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.
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.
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.
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.
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
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
