C. For both constructs the ideal concentration of IPTG is 0.5 mM. The higher amount of inducer does not lead to a higher expression of the protein of interest.

Since the expression is far more robust in the optimized plasmid backbone, we decided to perform future expressions in this construct. For this the expression at 18°C led to a higher protein amount than t = 37°C and an induction with 0.5mM led to higher expression than 1 mM. 0.2 mM had not been tested in this construct, but since 0.5mM proved to be the ideal expression concentration for the other construct at 18°C, we chose this to be our working concentration.

**So the ideal conditions were identified as:
Overnight, 18°C, 0.5mM IPTG, pOCC97\_optimized.**

#### 2. Purification

**5th October - Paula and Mara**

*In silico* analysis of our new fusion protein is presented below.

Theoretical analysis of the expected parameters of the new protein (determined with ExPASy ProtParam tool):

Extinction coefficient: 205750

Estimated half-life > 30 hours in mammalian cells, >20 hours in yeast, >10 hours in E.coli.

The same way that we had to identify the ideal expression conditions by trial and comparison, we also had to establish the purification protocol for this new protein. We did this first by purification on an amylose resin of two cultures with each 500 mL culture grown for 18 hours at 18°C with 0.5mM IPTG.

Buffer compositions were adapted from Alionas protocol for eGFP-dCas9 purification.

**Two different methods of binding to the column were investigated:**

First **binding in the column** was tested by loading the cell lysate on the resin in a purification column, as given in most purification protocols (Figure 34). The cell lysate was loaded at very low flow rate for approximately two hours column loading time.

![](https://static.igem.org/mediawiki/2019/5/58/T--TU_Dresden--notebook104.png)

Figure 34 - Purification of the full construct from pOCC97 optimized in Amylose Resin column

Second via **batch binding** (Figure 35). The resin was pipetted into a falcon and incubated with the cell lysate for 1.5 hours on a rotator at 4°C.

![](https://static.igem.org/mediawiki/2019/c/c7/T--TU_Dresden--notebook105.png)

Figure 35 - Purification of the full construct from pOCC97 optimized via batch

It can be seen that the band of interest is not visible before but well visible after induction. Additionally on both SDS-PAGES it is obvious that our protein has a good solubility, since the band of stronger intensity is in the supernatant and not in the pellet. Unfortunately, in both SDS-PAGES a strong band can be seen in the flow through, this results from overloading of the column. Since the column volume was only 1mL, the column was saturated and a lot of protein got lost. But the elutions show that only our protein eluted specifically with a lot of truncated versions that typically appear for very large proteins in E.coli, which is interrupting translation or transcription.

For the rest of the lab work after October 6th [DOWNLOAD THE PDF](https://static.igem.org/mediawiki/2019/b/b6/T--TU_Dresden--after6.pdf)

##### [Electromobility shift assays![](https://static.igem.org/mediawiki/2019/e/ef/T--TU_Dresden--flechaPau.png)](#answerFour)

#### 10th September - Nikitha and Sebastian

Materials:

-100 ng of PCR amplified *sry* gene
-200 ng of dCas9-GFP
-200 ng of guide RNA specifically targeting the amplified *sry* gene
-The 6 unique guide RNAs all targeting different regions of *sry* gene were designed. Using the online tool benchling and fasta sequence of *sry* gene, following guide RNAs were designed

![](https://static.igem.org/mediawiki/2019/b/be/T--TU_Dresden--notebook-30.png)
1 x Reaction buffer (20 mM Hepes buffer pH 7.2, 100 mM Nacl, 5 mM Mgcl2, 0.1 mM EDTA)

Methods:

We wanted to check if the overall efficiency of mobility shift increases when combinations of guide RNAs are used, so individual reactions with combinations of guide RNA were used.

Guide RNA, dCas9-GFP and *sry* gene was incubated in reaction buffer (respective amounts mentioned in the materials section) for 37 °C for 1 hour.

Post incubation, they were mixed with loading dye without SDS, 20 % glycerol in Orange G dye and loaded onto 4-20 % gradient acrylamide- TBE precast gel. Gel was run for 3 hours at 75V in 1 x TBE buffer.

Gel was then stained using ETBR with 1 : 20000 dilution in 1x TBE for 10 minutes. The loading order is presented below (Figure 19):

![](https://static.igem.org/mediawiki/2019/e/e8/T--TU_Dresden--notebook-31.png)
Figure 30 – SDS-PAGE results

Lane 1: 1 kb DNA ladder
Lane 2: *sry* gene
Lane 3: *sry* gene + dCas9
Lane 4: *sry* gene + dCas9 + guide RNA 1
Lane 5: *sry* gene + dCas9 + guide RNA 4, 2 and 6
Lane 6: *sry* gene + dCas9 + guide RNA 1, 5 and 6
Lane 7: *sry* gene + dCas9 + guide RNA 1, 4, 3 and 6
Lane 8: *sry* gene + dCas9 + guide RNA 4, 3 and 7
Lane 9: *sry* gene + dCas9 + guide RNA 1, 4 and 2

Lane 1 - There is a clear *sry* gene at 800 base pairs and when *sry* gene is incubated with only dCas9 (lane 2) there is no shift seen in the position of the gene. In lane 3, when guide RNA 1 was incubated with the dCas9 DNA reaction mix, we see a shift in the mobility, this is because of the protein DNA interaction and this binding is hindering the gene mobility. In lanes 5,6,7,8 and 9 combinations of guide RNAs were used. From lane 7 and 8 we see the highest mobility shift. From the electromobility shift assay performed above, we conclude that our expressed dCas9-GFP protein is functional and is able to successfully bind to gene with the help of appropriate guide RNAs.

#### 11th September - Nikitha and Sebastian

We wanted to check if guide RNA alone is able to cause change in the mobility of *sry* gene
Methods 1 to 4 was repeated and this gel ran for 3 hours in order to get rid of all the secondary structures of the RNA formed (seen at the bottom of the gels, where guide RNA was loaded) and following was the loading order (Figure 20):

![](https://static.igem.org/mediawiki/2019/e/e7/T--TU_Dresden--notebook-32.png)
Figure 31 – SDS-PAGE results

Lane 1: Marker
Lane 2: *sry* gene
Lane 3: *sry* gene + guide RNA 1
Lane 4: *sry* gene + guide RNA 2
Lane 5: *sry* gene + guide RNA 4
Lane 6: *sry* gene + guide RNA 7
Lane 7: *sry* gene + dCas9
Lane 8: *sry* gene + dCas9 + guide RNA 1, 4 and 2
Lane 9: *sry* gene +dCas9 + guide RNA 3, 4 and 7
Lane 10: *sry* gene + dCas9 + guide RNA 1
Lane 11: guide RNA 1
Lane 12: dCas9-eGFP

From lane 3 till 7 , we do not see any difference in the mobility of *sry* gene when only guide RNA is added to the reaction mix. In Lane 8, 9 and 10 we see mobility shift of the gene and in lane 11, when only guide RNA was loaded , we see no bands and in lane 12, we see dCas9 in stacking part of gel, owing to higher molecular weight.

#### 14th September – Nikitha and Sebastian

1. In order to characterize the functionality of our expressed full construct (dCas9-HRP), we performed gel shift assay using our expressed protein and specific guide RNAs targeting*sry* gene. Control for this experiment is dCas9-GFP (which was successful in binding and hindering the mobility of DNA).

2. Checking the binding efficiency of dCas9 protein to GFP with the help of guide RNA specific to eGFP. Here, K3037005 in K3037000 was cleaved with BsaI and NotI to cut the eGFP fragment out.

3. Proof of concept to demonstrate that our dCas9 protein is successfully able to bind to DNA immobilized on cellulose strip. Further before loading on gel, strip was washed with wash buffer (W1) to remove unbound proteins (Figure 41).

