Team:IISER Tirupati/Notebook
Notebook
May 2019
1-5-19
Yeast agglutination assay was done to check for the presence of fimbriae in DH5alpha but no agglutination was observed.
2-5-19
Yeast agglutination assay was repeated with modifications but still, no agglutination was observed
3-5-19
NEB DH5alpha, NEB BL21, DH5alpha and BL21 were grown on LB plate.
4-5-19
Yeast agglutination assay was again repeated following a different protocol, but still no agglutination was observed.
5-5-19 to 7-5-19
The different biobricks in the registry were studied and the required ones were selected
8-5-19
Yeast was grown in YPD
9-5-19
The yeast did not grow in YPD, hence media for growing yeast was prepared manually
Still, no growth of yeast was observed
10-5-19
Hemagglutination assay using NEB DH5 alpha and Pseudomonas was done but again agglutination was not observed
11-5-19
Hemagglutination assay using NEB DH5alpha, BL21 and pseudomonas was repeated following various suggestions from experts but no agglutination was observed
14-5-19
Fimbriae were isolated from DH5 alpha and SDS page was done but the bands corresponding to Fimbriae was not observed.
Culture for the preparation of DH5alpha competent cells was set up
20-5-19
DH5alpha competent cells were prepared
19-5-19
Biobrick was transformed into the competent cells to check the efficiency of the prepared competent cells
21-5-19
No colonies were observed after transformation
The protocol for the preparation of competent cell was modified based on various suggestions.
Culture for the preparation of competent cell was set up
22-05-19
Competent cells were prepared
Transformation was done to check the efficiency of the prepared competent cells
23-5-19
No colonies were observed
Culture for the preparation of ultracompetent cells was setup
Buffers required for the same were prepared
24-5-19
Ultracompetent cells were prepared
Transformation was done to check the efficiency of ultracompetent cells
Genomic DNA was isolated
26-5-19 to 31-5-19
No colonies appeared
The gblocks and the primers required for making the constructs were designed
June
1-6-19
Agglutination assay using new yeast was done but the results were negative
10-6-19
The required biobricks, pSB3k3 plasmid and pSB1C3 plasmid were transformed in NEB DH5alpha competent cells
13-6-19
Colonies appeared, so the cultures for the isolation of the plasmids were set up
Lactate assay was done using the lactate detection kit sponsored by Sigma Aldrich But unexpected results were obtained
14-6-19
All the ordered primers arrived
The plasmids were isolated
Genomic DNA was isolated
14-6-19 to 18-9-19
lldP, lldR and lldD were amplified from genomic DNA
20-6-19
Yeast agglutination assay was repeated using yeast from InStem but again no agglutination was observed
28-6-19
BL21 competent cells were prepared
29-6-19
pSB1C3 and pSB3K3 plasmids were co-transformed in BL21 and DH5alpha to check the compatibility of plasmids
29-6-19
Confirmation tests for co-transformation were performed.
30-6-19
Lactate assay was repeated but again the unexpected results were obtained
July
1-7-19 to 16-7-19
HPLC was done to detect the presence of lactate in cancer cells
17-7-19 to 21-7-19
Tried to isolate fimbriae using a protocol from literature but it failed again
August
1-8-19
gblocks arrived
3-8-19
lldP+lldR was SOEed with prefix-strong promoter-strong RBS+lldP flank and lldR flank-terminator-suffix
4-8-19
The above construct was digested using EcoR1 and Pst1 and ligated to digested pSB3K3
5-8-19
The ligated plasmids were transformed into DH5alpha
6-8-19
Colony PCR was done but positive colonies were not obtained
7-8-19
prefix-O1-P-O2-strong RBS-sfGFP flank was SOEed with sfGFP-terminator-suffix
prefix-constitutive promoter- strong RBS-sfGFP flank was SOEed with sfGFP-terminator-suffix
8-8-19
The above constructs were digested using EcoR1 and Pst1 and ligated with digested pSB1C3
9-8-19
Ligated plasmids were transformed into DH5alpha
10-8-19
Colonies appeared and Colony PCR was done
No positive colonies with plasmid containing prefix-O1-P-O2- strong RBS -sfGFP-terminator-suffix were obtained and positive colonies with plasmids containing prefix-constitutive promoter- strong RBS -sfGFP-terminator-suffix were obtained for
Culture for the isolation of positive plasmid was set up
11-8-9
