iGEM 2017 — Team DTU-Denmark

[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:DTU-Denmark/Notebook

# Lab Notebook

### 24 July 2017

#### AMC assay

*KK* and *AMH* worked on a standard curve for the AMC assay.

### 25-26 July 2017

#### AMC assay

*KK*, *AMH* and *CAL* did the AMC substrate experiment where they tested the protease activity of three different snake venoms on a three amino acid long peptide chain attached to the fluorescent molecule AMC.

### 28 July 2017

#### AMC assay

*KK*, *AMH*, *CAL* and *AFT* did a confirmation of the results by repeating the AMC substrate test with venom again.

### 8 August 2017

#### BioBrick assembly

*PHS* ordered the first gBlocks from IDT.

### 23 August 2017

#### BioBrick assembly

*PHS* ordered primers from IDT.

### 25 August 2017

#### BioBrick assembly

*PHS* ordered a promoter with the same sequence as BBa\_R0010 but in RFC[25].

### 26 August 2017

#### BioBrick assembly

*PHS* assembled amilCP with his-tag (BBa\_K2355002) with potential linkers. This assembly was unsuccessful.

### 27 August 2017

#### BioBrick assembly

*PHS* assembled amilCP with his-tag (BBa\_K2355002) with the ALK/G linker (BBa\_K2355001). This assembly was unsuccessful.

### 30 August - 2 September 2017

#### BioBrick assembly

*CMP* assembled amilCP with his-tag (BBa\_K2355002) with potential linkers this assembly was unsuccessful due malfunctioning restriction cutsites.

### 9 September 2017

#### BioBrick assembly

*PHS* ordered the first primers for the LacZ part.

### 14 September 2017

#### BioBrick assembly

*FBF* and *CMP* did a PCR to extend DNA fragments from IDT. The PCR was unsuccessful due to faulty primers.

### 14-16 September 2017

#### Substrate screening

*KK* and *AFT* screened 360 eight amino acid long peptide chains as potentially substrates for *Bitis arietans*, *Bitis gabonica* and *Naja nigricollis* venom.

### 16-17 September 2017

#### BioBrick assembly

*KK*, *CMP*, *CAL* and *AFT* ordered new sequences from IDT as the old ones had malfunctioning restriction cutsites.

*PHS* ordered the second round of primers for the LacZ part.

### 21 September 2017

#### Substrate screening

*KK* and *AFT* analysed the extensive amount of data obtained from the substrate screening experiment.

### 22 September 2017

#### BioBrick assembly

*CAL* and *CMP* ordered new DNA sequences from IDT, sequences used for the assembly of BBa\_K2355001, BBa\_K2355002 and BBa\_K2355302. Unfortunately the sequence for BBa\_K2355302 did not arrive until the 20th of October.

### 25 September 2017

#### BioBrick assembly

*CAL* and *CMP* ordered the test part that was used for testing of the single chain avidin binding. Unfortunately it did not arrive until the 20th of October.

### 29 September - 1 October 2017

#### BioBrick assembly

*CAL* and *CMP* did an assembly of unregistered composite part containing single chain avidin followed by a small peptide chain cleaved by snake venom that is linked to amilCP with his-tag. This assembly was unsuccessful.

### 2 October 2017

#### BioBrick assembly

*PHS* did a digest of the lacZ part (BBa\_K2355313).

#### Biobrick testing

*CAL* and *CMP* tested degradation of amilCP (BBa\_K592009) by snake venom. The absorbance of amilCP (BBa\_K592009) showed no change after mixing with venom.

### 3-6 October 2017

#### BioBrick assembly

*PHS* PHS did a ligation and transformation of an intermediate of the lacZ part (BBa\_K2355313), containing the ScAvidin and the emperical FRET linker part. The transformation was successful, and two pure cultures were made (Hereafter named P7C5).