![](https://static.igem.org/mediawiki/2019/c/c6/T--TU_Dresden--notebook-100.png)

Figure 41 - Proof of dCas9 and DNA bound to cellulose interaction (Lanes 10, 11 and 19)

Lane 1 - Marker
Lane 2- 100 ng of*sry* gene
Lane 3- *sry* + dCas9-HRP
Lane 4 -*sry* + dCas9-HRP+Guide RNA 1 + Guide RNA 4 +Guide RNA 2
Lane 5 -*sry* + dCas9-HRP+Guide RNA 1
Lane 6 -*sry* + dCas9-GFP+Guide RNA 1 + Guide RNA 4 +Guide RNA 2
Lane 7 - eGFP+dCas9-HRP+Guide RNA 7
Lane 8 - eGFP+dCas9-HRP+Guide RNA 7
Lane 9 - eGFP
Lane 10 -*sry* + dCas9-HRP+Guide RNA 1 + Guide RNA 4 +Guide RNA 2
(Here*sry* gene was immobilized on cellulose strip and addition of dCas9 proteins and guide RNA was all done in cellulose strip).
Lane 11 -*sry* + dCas9-GFP+Guide RNA 1 + Guide RNA 4 +Guide RNA 2
Lane 12 -*sry* gene loaded on cellulose strip.

In lane 2 when PCR amplified*sry* gene was loaded, a faint band is seen at 3 Kb, most likely that this band is visible is because we did not perform PCR gel clean up. In lane 3 - without the presence of guide RNA, dCas9 -HRP is not able to bind to*sry*. In lane 4 and 5 with the presence of specific guide RNA, dcas9-HRP is not able to bind to*sry* gene, indicating that our expressed full construct of dCas9-HRP is unable to bind to the DNA and is not functional.

In lane 6 - we have our positive control, where dCas9-GFP is successfully able to bind and hinder the mobility of*sry* gene.

In lane 7- eGFP was not pulled by dCas9-HRP, proving again the non-functionality of the expressed protein. In lane 8 - eGFP was successfully bound to dCas9-GFP and pulled up. In lane 8 we see the digested band of eGFP. In lane 11 - we again do not see the pull up of*sry* gene loaded on the cellulose strip with dCas9-HRP but in lane 12- when dCas9-GFP was loaded, we see the gene pull up showing the functionality and proof of concept of dCas-GFP functioning on the cellulose disc and this is specific interaction since all the unbound proteins were washed and removed out. Lastly, lane 12 just has*sry* gene loaded on the strip

#### 14th October – Nikitha and Sebastian

In this EMSA we invesigate for the first timne if our full construct has an able dCas9, wich means if it is able to bind to the *sry* gene and cause the shft in the EMSA assay.
Furthermore we investigated the different incubation times that Cas9 needs with its guideRNAs and the target DNA. The original protocol from the MPI stated that 1 hour of incubation was necessary ut we wanted to cut down this time.

![](https://static.igem.org/mediawiki/parts/6/64/T--TU_Dresden--complete.png)

Figure 42 - Proof that the dCas9 of our full construt is active (lane 3) and investigation of different incubation times (lane 7-12)

In lane three where our purified full construct was loaded in very high concentration the *sry* gene cannot even be seen anymore at the same height as the pure sry without shift. This could be due to several reasons.
One theory was that the concentration is so high and the pull so strong that no gene is visible anymore. Otherwise it could just have been a pipetting mistake.
The incubation time analysis showed that the ideal incubation time for our protein is not necessarily one hour, lowertime-points, even five minutes, showed identical shift.

## Results & Discussion

* We designed four new Biobricks and adapted an old one (HRP, K1800002) to the RCF25 standart. Each one of them was cloned into pBS1C3. The dCas9 sequence was mutated by site directed mutagenesis PCR to remove a forbidden EcoRI site in the middle of the coding sequence.
* We assembled a novel fusion protein from six BioBricks one by one as planned in silico and proved each intermediate step via sequencing.
* We designed our own expression plasmid, that we optimized for ideal fast and easy expression and made it compatible to the BioBrick standart. It is uploaded in the registry as [BBa\_K3037000](http://parts.igem.org/Part%3ABBa_K3037000).
* The final construct of all assembled BioBricks was cloned into the expression plasmid [BBa\_K3037000](http://parts.igem.org/Part%3ABBa_K3037000) and successfully expressed via IPTG induction
* **All parts, except one, of the final construct were separately proven to work as part of the fusion protein:**
  + MBP -> verified by purification on amylose resin
  + dCas9 -> verified by EMSA shift assay
  + HRP -> verified by activity assay compared to K18002
  + strep -> failed to verify via Strep-Tactin-Column purification (the old BioBrick [BBa\_K823038](http://parts.igem.org/Part%3ABBa_K823038))
  was designed for Western Blot analysis, not for strep-column purification)
* We have a functional eGFP-dCas9-fusion protein expressed, which is uploaded as a BioBrick in [BBa\_K3037000](http://parts.igem.org/Part%3ABBa_K3037005). We show that it is able to fluoresce and that it binds successfully to the *sry* gene with the help of guideRNA, that we designed and optimized.
* We showed that dCas9 on its own, without guideRNAs, is unable to bind to *sry* gene, proving that for binding guide RNA is required. Futheron we showed that guide RNAs on their own is unable to cause mobility shift of *sry* gene, proving that dCas9 is needed for it.

## Idea & Planning

The isolation of DNA from biological samples usually requires a relatively long amount of time and involves multiple intermediate steps in the procedure. To address this issue, Zou *et al.* (2017) established a novel nucleic acid extraction method that allows to rapidly capture nucleic acids on cellulose paper avoiding laborious steps in between, and was ready to use for PCR.[1] The main advantage of this procedure is the time of its performance which does only take 30 seconds in total. But the total amounts of DNA are very low and the method was mainly focused on plants, so that we had to develop our own method for the extraction of genomic DNA from human cells and microbes.

By dipping a cellulose disk into a lysis buffers - P1 (50 mM Tris-HCl, pH 8.0, 10 mM EDTA) and P2 (200 mM NaOH, 1% SDS), with a following washing step, DNA was successfully extracted from A. thaliana in (Zou *et al.*, 2017) and subsequently amplified by PCR.[1]

The aforementioned method was modified in our experiment, in order to evaluate the role of salt concentration on the efficacy of DNA binding to the cellulose paper which leads to the higher yield of the final product. The samples were treated with different solutions of NaCl (50 mM, 100 mM and 500 mM) aiming to determine the most optimal concentration for DNA extraction procedure.

In our experiment we used  GB05 for testing the genomic DNA extraction and GB05 with pSB1C3 (BBa\_J04450) for plasmid DNA extraction. The cells were lysed with the buffers P1 (50 mM Tris-HCl, pH 8.0, 10 mM EDTA) and P2 (200 mM NaOH, 1% SDS), followed by simple dipping of the cellulose disks into the cell lysate to extract genomic and plasmid DNAs. The disks were washed with buffer W1 (100mM Tris, 0.1% Tween20) to selectively remove proteins and cell debris while leaving DNA bound to the cellulose matrix. The bound DNA is ready for follow up experiments.

The opportunity to shorten the time for the DNA extraction procedure and by using affordable materials, also comes along with a great advantage to apply this technique in limited resource settings which make it achievable for many people around the world.