*FBF*, *CAL* and *CMP* tried to verify assembly of unregistered composite part with PCR and digestion. Assembly assessed unsuccessful.
![Graph of measurement data](https://static.igem.org/mediawiki/2017/thumb/e/e8/T--DTU-Denmark--labnotebook-1.png/800px-T--DTU-Denmark--labnotebook-1.png.jpeg)

Figure 1: shows colony PCR with no specific bands the ladder is 1Kb. Conclusion unsuccessful assembly.

### 6 October 2017

#### BioBrick assembly

*PHS* did OD600 measurements of the pure culture from the P7C5, and thereafter he did a plasmid prep of the intermediate part. The part was digested, and confirmed to be assembled correctly.

### 8 October 2017

#### BioBrick assembly

*PHS* did a transformation of part BBa\_K823016 from the distribution.

### 9 October 2017

#### BioBrick assembly

*CAL* and *CMP* ordered the linkers as primer sequences from IDT.

*PHS* did an assembly of promoter (BBa\_R0010) to the plasmidprepped P7C5 part. At the same time, a pure culture of the BBa\_K823016 lacZ part was made.

### 10 October 2017

#### BioBrick assembly

*PHS* did a transformation of the assembly started on the 9th of October.

### 11 October 2017

#### BioBrick assembly

*PHS* verified that the assembly did not work. There wasn’t accounted for the religation of plasmids. *PHS* was not able to verify the insert of the promoter (BBa\_R0010) from a cPCR, he made an overnight culture.

### 12 October 2017

#### BioBrick assembly

*CAL* and *CMP* did an assembly of an unregistered composite part and amilCP (BBa\_K592009) with promoter-RBS (BBa\_K608003).

### 13 October 2017

#### BioBrick assembly

*PHS* checked the culture from the 11 of october by OD measurements and a plasmid prep was done. To verify if the assembly worked *PHS* did a digest, unfortunately the digest showed that the assembly had not worked. *PHS* decided to do a PCR to amplify the part of the assembly that had worked two different linkers without the backbone.

*CMP* and *CAL* verified that the assembly of BBa\_K592009 with BBa\_K608003 was unsuccessful assembly due to use of wrong restriction enzymes.

### 16 October 2017

#### BioBrick assembly

*CAL* and *CMP* tried to verify the assembly of the unregistered composite part with digestion. Unsuccessful assembly.

*CAL* and *CMP* made a 3A assembly of amilCP (BBa\_K592009) with promoter-RBS (BBa\_K608003). Successful assembly.

*PHS* ordered a new primer for the LacZ part.
![Graph of measurement data](https://static.igem.org/mediawiki/2017/9/9b/T--DTU-Denmark--labnotebook-2.png)

Figure 2: 1Kb ladder. Unregistered composite part double cut with EcoRI & PstI and uncut in duplicate. Conclusion unsuccessful assembly.

![Graph of measurement data](https://static.igem.org/mediawiki/2017/7/75/T--DTU-Denmark--labnotebook-3.png)

Figure 3: 1Kb ladder. Verification of promoter-RBS (BBa\_K608003) with amilCP without a his-tag (BBa\_K592009). Well 2 double cut with EcoRI & PstI. Well 3-4 single cut with EcoRI or PstI. Well 5 uncut. This showed a successful assembly.

### 17 October 2017

#### BioBrick assembly

*CAL*, *AFT* and *CMP* made a 3A assembly of amilCP (BBa\_K592009) with promoter-RBS (BBa\_K608003). After digest the fragments were gel purified. The ligation was performed with equal amounts of plasmid and insert. Unsuccessful assembly - used a non optimal ratio of plasmid to insert in the ligation.

*CAL* and *CMP* made a 3A assembly of amilCP with his-tag (BBa\_K2355002) with promoter-RBS (BBa\_K608003).

*CAL* and *CMP* ordered primers for amplifying the unregistered composite part from IDT.