### References

[1] Zou Y, Mason MG, Wang Y, Wee E, Turni C, et al. (2018) Correction: Nucleic acid purification from plants, animals and microbes in under 30 seconds. PLOS Biology 16(5): e1002630. https://doi.org/10.1371/journal.pbio.1002630

## Labwork & Protocols

##### [Quantifying the amount of DNA extracted with the help of Whatman(cellulose disc) ![](https://static.igem.org/mediawiki/2019/e/ef/T--TU_Dresden--flechaPau.png)](#answerFour1)

#### 5th July – Nikitha and Sebastian

For extracting DNA from  cells, we followed the DNA extraction protocol.

#### 7th July – Nikitha and Sebastian

1. Optimizing the cell lysis time for higher elution of DNA from Whatman cellulose disc. We quantified the concentration of DNA that we eluted from the Whatman cellulose disc. We performed the reaction in duplicates and found out that 1 minute of cell lysis works the best with lower variations in the concentration of duplicates. Concentrations of DNA with 1 minute lysis time is 30 - 40 ng/uL – (Graph 1).

![](https://static.igem.org/mediawiki/2019/1/13/T--TU_Dresden--notebook-33.png)

Graph 1 - Nanodrop quantification of DNA eluted with varying lysis time

2. Optimizing lysis time and checking the optimal elution temperature for bound DNA from cellulose disc

![](https://static.igem.org/mediawiki/2019/0/01/T--TU_Dresden--notebook-34.png)

Graph 2 – Concentrations of DNA eluted with varying lysis time and elution done at room temperature and the second part of the graph (legend grey) with same lysis time with elution done at 55 °C

From graph 2, we conclude that elution at 55 °C yielded more DNA but however, with higher variations in the duplicates.

3. Measuring the purity of eluted DNA

![](https://static.igem.org/mediawiki/2019/7/76/T--TU_Dresden--notebook-35.png)

Graph 3 - Absorbance ratio at 260/280 nm to measure protein contamination

From Graph 3, we conclude that when elution is done at RT the ratio of absorbance at 260/280 nm is above 1.8 indicating no protein contamination, and also that they do not interfere with the concentration of DNA quantified earlier (Graphs 1 and 2).

##### [Improving the yield of DNA extraction and quantifying this extracted DNA![](https://static.igem.org/mediawiki/2019/e/ef/T--TU_Dresden--flechaPau.png)](#answerFour2)

#### 23rd August – Nikitha and Sebastian

Taken as a basis cellulose paper based DNA and RNA extraction method established by Y. Zou *et al.* was modified in our experiment. A genomic DNA was extracted from the overnight grown  GB05 cultures with the following DNA extraction procedures [1], [2].
To determine the relation between salt concentration used and the yield of DNA after the extraction procedure, different solutions of NaCl (50 mM, 100 mM and 500 mM) were applied in DNA precipitation step (Figure 1). The most intensive band was observed in the samples extracted with the addition of 100 mM of NaCl.

![](https://static.igem.org/mediawiki/2019/e/ea/T--TU_Dresden--notebook-36.png)

Figure 1 – Agarose gel electrophoresis results: salt concentration affects the yield of the extracted DNA.
 A. DNA – nitrocellulose interaction; B. DNA – cellulose interaction (single washing step)

Additionally, to compare the efficacy of DNA interaction with different types of paper, prior to the agarose gel electrophoresis, the extracted DNA was loaded on nitrocellulose and cellulose discs. It was shown that DNA have slightly stronger interaction with nitrocellulose paper rather than with cellulose which resulted in the bands of much lower intensity on the agarose gel (Figure 1A).

In the separate experiment an additional washing step was performed after the nitrocellulose – and cellulose - DNA binding step. The obtained results are depicted in the Figure 2.

![](https://static.igem.org/mediawiki/2019/5/5f/T--TU_Dresden--notebook-37.png)

Figure 2 – Agarose gel electrophoresis results: salt concentration affects the yield of the extracted DNA.
 A. DNA – nitrocellulose interaction; B. DNA – cellulose interaction (two washing steps)

In case of the DNA – nitrocellulose interaction, the faint bands are observed only in the lanes 3 and 4, where there was no salt treatment performed. Meanwhile, in the rest of the lanes, almost no bands could be noticed, which might be due to very strong binding between the DNA and the nitrocellulose disc.

In order to get rid of any residual salts present in the samples, an additional washing step was performed with the buffer containing 10 mM Tris-HCl and 80% ethanol. It was expected that ethanol would remove residual salts bound to the cellulose strip. However, in the following experiment all DNA was eluted, and almost no band was observed.

## Results & Discussion

* We were able to successfully extract pure genomic DNA from  (GB05) cells using a paper based fast isolation method.
* We optimized buffer conditions for lysis and washing buffers, establishing a robust, well working protocol.
* Concentration of DNA isolated is 30 - 40 ng/uL with a 260/280 ratio of ~1.8 (indicating very low amount of contaminating proteins)
* we found that 100 mM NaCl enhances DNA binding to cellulose paper. This is probably due to the electrotatic interaction between the -OH groups of the cellulose and the negative phosphate backbone of the DNA. The interaction between the two is mediated between cations (here Na+).
* Nitrocellulose discs established much stronger DNA binding than cellulose paper.
* If more than one washing step is performed the DNA starts to elude and concentration is lost. Therefore one washing step is ideal.
* DNA extraction from plant cells works well as described by Zou *et al.* 2017 and was used by our team for all the PCRs performed for our secodn project  [Sweet Spirulina](https://2019.igem.org/Team%3ATU_Dresden/Spirulina)

## Idea & Planning

From the very beginning of our project we had access to a well functioning dCas9 –GFP fusion protein, which we could use for our experiments while the cloning for the fusion protein that we desined was still in the making. But eGFP as a reporter molecule requires a plate reader or other advanced technology that is able to excite the molecule at a certain defined wavelength. But the idea behind our DipGene project was to express dCas9–HRP in order to have a visual read out visible to the naked eye. But while our second construct was in the cloning phase, we started with characterizing cellulose dCas9-GFP interaction to understand fusion protein behaviour with cellulose.

In order to characterize the behaviour of dCas9 fusion protein binding with nitrocellulose membrane we designed following experiments:

**1. To check if dCas9 binds to bacterial DNA (GB05 cells) immobilized on strip without the help of guide RNAs.**

**2. If dCas9 binds to target plasmid DNA immobilized on strip with the help of guide RNAs specifically targeting eGFP (which is coded for on the plasmid).**

**3. To check if dCas9 can bind to human target DNA, since our genomic DNA extraction from buccal swap was not established yet we amplified the target gene in PCR and used that as a target.**

**4. We checked different blockers to stop unspecific binding od proteins from the cell lysate on the membrane and later check the efficiency of blocking by running on the 12.5 % SDS- acrylamide gel.**

In order to find if dCas9 is bound to the nitrocellulose strip or it elutes out of the strip post washing we ran incubated strips on 12.5 % SDS- acrylamide gel.

## Labwork & Protocols

Aim of all the experiments performed below was to investigate the interaction between dCas9 fusion proteins and cellulose. It was of specific interest to find out if dCas9 would unspecifically bind to the cellulose. And if we could identify blokers or washing conditions that would specifically wash away the unspecifically bound dCas9.

#### 10-20 th May – Nikitha and Sebastian

The first problem was to find a way of quantifying the dCas9 bound to the cellulose. The first idea was to follow the glowing of eGFP from the dCas9-eGFP fusion protein. Different plate readers in our institution and in the MPI-CBG were tried, but the intrinsic glowing of cellulose and nitrocellulose by itself gave such a strong backgrund signal, that it was not possible to investigate the dCas9-cellulose interaction this way.
Antibodies targeting dCas9 or eGFP were unfortunately not available in the institution and could not be ordered due to limited funding, therefor a different way had to be found.

#### 6th June – Nikitha and Sebastian

Aim of this experiment was to check if dCas9 is bound to the nitrocellulose strip or it elutes out of the strip post washing.

We followed the DNA extraction protocol. Following the extraction using the nitrocellulose strip, extracted DNA was incubated with the dCas9-GFP to understand the interaction between the protein and DNA bound to nitrocellulose. Samples were loaded onto the gel according to following loading order (Figure 1):

![](https://static.igem.org/mediawiki/2019/1/1a/T--TU_Dresden--notebook-40.png)

Figure 1 – SDS-PAGE results of dCas9 interation wth the nitrocellulose strip

Lane 1: 6 ul of protein marker
Lane 2: 20 ul of bacterial cell lysate
Lane 3: 20 ul Wash 1 after dipping in bacterial cell lysate
Lane 4: 20 ul Wash 1 from control
Lane 5: 20 ul Wash 1 after dCas9 incubation
Lane 6: 20 ul Wash 1 after dCas9 incubation from control
Lane 7: 20 ul Wash 2 from cell lysate
Lane 8: 20 ul Wash 2 from control
Lane 9: dCas9 protein from the nitrocellulose bound DNA strip
Lane 10: 20 ul of control

Conclusion: We see the dCas9 protein band at approximately at 170 kDA.

#### 7th July – Nikitha and Sebastian

![](https://static.igem.org/mediawiki/2019/3/32/T--TU_Dresden--notebook-41.png)

Figure 2 – SDS-PAGE results of dCas9 binding to GFP with the help of gRNA

Steps 1 to 5 for DNA extraction from the cell lysate was repeated along with following changes.

Post blocking (in 0.5 % Gelatin + 0.05 % tween 20 in 1X PBS) for 20 minutes at RT, the nitrocellulose strip was dipped in Wash 1 and then transferred to a new 1.5 mL tube, where it incubated with guide RNA targeting the GFP expressed and dcasp-GFP for 10 minutes at 37 °C. Strip now was again dipped in wash buffer (W1) to remove unbound dCas9-GFP fusion protein and then dipped again in wash buffer (W2) to elute the bound DNA-dCas9 bound complex. Now all the elutes along with respective controls was loaded onto the 12.5 % SDS-PAGE gel according to the following scheme (Figure 2). Note: control did not have DNA.

![](https://static.igem.org/mediawiki/2019/3/3d/T--TU_Dresden--notebook-42.png)

Figure 3 – SDS-PAGE results of the respective control

Lane 1: 6 ul of Protein marker
Lane 2: Cell lysate
Lane 3: Wash 1 after nitrocellulose strip dipping in cell lysate
Lane 4: Blocking solution post incubation with strip for 20 minutes.
Lane 5: Wash 1 from the strip, post blocking.
Lane 6: Guide RNA - dCas9 GFP complex (200 ng equi molar concentration)
Lane 7: Guide RNA - dCas9 GFP complex solution in which the membrane was incubated.
Lane 8: W1 of the strip after dCas9-guide RNA incubation.
Lane 9: W2 of the strip after dCas9-guide RNA incubation.
Lane 10: Nitrocellulose strip loaded onto the gel

Conclusion: dCas9- guide RNA is not seen most likely due to less amount loaded onto the gel (Lane 6) and the gelatin blocker gave a smear throughout the lane. For further characterization, gelatin blocker will not be used.

#### 8th July – Nikitha and Mara

The aim of the experiment was to check different blocker interaction with nitrocellulose strip.

Blockers used - 1) 3 % BSA + Tween 20 in 1x PBS and 2) 2 % skimmed milk in 1x PBS.
Nitrocellulose strip was dipped in respective blocking solution and incubated for 20 minutes at RT, with shaking.

Post blocking, strips were washed using W1 buffer and incubated with dCas9 solution (in 1x reaction buffer) for 10 minutes at 37 °C. The loading scheme was (Figure 4):

![](https://static.igem.org/mediawiki/2019/6/60/T--TU_Dresden--notebook-43.png)

Figure 4 – SDS-PAGE results of different blocker interaction with nitrocellulose strip

Lane 1: dCas9 reaction buffer solution from skimmed milk blocked nitrocellulose strip
Lane 2: dCas9 reaction buffer solution from BSA blocked nitrocellulose strip
Lane 3: dCas9 (200 ng)
Lane 4: dCas9 incubated with nitrocellulose membrane.
Lane 5: Nitrocellulose strip incubated with skimmed milk blocker
Lane 6: Skimmed milk blocked nitrocellulose membrane incubated with dCas9
Lane 7: Skimmed milk
Lane 8: Nitrocellulose strip incubated with BSA blocker
Lane 9: BSA blocked nitrocellulose membrane incubated with dCas9
Lane 10: BSA

Conclusion: Concentration of the blockers used was too high and was diluted 100 times and the above experiment was repeated again (Figure 5).

#### 9th July – Nikitha and Mara

![](https://static.igem.org/mediawiki/2019/7/79/T--TU_Dresden--notebook-44.png)

Figure 5 – SDS-PAGE results of diluted blockers

Lane 1: Marker
Lane 2: dCas9
Lane 3: dCas9 incubated with nitrocellulose strip, strip was loaded onto the lane
Lane 4: solution of Skimmed milk (1 : 100) blocked strip
Lane 5: Skimmed milk blocked (1 : 100 dilution) strip, washed and incubated with dCas9 , strip was loaded onto the lane
Lane 6: Solution from dCas9 incubated strip which was blocked with skimmed milk
Lane 7: Skimmed milk
Lane 8: BSA blocked (1 : 100 dilution) strip, washed and incubated with dCas9 , strip was loaded onto the lane
Lane 9: Solution from dCas9 incubated strip which was blocked with BSA
Lane 10: BSA

We hypothesized that from lane 2 and 3 that faint band of dCas9 is visible when loaded onto the gel directly and when loaded with nitrocellulose strip, the binding is very strong and it is not leaving the strip.

#### 20th July – Nikitha and Mara

A method was finally found to investigate the method of dCas9-cellulose interaction. Via an EMSA-shift assay the binding of dCas9 to its target DNA can be studied. If we immobilize DNA on a cellulose strip, then incubate it with dCas9-sgRNA and load the paper-strp directly into the EMSA well, the dCas9 will still not be visible but indirectly its effect on the DNA can be seen. That means the shift that DNA-binding causes will be visible. This way the dCas9-cellulose interaction can indirectly be studie

## Results & Discussion

* dCas9-GFP can only be detected in the SDS-PAGE when loaded directly from solution. It was therefore not possible to investigate the dCas9 celluloe interaction with SDS-PAGES.
* We speculate that dCas9 nitrocellulose interaction is very strong and does not elute from strip.

## our great sponsors

We are proudly sponsored by the following partners in science:

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### CONTACT US:

Email:
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2019. iGEM 2019 Team TU-Dresden.