### 18-19 October 2017

#### BioBrick assembly

*CAL*, *AFT* and *CMP* repeated ligation and transformation with the digest from amilCP (BBa\_K592009) and promoter-RBS (BBa\_K608003) along with amilCP with his-tag (BBa\_2355002). The ligation protocol was changed to a ratio of 1:3 plasmid to insert. Also two incubation times were used. One at 30 min and one over night. The assembly of both amilCP with and without his-tag was successful.

*PHS* introduced a RFC[25] restriction sites into the LacZ part (BBa\_2355313) by doing a PCR with restriction sites in the tails. The PCR was done only for the lacZ part of the BBa\_2355313 composite part (Effectively amplifying the BBa\_I732005 part).
*PHS* also did an assembly and transformation of the gel purified P7C5 part with the R0010 gBlock.

*CAL* and *CMP* ordered linkers as primer sequences in RFC[10].

*PHS* did a gel purification of the PCR amplified BBa\_I732005 with RFC[25] sites, and as the assembly from the day before worked, he also did an overnight culture of some of the succesful transformants.
![](https://static.igem.org/mediawiki/2017/a/ac/T--DTU-Denmark--labnotebook-4.png)

Figure 4: Digested results of promoter and RBS (BBa\_K608003). Well 2 is cut with EcoRI and SpeI. Well 3 shows the undigested promoter and RBS (BBa\_K608003).

### 20-26 October 2017

#### BioBrick assembly

*KK* did a amplification of our DNA sequences by PCR.

*PHS* did a plasmid prep and also digested to verify. He also spreaded a solution of X-gal (10 mM) and IPTG onto LB-Cam plates.

### 21 October 2017

#### BioBrick assembly

*CMP* and *AFT* did an assembly of test part (p032) that consist of single chain avidin and amilCP with his-tag. This was done with two different protocols. Protocol #1 - ligation was done straight from the digest. It showed too many colonies and had to be restricted. Protocol #2 - no colonies appeared when digest was extracted from a gel.

*CMP* and *AFT* did an assembly of a part that consists of single chain avidin and amilCP with his-tag flanking a double cut site (BBa\_K2355302) with two protocols. Protocol #1 ligation with the digested fragments. Protocol #2 ligation with gel extracted digest.

*PHS* ligated the part from the 20th of October, it was later transformed onto a X-gal + IPTG LB Cam plate.
The plates with X-gal showed blue colonies for the LacZ part that contained the promoter and RBS (BBa\_K608003).

### 22 October 2017

#### BioBrick assembly

*KK* and *CMP* ordered new primers for verification and amplification of all parts.

*CAL* and *CMP* did a restreaking of test part (p032).

*CAL*, *CMP* and *AFT* did a digest verification of 3A assembly of amilCP (BBa\_K592009) and promoter-RBS (BBa\_K608003). It was a success the part was present.

*CAL*, *CMP* and *AFT* did a digest verification of 3A assembly of amilCP with his-tag (BBa\_2355002) and promoter-RBS (BBa\_K608003). It was a success.

*PHS* saw blue colonie for the full LacZ part, the plates was very overgrown which made *PHS* do a restreaking of the colonieson new X-gal+IPTG plates.
![](https://static.igem.org/mediawiki/2017/8/8a/T--DTU-Denmark--labnotebook-5.png)

Figure 5: Digest verification of promoter-RBS (BBa\_K608003) and amilCP with his-tag (BBa\_K2355002). 1Kb ladder, well 2 double cut with EcoRI and PstI, well 3-4 single cuts with EcoRI or PstI, well 5 uncut.

![](https://static.igem.org/mediawiki/2017/d/d5/T--DTU-Denmark--labnotebook-6.png)

Figure 6: Digest verification of promoter-RBS (BBa\_K608003) and amilCP without his-tag (BBa\_K592009).1Kb ladder, well 2 double cut with EcoRI and PstI, well 3-4 single cuts with EcoRI or PstI, well 5 uncut.

### 23 October 2017

#### BioBrick assembly

*CAL*, *CMP* and *AFT* made a batch of digested pSB1C3 backbone.

*CAL* and *CMP* did a digest verification of test part (p032) that consist of single chain avidin and amilCP with his-tag. This part will be used to test the single chain avidin binding with biotin beads and how it is affected by snake venom.
*CAL* and *CMP* made an assembly of unregistered composite part this time with a different ratio in the ligation. Unsuccessful assembly.

*CAL* and *CMP* annealed primer sequences from IDT in order to make double stranded DNA coding different peptide chains cleaved by different snake venoms. It was a success.

*CAL* and *CMP* did assembly of all peptide chains parts within the backbone. These are the following parts BBa\_K2355006, BBa\_K2355008, BBa\_K2355014, BBa\_K2355015, BBa\_K2355025, BBa\_K2355026, BBa\_K2355032, BBa\_K2355033,
BBa\_K2355037 and BBa\_K2355039.

*CAL* and *CMP* did an assembly of the peptide chain part found in AMC assay (BBa\_K2355001) within the backbone.

*PHS* saw a lot of blue colonies from the full LacZ part that he restreaked on the 22th of October. These colonies was then used for cPCR and also an overnight culture. Unfortunately the cPCR did not confirm that the part was put together properly as the bands indicated that the insert was too small.

### 24 October 2017

#### BioBrick assembly

*CAL* and *CMP* put on an overnight cultures of: (BBa\_K2355006, BBa\_K2355008, BBa\_K2355014, BBa\_K2355015, BBa\_K2355025, BBa\_K2355026, BBa\_K2355032, BBa\_K2355033, BBa\_K2355037 and BBa\_K2355039, BBa\_K2355001, BBa\_K2355302).

*PHS* plasmid prepped the overnight culture from the 23th of October (BBa\_K2355313). It was then digested for verification. The bands indicated that the single chain avidin was present. That, along with the lacZ activity, indicates that the part was assembled as planned.

### 25-26 October 2017

#### BioBrick assembly

*CAL* and *CMP* did a verification of the assembly of all peptide linkers within the backbone by first doing a plasmid prep of the overnight cultures from the 24 of October and running a digest. Which showed great success. They also did a verification of peptide chain part found in AMC assay (BBa\_K2355001) within the backbone by digest.

*PHS* and *KK* send all linkers (BBa\_K2355006, BBa\_K2355008, BBa\_K2355014, BBa\_K2355015, BBa\_K2355025, BBa\_K2355026, BBa\_K2355032, BBa\_K2355033, BBa\_K2355037 and BBa\_K2355039), the ALK/G linker (BBa\_K2355001) and our part BBa\_K2355302 to sequencing.

*KK* also ran a verification of the parts (BBa\_K2355006, BBa\_K2355008, BBa\_K2355014, BBa\_K2355015, BBa\_K2355025, BBa\_K2355026, BBa\_K2355032, BBa\_K2355033, BBa\_K2355037 and BBa\_K2355039, BBa\_K2355001, BBa\_K2355302, BBa\_K2355313).

#### Verification by digestion

![](https://static.igem.org/mediawiki/2017/7/73/T--DTU-Denmark--labnotebook-7.png)

Figure 7: Digest verification of linkers: BBa\_K2355006 well 2-7, BBa\_K2355008 well 8-13 and BBa\_K2355014 well 14-19. 1Kb ladder. Well 4, 7, 10, 13, 16 and 19 uncut. The rest of the wells are single cuts with either EcoRI or PstI.

![](https://static.igem.org/mediawiki/2017/thumb/5/57/T--DTU-Denmark--labnotebook-8.png/800px-T--DTU-Denmark--labnotebook-8.png.jpeg)

Figure 8: Digest verification of linkers: BBa\_K2355015 well 2-7, BBa\_K2355025 well 8-13 and BBa\_K2355016 well 14-19. 1Kb ladder. Well 4, 7, 10, 13, 16 and 19 uncut. The rest of the wells are single cuts with either EcoRI or PstI.