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:TU Dresden/Experiments

Team TU-Dresden | the DipGene project

![Preloader](https://static.igem.org/mediawiki/2019/0/08/T--TU_Dresden--preloader.gif)

### Beaming BioBricks from Space...

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[# ![TU Dresden 2019](https://static.igem.org/mediawiki/2019/c/cb/T--TU_Dresden--logo3.png)](https://2019.igem.org/Team%3ATU_Dresden)

* Project
  + [Motivation](https://2019.igem.org/Team%3ATU_Dresden/Motivation)
  + [Description](https://2019.igem.org/Team%3ATU_Dresden/Description)
  + [Design](https://2019.igem.org/Team%3ATU_Dresden/Design)
  + [Results](https://2019.igem.org/Team%3ATU_Dresden/Results)
  + [Demonstrate](https://2019.igem.org/Team%3ATU_Dresden/Demonstrate)
  + [Sweet Spirulina](https://2019.igem.org/Team%3ATU_Dresden/Spirulina)
* Lab
  + [Notebook](https://2019.igem.org/Team%3ATU_Dresden/Notebook)
  + [Parts](https://2019.igem.org/Team%3ATU_Dresden/Parts)
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  + [Protocols](https://2019.igem.org/Team%3ATU_Dresden/Experiments)
  + [Safety](https://2019.igem.org/Team%3ATU_Dresden/Safety)
* Human Practices
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  + [Public Engagement](https://2019.igem.org/Team%3ATU_Dresden/Public_Engagement)
* [Collaborations](https://2019.igem.org/Team%3ATU_Dresden/Collaborations)
* Team
  + [Our Team](https://2019.igem.org/Team%3ATU_Dresden/Team)
  + [Attributions](https://2019.igem.org/Team%3ATU_Dresden/Attributions)
  + [Sponsors](https://2019.igem.org/Team%3ATU_Dresden/Sponsors)
  + [Crowdfunding](https://www.indiegogo.com/projects/sweet-spirulina-dipgene?fbclid=IwAR3LKhhbd6dBNbMVXAZZ6Uh9-k_P-o5r7Lz0Ba6JeimMBrkAQ_H4mlPYBZo#/)
* [Judging](https://2019.igem.org/Team%3ATU_Dresden/Judging)

## Protocols

![](https://static.igem.org/mediawiki/2019/e/eb/T--TU_Dresden--notebook_icon.jpg)

[Use a BioBrick from the Disctribution kit](https://static.igem.org/mediawiki/2019/f/ff/T--TU_Dresden--protocols1.pdf)

[The fastest gDNA extraction protocol for PCR with cellulose](https://static.igem.org/mediawiki/2019/1/18/T--TU_Dresden--protocols2.pdf)

[Make a stock of electrocompetent cells from E. coli GB05 with flash freezing](https://static.igem.org/mediawiki/2019/d/da/T--TU_Dresden--protocols3.pdf)

[Electroporate E. coli GB05](https://static.igem.org/mediawiki/2019/d/d8/T--TU_Dresden--protocols4.pdf)

[Heat shock transformation with NEB E.coli 5α](https://static.igem.org/mediawiki/2019/3/3f/T--TU_Dresden--protocols5.pdf)

[Optimized miniprep protocol for ZymoPURE Plasmid Miniprep Kit](https://static.igem.org/mediawiki/2019/7/75/T--TU_Dresden--protocols6.pdf)

[No-Kit miniprep protocol](https://static.igem.org/mediawiki/2019/0/0e/T--TU_Dresden--protocols7.pdf)

[Enzyme Saver Mastermix for gel electrophoresis analysis](https://static.igem.org/mediawiki/2019/1/15/T--TU_Dresden--protocols8.pdf)

[Agarose gel preparation and run](https://static.igem.org/mediawiki/2019/4/4e/T--TU_Dresden--protocols9.pdf)

[BioBrick expression protocol in E. coli pRARE T7](https://static.igem.org/mediawiki/2019/d/d2/T--TU_Dresden--protocols10.pdf)

[Cell lysis and Purification of HRP-Strep](https://static.igem.org/mediawiki/2019/d/d2/T--TU_Dresden--protocols10.pdf)

[Expression and purification of dCas9 fusion proteins via Amylose Resin and Strep-tag](https://static.igem.org/mediawiki/2019/2/2a/T--TU_Dresden--protocols12.pdf)

[Activity assay for HRP with TMB](https://static.igem.org/mediawiki/2019/7/72/T--TU_Dresden--protocols13.pdf)

## our great sponsors

We are proudly sponsored by the following partners in science:

[![CMCB Dresden](https://static.igem.org/mediawiki/2019/b/b0/T--TU_Dresden--SPONSORcmcb.png)](https://tu-dresden.de/cmcb)
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[![BioZym](https://static.igem.org/mediawiki/2019/e/ef/T--TU_Dresden--SPONSORbiosym.jpg)](https://www.biozym.com)
[![Snapgene](https://static.igem.org/mediawiki/2019/b/bf/T--TU_Dresden--snapgene.png)](https://www.snapgene.com)
[![IDT Technologies Inc](https://static.igem.org/mediawiki/2019/1/1d/T--TU_Dresden--idt.png)](https://eu.idtdna.com/)

[![New England Biolabs GmBH](https://static.igem.org/mediawiki/2019/0/05/T--TU_Dresden--neblogo.png)](https://www.neb-online.de/)
[![New England Biolabs GmBH](https://static.igem.org/mediawiki/2019/e/eb/T--TU_Dresden--jenna.gif)](https://www.jenabioscience.com/)
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[![ZYMO Research](https://static.igem.org/mediawiki/2019/5/5c/T--TU_Dresden--zymologo.png)](https://www.zymoresearch.de/)

[![Arbor Biosciences](https://static.igem.org/mediawiki/2019/8/87/T--TU_Dresden--arborlogo.png)](https://arborbiosci.com)
[![TUD GFF e.V](https://static.igem.org/mediawiki/2019/0/0d/T--TU_Dresden--sponsor_gff.jpg)](https://tu-dresden.de/tu-dresden/profil/freunde-foerderer/gff?set_language=en)
[![School of Science - TU Dresden](https://static.igem.org/mediawiki/2019/1/1f/T--TU_Dresden--sponsor_ultimo.jpg)](https://tu-dresden.de/mn/der-bereich)
[![School of Science - TU Dresden](https://static.igem.org/mediawiki/2019/8/8c/T--TU_Dresden--eurofins.png)](https://www.eurofins.com/)

### CONTACT US:

Email:
tudigem2019@gmail.com

[![](https://static.igem.org/mediawiki/2019/f/fa/T--TU_Dresden--twitter.png)](https://twitter.com/igemtudy/status/1139579162954477569)
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2019. iGEM 2019 Team TU-Dresden.

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:TU Dresden/Results

Team TU-Dresden | the DipGene project

![Preloader](https://static.igem.org/mediawiki/2019/0/08/T--TU_Dresden--preloader.gif)

### Beaming BioBricks from Space...

Toggle nav
![](https://static.igem.org/mediawiki/2019/7/7e/T--TU_Dresden--bars-solid.png)

[# ![TU Dresden 2019](https://static.igem.org/mediawiki/2019/c/cb/T--TU_Dresden--logo3.png)](https://2019.igem.org/Team%3ATU_Dresden)

* Project
  + [Motivation](https://2019.igem.org/Team%3ATU_Dresden/Motivation)
  + [Description](https://2019.igem.org/Team%3ATU_Dresden/Description)
  + [Design](https://2019.igem.org/Team%3ATU_Dresden/Design)
  + [Results](https://2019.igem.org/Team%3ATU_Dresden/Results)
  + [Demonstrate](https://2019.igem.org/Team%3ATU_Dresden/Demonstrate)
  + [Sweet Spirulina](https://2019.igem.org/Team%3ATU_Dresden/Spirulina)
* Lab
  + [Notebook](https://2019.igem.org/Team%3ATU_Dresden/Notebook)
  + [Parts](https://2019.igem.org/Team%3ATU_Dresden/Parts)
  + [Characterization](https://2019.igem.org/Team%3ATU_Dresden/Characterization)
  + [Protocols](https://2019.igem.org/Team%3ATU_Dresden/Experiments)
  + [Safety](https://2019.igem.org/Team%3ATU_Dresden/Safety)
* Human Practices
  + [Integrated Human Practices](https://2019.igem.org/Team%3ATU_Dresden/Human_Practices)
  + [Public Engagement](https://2019.igem.org/Team%3ATU_Dresden/Public_Engagement)
* [Collaborations](https://2019.igem.org/Team%3ATU_Dresden/Collaborations)
* Team
  + [Our Team](https://2019.igem.org/Team%3ATU_Dresden/Team)
  + [Attributions](https://2019.igem.org/Team%3ATU_Dresden/Attributions)
  + [Sponsors](https://2019.igem.org/Team%3ATU_Dresden/Sponsors)
  + [Crowdfunding](https://www.indiegogo.com/projects/sweet-spirulina-dipgene?fbclid=IwAR3LKhhbd6dBNbMVXAZZ6Uh9-k_P-o5r7Lz0Ba6JeimMBrkAQ_H4mlPYBZo#/)
* [Judging](https://2019.igem.org/Team%3ATU_Dresden/Judging)