![](https://static.igem.org/mediawiki/2017/thumb/c/c6/T--DTU-Denmark--labnotebook-9.png/800px-T--DTU-Denmark--labnotebook-9.png.jpeg)

Figure 9: Digest verification of linkers: BBa\_K2355032 well 2-7, BBa\_K2355033 well 8-13 and BBa\_K2355037 well 14-19. 1Kb ladder. Well 4, 7, 10, 13, 16 and 19 uncut. The rest of the wells are single cuts with either EcoRI or PstI.

![](https://static.igem.org/mediawiki/2017/thumb/6/66/T--DTU-Denmark--labnotebook-10.png/800px-T--DTU-Denmark--labnotebook-10.png.jpeg)

Figure 10: Digest verification of linkers: BBa\_K2355039 well 2-7 and BBa\_K2355001 well 8-13. 1Kb ladder. Well 4, 7, 10 and 13 uncut. The rest of the wells are single cuts with either EcoRI or PstI.

![](https://static.igem.org/mediawiki/2017/thumb/1/1e/T--DTU-Denmark--labnotebook-11.png/800px-T--DTU-Denmark--labnotebook-11.png.jpeg)

Figure 11: Digest verification of 6 potential colonies of composite part BBa\_K2355302. 1Kb ladder. Wells 2,5,8,11,14,17 double cut with EcoRI and PstI. wells 3,6,9,12,15,18 single cut with EcoRI. The rest of the wells are uncut.

![](https://static.igem.org/mediawiki/2017/thumb/2/24/T--DTU-Denmark--labnotebook-13.png/800px-T--DTU-Denmark--labnotebook-13.png.jpeg)

Figure 12: Verfication of linkers by PCR. 100 bp ladder. Linkers: BBa\_K2355006 well 2-3, BBa\_K2355008 well 4-5, BBa\_K2355014 6-7, BBa\_K2355015 8-9, BBa\_K2355025 10-11. Wells 12-16 negative controls.

![](https://static.igem.org/mediawiki/2017/thumb/f/f1/T--DTU-Denmark--labnotebook-12.png/800px-T--DTU-Denmark--labnotebook-12.png.jpeg)

Figure 13: Verfication of linkers by PCR. 100 bp ladder. Linkers BBa\_K2355026 well 2-3, BBa\_K2355032 well 4-5, BBa\_K2355033 well 6-7, BBa\_K2355037 well 8-9, BBa\_K2355039 well 10-11, BBa\_K2355001 well 12-13. Wells 14-16 negative controls.

![](https://static.igem.org/mediawiki/2017/thumb/d/d0/T--DTU-Denmark--labnotebook-14.png/800px-T--DTU-Denmark--labnotebook-14.png.jpeg)

Figure 14: Verification of parts by PCR. 1 kb ladder. Six colonies of an unregistered composite part well 2-7, six colonies of part BBa\_K2355302 well 9-14 and two colonies of part BBa\_2355002.

### 27 October 2017

#### BioBrick assembly

We received the sequencing results from the 26 of October. The parts that were sent to sequencing are the following: all linkers (BBa\_K2355006, BBa\_K2355008, BBa\_K2355014, BBa\_K2355015, BBa\_K2355025, BBa\_K2355026, BBa\_K2355032, BBa\_K2355033, BBa\_K2355037 and BBa\_K2355039), the ALK/G linker (BBa\_K2355001) and our part BBa\_K2355302 to sequencing.

### 29 October 2017

#### BioBrick assembly

*PHS* did a his-tag purification of amilCP with his-tag (BBa\_K2355002).

### 30 October 2017

#### BioBrick assembly

*CAL* and *CMP* lysed the cells by sonication.

*KK* ran a PCR of BBa\_K2355313 and BBa\_K2355302.

*PHS* tested the single chain avidin binding by using the test part assembled the 21st of October (LacZ + promoter) and BBa\_K2355313.

*KK* and *PHS* also tested the if the LacZ was degraded by the snake venom.

The composite part BBa\_K2355313 was tested with venom by *PHS*.