## Results

### **Project Achievements - Overview**

* We successfully designed our own fusion protein in silico, ordered all the parts as separated BioBricks or used old ones from the registry and assembled them to our Full Construct

* We proved that every single part of our Full Construct is working
  + MBP → verified via amylose resin purification
  + dCas9 → verified via Electrophoretic Mobility Shift Assay (EMSA)
  + HRP → verified via TMB-conversion activity assay

* The only part of our Full Construct that is not working was the Strep-tag. It was designed for Western Blot analysis and not for Strep-column purification. (Read more about it  [here](http://parts.igem.org/Part%3ABBa_K823038))

* We successfully designed our own tailored DNA extraction method from bacteria in which nucleic acids of high purity are immobilized in a cellulose paper strip.

* With this simple, fast and easy method we proved that dCas9 can bind to DNA that is immobilized on a paper strip

* We proved that dCas9 is not binding unspecifically to the paper

##### [Results of the novel fusion protein design ![](https://static.igem.org/mediawiki/2019/e/ef/T--TU_Dresden--flechaPau.png)](#answerOne1)

#### 1. Cloning Full Construct

**Final construct in pSB1C3**

We successfully cloned the Full Construct in pSB1C3. We checked that using PCR induced mutagenesis we were successfully able to remove the illegal restriction site. Full Construct i.e. MBP-dCas9- Linker-HRP-Strep is 6 kb fragment (Figure 1).

![](https://static.igem.org/mediawiki/2019/f/ff/T--TU_Dresden--results1.png)

Figure 1 - Restriction digest of pSB1C3 with EcoRI and PstI showing Full Construct at 6 kb.

**Final construct in pOCC97**

We checked in silico the best restriction enzymes for detecting the correct insertion of our Full Construct (Figure 2). It can be seen in the first lane (digested with XbaI and PstI) that the difference in size between the plasmid backbone (5 kb) and the Full Construct (6 kb) is not sufficient to be properly separated on an agarose gel. Therefore, a new restriction digest had to be prepared in which a triple digest with XbaI, PstI and PmlI was set up. The fragments of different size make it easy to distinguish a successful insertion of the final construct in the expression plasmid (Figure 3).

![](https://static.igem.org/mediawiki/2019/9/9c/T--TU_Dresden--results2.png)

Figure 2 - In silico restriction digest of Full Construct. Digestion with XbaI and PstI in the lane 1. Digestion with XbaI, PstI and PmlI in the lane 2.

![](https://static.igem.org/mediawiki/2019/7/71/T--TU_Dresden--sophie1.jpg)

Figure 3 - Restriction digest of full construct (BBa\_K3037003) in pOCC97 (BBa\_K3037000) with XbaI, PstI and PmlI. The lanes marked with the red X correspond to the clones with the correct insert. The background bands shown in the lanes correspond to the T7 expression plasmid transformed into the E. coli pRARE strain from NEB.

#### 2. Expression and Purification of the Full Construct

We used the ExPASy ProtParam tool (https://web.expasy.org/protparam/) to calculate physical and chemical parameters for our protein:

**Number of amino acids**: 2106
**Molecular weight:** 238793.94
**Theoretical pI:** 8.39
**Ext. coefficient\*:** 205750
**Abs** 0.1% (=1 g/l) 0.862, assuming all pairs of Cys residues form cystines

\*Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.

**Characterization of different expression conditions**

We tried expression of our construct in our expression plasmid  [BBa\_K3037000](http://parts.igem.org/Part%3ABBa_K3037000) under different IPTG concentrations and two temperatures after induction (Figure 4 and 5). By comparing the expression levels (Figure 5) we determined that the optimal conditions for the expression of  [BBa\_K3037003](http://parts.igem.org/Part%3ABBa_K3037003) in pOCC97 (BBa\_K3037000) are 18 ºC and 0.5 mM IPTG. The expression seems to be more stable over time for the optimized plasmid than for the non-optimized.

![](https://static.igem.org/mediawiki/2019/6/61/T--TU_Dresden--results4.png)

Figure 4 - Expression of the Full Construct in pOCC97 optimized at different temperatures and IPTG concentrations

![](https://static.igem.org/mediawiki/2019/c/c2/T--TU_Dresden--results5.png)

Figure 5 - Expression of the Full Construct in optimized pOCC97 under different conditions.

**MBP affinity tag purification**

After proving that the Full Construct was expressed properly in our plasmid and improved its expression conditions, it was purified by using amylose Resin to bind its MBP affinity tag. Our Full Construct was present in all the elution fractions after purification with the amylose resin (Figure 6). The concentration of the purified protein was 4.56 mg/mL.

![](https://static.igem.org/mediawiki/2019/1/11/T--TU_Dresden--results6.png)

Figure 6 - MBP affinity tag chromatography purification of Full Construct using amylose resin.

**Strep-tag purification**

After purification with the N-terminus MBP affinity tag, we tried to purify our Full Construct using our C-terminus Strep-tag. However, our protein was not able to bind to the column resin and all of our protein fraction is found in the column flowthrough and none in the eluates (Figure 7). Apparently, the Strep-tag BioBrick [BBa\_K823038](http://parts.igem.org/Part%3ABBa_K823038) can be only used for Western Blot detection of the tagged protein.

![](https://static.igem.org/mediawiki/2019/1/1b/T--TU_Dresden--results7.png)

Figure 7 - Strep-tag affinity chromatography of eluates from amylose resin purification.

**gRNA design**

We used the CRISPR Guide RNA Design from Benchling (https://www.benchling.com/crispr/) to design six gRNA which target the *sry* locus and one gRNA that targets our eGFP BioBrick. The gRNA with the PAM sequence and the context are specified in the following table 1.

Table 1 - The guide RNA with the PAM sequence
![](https://static.igem.org/mediawiki/2019/7/77/T--TU_Dresden--results-tablaADN.png)

**dCas9 activity detecting the target sequence**

We wanted to check if the best gRNA combination for binding dCas9 to the target DNA sequence. We used our eGFP-dCas9 fusion protein ( [BBa\_K3037005)](http://parts.igem.org/Part%3ABBa_K3037005)) for testing the enzyme ability of binding DNA. The mobility shift works very well with gRNA 1, and it increases when combinations of guide RNAs are used. The best gRNA combination is 1,4 and 2 (Figure 8). We also included negative controls loading only the *sry* amplicon with the gRNA's to test if the mobility shift that we saw in Figure 9 was influenced by a secondary structure formation of the gRNA and not by the dCas9 binding to the target sequence.

![](https://static.igem.org/mediawiki/2019/8/88/T--TU_Dresden--results8.png)

Figure 8 - Electrophoretic Mobility Shift Assay of *sry* targeted with dCas9 and different gRNA combinations

Lane 1: 1 kb DNA ladder
Lane 2: *sry* gene
Lane 3: *sry* gene + dcas9
Lane 4: *sry* gene + dcas9 + guide RNA 1
Lane 5: *sry* gene + dcas9 + guide RNA 4, 2 and 6
Lane 6: *sry* gene + dcas9 + guide RNA 1, 5 and 6
Lane 7: *sry* gene + dcas9 + guide RNA 1, 4, 3 and 6
Lane 8: *sry* gene + dcas9 + guide RNA 4, 3 and 7
Lane 9: *sry* gene + dcas9 + guide RNA 1, 4 and 2

![](https://static.igem.org/mediawiki/2019/2/2c/T--TU_Dresden--results9.png)

Figure 9 - Electrophoretic Mobility Shift Assay of *sry* targeted with dCas9 and different gRNA combinations

We also tested if our eGFP-dCas9 can bind plasmid DNA targeted with the gRNA’s. In Figure 9a lane 8, we could see that the band from the digested plasmid DNA (lane 9) is completely prevented from entering the gel. This shows a high binding affinity of our eGFP-dCas9 with our gRNA to large plasmid fragments.

![](https://static.igem.org/mediawiki/2019/b/b7/T--TU_Dresden--results-CAS91.png)

Figure 9a - EMSA showing mobility shift of plasmid DNA targeted with gRNA’s.

Lane 1 - Marker
Lane 2- 100 ng of Sry gene
Lane 3- Sry + dCas9-HRP
Lane 4 - Sry + dCas9-HRP+Guide RNA 1 + Guide RNA 4 +Guide RNA 2
Lane 5 - Sry + dCas9-HRP+Guide RNA 1
Lane 6 - Sry + dCas9-GFP+Guide RNA 1 + Guide RNA 4 +Guide RNA 2
Lane 7 - eGFP+dCas9-HRP+Guide RNA 7
Lane 8 - eGFP+dCas9-HRP+Guide RNA 7
Lane 9 - eGFP
Lane 10 - Sry + dCas9-HRP+Guide RNA 1 + Guide RNA 4 +Guide RNA 2 (Here Sry gene was immobilized on cellulose strip and addition of dCas9 proteins and guide RNA was all done in cellulose strip).
Lane 11 - Sry + dCas9-GFP+Guide RNA 1 + Guide RNA 4 +Guide RNA 2
Lane 12 - Sry gene loaded on cellulose strip.

With all of the previous experiments we were ready to test the activity of our full construct with the best working gRNA combination. In Figure 9b lane 2 we see the sry gene as a control, when we add a high concentration of our full construct dCas9-HRP (BBa\_K3037003) we can see that the dCas9-HRP fusion protein completely binds all the sry PCR amplicon, preventing it from entering the TBE-PAGE. We can confirm that the shift that we see in lane 2 is produced by the binding of our full construct when we serially dilute our protein. Starting in lane 9, and then from lanes 6 to 8 we see that the intensity of the shifted bands decreases with lower dCas9-HRP concentration.

![](https://static.igem.org/mediawiki/2019/6/6d/T--TU_Dresden--results-CAS92.png)

Figure 9b - EMSA performed with the full construct (BBa\_K3037003) targeting the sry gene.

Lane 1 - 1 kb plus ladder
Lane 2 - sry gene
Lane 3 - 4.56 ug of dcas9-HRP + guide RNA (1,4 and 2) + sry gene
Lane 4 - 4.56 ug of dcas9-eGFP + guide RNA (1,4 and 2 ) + sry gene
Lane 5 - 600 ng of dcas9-HRP + guide RNA (1, 4 and 2 ) + sry gene
Lane 6 - 2.56 ug of dcas9-HRP + guide RNA (1,4 and 2) + sry gene
Lane 7 - 1.28 ug of dcas9-HRP + guide RNA (1,4 and 2) + sry gene
Lane 8 - 0.64 ug of dcas9-HRP + guide RNA (1,4 and 2) + sry gene
Lane 9 - 8.56 ug of dcas9-HRP + guide RNA (1,4 and 2) + sry gene

**Testing our HRP BioBrick using Freiburg’s fusion protein standard (RFC25)**

We were able to successfully express the HRP protein which was adapted to the RFC25 BioBrick standard from the BioBrick  [BBa\_K1800002](http://parts.igem.org/Part%3ABBa_K1800002), expression was performed using our plasmid backbone BioBrick pOCC97 ( [BBa\_K3037000](http://parts.igem.org/Part%3ABBa_K3037000)). The HRP expression increased over time only after induction of the cells with IPTG (Figure 10).

![](https://static.igem.org/mediawiki/2019/6/6d/T--TU_Dresden--results10.png)

Figure 10 - HRP expression after induction over time. HRP is marked with a black arrow pointing left.

The activity of HRP in the Full Construct was measured in a dynamic assay. We confirmed that our HRP from our Full Construct is active and is able to catalyze the conversion of TMB to its oxidized blue colored product (Figure 11). The absorbance measured at 650 nm over time confirmed the oxidation of TMB when comparing the clarified cell lysate from the *E.coli* cells that expressed the Full Construct with the HRP, versus the cells without our Full Construct plasmid (Figure 10).

![](https://static.igem.org/mediawiki/2019/2/28/T--TU_Dresden--results11.png)

Figure 11- Conversion of TMB substrate by HRP over time using clarified cell lysate from *E.coli*
 expressing the HRP BioBrick  [BBa\_K1800002](http://parts.igem.org/Part%3ABBa_K1800002) and our Full Construct  [BBa\_K3037003](http://parts.igem.org/Part%3ABBa_K3037003). The HRP
activity of our Full Construct was compared to the HRP BioBrick  [BBa\_K1800002](http://parts.igem.org/Part%3ABBa_K1800002) and *E.coli* pRARE cell lysate.

##### [Results of the cellulose-based DNA extraction ![](https://static.igem.org/mediawiki/2019/e/ef/T--TU_Dresden--flechaPau.png)](#answerOne2)

**Amount and purity of nucleic acids extracted using the cellulose disk extraction method**

We quantified the concentration of DNA that we eluted from the Whatman cellulose disk using a NanoDrop spectrophotometer. We performed the reaction in duplicates and found out that 1 minute of cell lysis at room temperature works the best (Figure 12). Extended lysis times led to unnecessary waiting which did not improve the extracted DNA yield.

![](https://static.igem.org/mediawiki/2019/b/b1/T--TU_Dresden--results12.png)

Figure 12 - HRP expression after induction over time. HRP is marked with a black arrow pointing left.

From Figure 13 we conclude that when we perform cell lysis at room temperature and extract DNA with cellulose disk, it yields a higher purity in the eluted sample. The absorbance ratio measured at 260/280 nm is between 1.8 - 2.0, which indicates that there is little to none protein contamination from the cell lysate.

![](https://static.igem.org/mediawiki/2019/d/d6/T--TU_Dresden--results13.png)

Figure 13 - Absorbance ratio at 260/280 nm to measure protein contamination

**Agarose gel electrophoresis of cellulose disks**

We wanted to find the best matrix that would be able to selectively bind DNA from a cell lysate and that will allow us to perform our dCas9 gene targeting without nonspecific interactions. In order to understand which type of nucleic acids were extracted from the aforementioned cell lysates, we decided to load the cellulose disks into an agarose gel electrophoresis.

**Choosing the best matrix for DNA extraction**

Since we were trying to develop an affordable method for diagnostics we chose Whatman filter paper because of it's low cost. As a control we used a nitrocellulose membrane that according to Zou et al. (2017) had a stronger binding to nucleic acids. Since they also mentioned that higher salt concentrations increase the electrostatic interactions between the cellulose and the DNA.

We found that by adding 100 mM NaCl to the cell lysates we were able to extract a good amount of genomic DNA using cellulose disks (Figure 14). The nitrocellulose membrane had a great binding capacity of DNA; however, as mentioned by Zou et al. (2017) the interactions between the nitrocellulose and the DNA were too strong and the nucleic acids were not leaving the membrane in the agarose gel electrophoresis (Figure 15). This high electrostatic interactions could interfere with the ability of our dCas9-HRP to bind DNA on top of the matrix.