# Abbreviations

AFT: Andreas Frederik Treschow

AMH: Aleksander Moldt Haack

CAL: Cathrine Agnete Larsen

CMP: Chrysillis Magaard Polhaus

FBF: Frederik Bayer Frøhling

KK: Konstantinos Kalogeropoulos

PHS: Philip George Hau Sørensen

* [Lab notebook](#notebook)
* [Abbreviations](#abbreviations)

FIND US AT

![Mail](https://static.igem.org/mediawiki/2017/e/e6/T--DTU-Denmark--mail_logo.svg)
[![Instagram](https://static.igem.org/mediawiki/2017/9/9f/T--DTU-Denmark--instagram_logo.png)](https://www.instagram.com/igem_dtubiobuilders/?hl=da)
[![Facebook](https://static.igem.org/mediawiki/2017/2/20/T--DTU-Denmark--facebook_logo.png)](https://www.facebook.com/dtubiobuilders/?fref=ts)
[![Twitter](https://static.igem.org/mediawiki/2017/7/7d/T--DTU-Denmark--twitter_logo.png)](https://twitter.com/iGEM_DTU)

MAIN SPONSORS

[![Otto Mønsted](https://static.igem.org/mediawiki/2016/f/f3/T--DTU-Denmark--OM_logo.jpg)](http://www.omfonden.dk/information/)
[![Lundbeck fundation](https://static.igem.org/mediawiki/2016/4/45/T--DTU-Denmark--nnfond_logo.png)](http://www.novonordiskfonden.dk/)

[![](https://static.igem.org/mediawiki/2017/b/bf/HQ_menu_logo.jpg)](https://2017.igem.org)


[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:DTU-Denmark/Experiments

[![](http://placehold.it/350x150)](https://2017.igem.org/Team%3ADTU-Denmark)

MENU

[HOME](https://2017.igem.org/Team%3ADTU-Denmark)

TEAM

[Team](https://2017.igem.org/Team%3ADTU-Denmark/Team)
[Collaborations](https://2017.igem.org/Team%3ADTU-Denmark/Collaborations)

PROJECT

[Description](https://2017.igem.org/Team%3ADTU-Denmark/Description)
[Design](https://2017.igem.org/Team%3ADTU-Denmark/Design)
[Experiments](https://2017.igem.org/Team%3ADTU-Denmark/Experiments)
[Notebook](https://2017.igem.org/Team%3ADTU-Denmark/Notebook)
[InterLab](https://2017.igem.org/Team%3ADTU-Denmark/InterLab)
[Contribution](https://2017.igem.org/Team%3ADTU-Denmark/Contribution)
[Model](https://2017.igem.org/Team%3ADTU-Denmark/Model)
[Results](https://2017.igem.org/Team%3ADTU-Denmark/Results)
[Demonstrate](https://2017.igem.org/Team%3ADTU-Denmark/Demonstrate)
[Improve](https://2017.igem.org/Team%3ADTU-Denmark/Improve)
[Attributions](https://2017.igem.org/Team%3ADTU-Denmark/Attributions)

PARTS

[Parts](https://2017.igem.org/Team%3ADTU-Denmark/Parts)
[Basic Parts](https://2017.igem.org/Team%3ADTU-Denmark/Basic_Part)
[Composite Parts](https://2017.igem.org/Team%3ADTU-Denmark/Composite_Part)
[Part Collection](https://2017.igem.org/Team%3ADTU-Denmark/Part_Collection)

[SAFETY](https://2017.igem.org/Team%3ADTU-Denmark/Safety)

HUMAN PRACTICES

[Silver HP](https://2017.igem.org/Team%3ADTU-Denmark/HP/Silver)
[Integrated and Gold](https://2017.igem.org/Team%3ADTU-Denmark/HP/Gold_Integrated)
[Public Engagement](https://2017.igem.org/Team%3ADTU-Denmark/Engagement)