For the aforementioned reason we decided to use the cellulose disks for DNA extraction with 100 mM NaCl added to the cell lysates.

![](https://static.igem.org/mediawiki/2019/8/87/T--TU_Dresden--results14.png)

Figure 14 - Varying salt concentration and the impact on DNA yield extracted from *E.coli* GB05 cell lysate using cellulose disc

![](https://static.igem.org/mediawiki/2019/1/10/T--TU_Dresden--results15.png)

Figure 15 - Varying salt concentration and the impact on DNA yield extracted from *E.coli* GB05 cell lysate using nitrocellulose disc

**Differentiating between gDNA and plasmid DNA**

We can identify the extracted genomic DNA from the bacteria cell lysate from the bands shown in the agarose gel electrophoresis above the 10 kb band from the marker; in addition, there is a band smear at the expected plasmid size in Figure 16 lane 3. We could only extract plasmid DNA when no extra salts were added to the cell lysate.

![](https://static.igem.org/mediawiki/2019/3/38/T--TU_Dresden--results16.png)

Figure 16 - Varying salt concentration and the impact on plasmid DNA yield extracted from *E.coli*
 GB05 (pSB1C3 expressing RFP;  [BBa\_J04450)](http://parts.igem.org/Part%3ABBa_J04450) cell lysate using nitrocellulose disc

In order to get genetic material from buccal swab samples to test *sry* - positive or *sry* - negative cells with our DipGene method, we tried to apply the same concept for DNA extraction that we proved works very well for bacterial cells. However, the amount of starting material that we got the buccal epithelial cells was not enough for visualizing nucleic acids in agarose gel electrophoresis (Figure 17).

![](https://static.igem.org/mediawiki/2019/6/6c/T--TU_Dresden--results17.png)

Figure 17 - Agarose gel electrophoresis of cell lysate from buccal epithelial cells. The cells were lysed using NEB's Monarch gDNA Cell lysis buffer at room temperature and at 95 °C. The attempt to bind DNA from the cell lysate using the cellulose disks was not noticeable due to low DNA concentration in the lysate.

**Direct PCR of mammalian cells and cyanobacteria (Spirulina) using cellulose disk**

Following the idea of the fast DNA extraction method from Zou et al. (2017), we decided to create and optimize cell lysis buffers tailored for mammalian cells that can be used for PCR amplification.
We successfully amplified the *sry* gene from *sry* positive cells lysed with NEB's Monarch gDNA Cell lysis buffer (Figure 18).

![](https://static.igem.org/mediawiki/2019/f/fd/T--TU_Dresden--results18.png)

Figure 18 - PCR of *sry* gene using the cellulose disk DNA extraction method.

We tried the same buffers that we used for bacterial cell lysis (*E.coli* GB05) to check if we could extract gDNA from Spirulina (cyanobacteria used in our other project, [read more about it](https://2019.igem.org/Team%3ATU_Dresden/Spirulina)) and use it to include homology arms for Homologous recombination. The target region was successfully amplified via PCR with the cellulose disk method (Figure 19).

![](https://static.igem.org/mediawiki/2019/1/12/T--TU_Dresden--results19.png)

Figure 19 - PCR of locus with homology arms used for homologous recombination in Spirulina using the cellulose disk DNA extraction method.

##### [Results of dCas9-cellulose interaction ![](https://static.igem.org/mediawiki/2019/e/ef/T--TU_Dresden--flechaPau.png)](#answerOne3)

A critical part of our project design was to develop an easy method to selectively immobilize DNA on a cellulose matrix, and tailor buffers that can be used to wash away proteins and debris that could interfere with our dCas9-HRP fusion protein in binding to the target DNA region. We optimized the wash buffer composition to selectively wash proteins out of our cellulose matrix, while leaving nucleic acids immobilized in the disks. Tests were performed using common western blot blocker proteins to check which buffers work best for removing proteins that adhere to the cellulose matrix.

We decided to saturate the cellulose disk with eGFP-dCas9 fusion protein (with gRNA’s targeting the sry gene) and incubate them at 37 °C for 30 minutes. The disks were washed with our tailored wash buffer and dipped into a solution containing the sry PCR amplicon, followed by another incubation at 37 °C for 30 minutes. The disks were loaded into a TBE-PAGE to check for a mobility shift (in an EMSA gel) which will only happen if the eGFP-dCas9 was not successfully washed away from the disk. In Figure 20 lane 4 and 5 we don’t see a band shift, which means that the eGFP-dCas9 was completely removed from the cellulose disk.

![](https://static.igem.org/mediawiki/2019/1/1f/T--TU_Dresden--results-CAS93.png)

Figure 20 - https://2019.igem.org/Wiki/images/1/1b/T--TU\_Dresden--results-fin1.pngFigure 20 - EMSA showing that the eGFP-dCas9 does not bind nonspecifically to the cellulose disks and can be washed away with the W1 buffer.

Lane 1 - Marker
Lane 2 - sry
Lane 3 - dCas9-HRP + (1,4,2 guide RNA) + sry
Lane 4 - dCas9 + gRNA (1,4 and 2) first incubated and then washed once and then sry gene.
Lane 5 - dCas9 + gRNA (1,4 and 2) first incubated and then washed twice and then sry gene.
Lane 6 - sry loaded on cellulose
Lane 7 - (200 ng) dCas9-eGFP + gRNA + sry - 1 minute incubation
Lane 8 - (200 ng) dCas9-eGFP + gRNA + sry - 5 minutes incubation
Lane 9 - (200 ng) dCas9-eGFP + gRNA + sry - 10 minutes incubation
Lane 10 - (200 ng) dCas9-eGFP + gRNA + sry - 30 minutes incubation
Lane 11 - (200 ng) dCas9-eGFP + gRNA + sry - 60 minutes incubation
Lane 12 - (400 ng) dCas9-eGFP + gRNA + sry - 60 minutes incubation

Moreover, when the eGFP-dCas9 is incubated in the cellulose disk in the presence of DNA, the fusion protein can bind to the DNA and remain bound after washes with the tailored buffers. This can be seen when we loaded the cellulose disks inside of the TBE-PAGE, there is a faint band shift (Figure 21, lane 11) at the same height as the control eGFP-dCas9 without the cellulose matrix (Figure 21, lane 6).

![](https://static.igem.org/mediawiki/2019/5/5c/T--TU_Dresden--results-CAS94.png)

Figure 21 - EMSA showing that the eGFP-dCas9 binds the target DNA sequence on top of the cellulose disks and remains bound after the washes with the tailored W1 buffer.

Lane 1 - Marker
Lane 2- 100 ng of Sry gene
Lane 3- Sry + dCas9-HRP
Lane 4 - Sry + dCas9-HRP+Guide RNA 1 + Guide RNA 4 +Guide RNA 2
Lane 5 - Sry + dCas9-HRP+Guide RNA 1
Lane 6 - Sry + dCas9-GFP+Guide RNA 1 + Guide RNA 4 +Guide RNA 2
Lane 7 - eGFP + dCas9-HRP+Guide RNA 7
Lane 8 - eGFP + dCas9-HRP+Guide RNA 7
Lane 9 - eGFP
Lane 10 - Sry + dCas9-HRP+Guide RNA 1 + Guide RNA 4 +Guide RNA 2 (Here Sry gene was immobilized on cellulose strip and addition of dCas9 proteins and guide RNA was all done in cellulose strip).
Lane 11 - Sry + dCas9-GFP+Guide RNA 1 + Guide RNA 4 +Guide RNA 2
Lane 12 - Sry gene loaded on cellulose strip.

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2019. iGEM 2019 Team TU-Dresden.