AWARDS

[Applied Design](https://2017.igem.org/Team%3ADTU-Denmark/Applied_Design)
[Entrepreneurship](https://2017.igem.org/Team%3ADTU-Denmark/Entrepreneurship)
[Hardware](https://2017.igem.org/Team%3ADTU-Denmark/Hardware)
[Measurement](https://2017.igem.org/Team%3ADTU-Denmark/Measurement)
[Model](https://2017.igem.org/Team%3ADTU-Denmark/Model)
[Plant](https://2017.igem.org/Team%3ADTU-Denmark/Plant)
[Software](https://2017.igem.org/Team%3ADTU-Denmark/Software)

[JUDGING FORM](https://igem.org/2017_Judging_Form?team=DTU-Denmark)

# Experiments

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

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

##### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

##### Inspiration

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

[![](https://static.igem.org/mediawiki/2017/b/bf/HQ_menu_logo.jpg)](https://2017.igem.org)

[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:DTU-Denmark/Results

# Results

The AMC experiment showed that we are able to detect a significant difference between the Bitis species and the *Naja nigricollis*. The initial AMC substrate experiment led to a more comprehensive substrate screening experiment that resulted in multiple substrate candidates. We managed to assemble the peptide sequences into the plasmid backbone (pSB1C3). The venom degradation test of the reporter molecules amilCP and β-galactosidase showed no reduction in the colorimetric or enzymatic properties. We improved part BBa\_K592009 by adding a His-tag, the expression of color was not reduced and the color protein could easily be retained by a His-tag purification. To further improve our diagnostic device a reduction on the response time for the result was undertaken. Instead of the amilCP the reporter enzyme β-galactosidase were to be attached to the substrate linker. However, there were no successful assembly of ScAvidin with the linker to the β-galactosidase.

Visit [experimental design page](https://2017.igem.org/Team%3ADTU-Denmark/ExperimentalDesign) for the theory and design behind our experiments, and [protocols](https://2017.igem.org/Team%3ADTU-Denmark/Protocols) for the protocols of our experiments.

* [Results](#results)
* [AMC Experiment](#amc)
* [Peptide Substrate Screening](#peptidesubstratescreening)
* [Assembly of
   amilCP with and without His-tag](#assemblyamilCP)
* [AmilCP with His-tag purification test](#hispurification)
* [AmilCP degradation by venom](#amilcpdegradation)
* [Test of lacZ
   degradation by venom](#laczdegradation)
* [Assembly of
  linkers derived
  from substrate
  screening and
   literature](#linkerassembly)
* [Assembly of
   composite part](#composite)
* [Assembly of
   composite lacZ
  part (BBa\_2355313)](#laczassembly)
* [Test of
   composite lacZ
   part: ScAvidin
   binding to biotin](#lacztest)
* [Comparison of
   composite part
   with β-galactosidase](#laczcomparison)

FIND US AT

![Mail](https://static.igem.org/mediawiki/2017/e/e6/T--DTU-Denmark--mail_logo.svg)
[![Instagram](https://static.igem.org/mediawiki/2017/9/9f/T--DTU-Denmark--instagram_logo.png)](https://www.instagram.com/igem_dtubiobuilders/?hl=da)
[![Facebook](https://static.igem.org/mediawiki/2017/2/20/T--DTU-Denmark--facebook_logo.png)](https://www.facebook.com/dtubiobuilders/?fref=ts)
[![Twitter](https://static.igem.org/mediawiki/2017/7/7d/T--DTU-Denmark--twitter_logo.png)](https://twitter.com/iGEM_DTU)

MAIN SPONSORS

[![Otto Mønsted](https://static.igem.org/mediawiki/2016/f/f3/T--DTU-Denmark--OM_logo.jpg)](http://www.omfonden.dk/information/)
[![Lundbeck fundation](https://static.igem.org/mediawiki/2016/4/45/T--DTU-Denmark--nnfond_logo.png)](http://www.novonordiskfonden.dk/)

[![](https://static.igem.org/mediawiki/2017/b/bf/HQ_menu_logo.jpg)](https://2017.igem.org)