The plasmid containing prefix-constitutive promoter- strong RBS -sfGFP-terminator-suffix was isolated
Plasmid linearization and insert release for prefix-constitutive promoter- strong RBS -sfGFP-terminator-suffix was done using prefix and VR2
The released inserts were of appropriate size
12-8-19
lldP+lldR was SOEed with prefix-strong promoter-strong RBS-lldP flank and lldR flank-terminator-suffix
The above construct was digested using EcoR1 and PSt1
13-8-19
The above construct was ligated to pSB3K3
The ligated plasmid was transformed into DH5alpha
14-8-19
Colony PCR was done for the above construct but positive colonies were not obtained
lldP was SOEed with lldD
15-8-19
lldP+lldD was SOEed with prefix-strong promoter-strong RBS-lldP flank and lldD flank-terminator-suffix but the end construct after SOEing did not give band of the required length
FimB biobrick was transformed in DH5alpha
16-8-19
The culture was set up for plasmid isolation
17-8-19
Plasmid containing FimB was isolated
lldD was SOEed with prefix- constitutive strong promoter-strong RBS -lldD flank and lldD flank-terminator-suffix The end construct did not give band of the required length
The SOEing was repeated but still, the band of required length was not obtained
18-8-19
FimB plasmid was transformed into BL21
lldR+lldD was SOEed with prefix-constitutive strong promoter-strong RBS-lldR flank and lldD flank-terminator-suffix, but the end construct obtained was not of the required length
19-8-19
DH5alpha and BL21 transformed with FimB plasmid and normal DH5alpha and BL21 were send to IISER Bhopal for TEM
FimHRPMrel was amplified from the biobrick plasmid. prefix-O1-P-O2-strong RBS-sfGFP flank was soed with sfGFP-terminator-suffix
20-8-19
FimHRPMrel was SOEed with prefix-string promoter-strong RBS-FimHRPMrel flank and FimHRPMrel flank- term-suffix
The prefix-strong promoter-strong RBS -FimHRPMrel-terminator-suffix construct was digested with EcoR1 and Pst1
The prefix-O1-P-O2-strong RBS-sfGFP-terminator-suffix was digested with EcoR1 and Pst1
21-8-19
FimHRPMrel construct was ligated with pSB1C3
Transformation of the plasmid containing FimHRPMrel was done
The digested prefix-O1-P-O2 - strong RBS -sfGFP- terminator-suffix construct was ligated with pSB1C3 plasmid
Transformation of the plasmid containing the above construct was done
22-8-19
Colony PCR was done for the colonies transformed with prefix-O1-P-O2 - strong RBS-sfGFP - term-suffix and positive colonies were obtained
The 2 constructs of IL12 were SOEed together
23-8-19
Plasmid containing prefix-O1-P-O2 -str RBS-sfGFP-term-suffix was isolated from the positive colonies. Linearisation and insert release were done and bands of required length were obtained
Colony PCR was done for the colonies transformed with FimHRPMrel, positive colonies were not observed
24-8-19
Colony PCR was again done for the colonies transformed with plasmid containing FimHRPmrel using different set of primers but no positive colonies were observed
IL12 was SOEed with prefix-const strong promoter-strong RBS-IL12 flank and IL12 flank-terminator-suffix, but the construct obtained was not of the required length
25-8-19
lldR+lldD was SOEed with prefix-strong promoter-strong RBS -lldR flank and lldD flank-terminator-suffix
The above construct was digested with Ecor1 and Pst1
26-8-19
The above construct was ligated with pSB3K3
The ligated product was transformed into DH5alpha
27-8-19
colony PCR was done, but again no positive colonies were obtained
29-8-19
lldP+lldR was again SOEed with prefix-const strong promoter-strong RBS-lldP flank and lldR flank-terminator-suffix
The above construct was digested with EcoR1 and Pst1
30-8-19
The digested construct was ligated with digested pSB3K3 plasmid
The above construct was transformed into DH5alpha
31-8-19
Colony PCR was done but no positive colonies were obtained
September
1-9-19
IL12 was soed with prefix-strong promoter-strong RBS-IL12 flank and IL12 flank-terminator-suffix
The above construct was digested with EcoR1 and pst1
2-9-19
The SOEed construct was ligated to pSB1C3
The ligated product was transformed into DH5alpha
3-9-19
Colony PCR was done and positive colonies were obtained
Culture was setup to isolate plasmid from the positive colonies
4-9-19
Plasmids containing IL12 construct was isolated
The isolated plasmid was transformed into BL21
5-9-19
prefix-O1-P-O2 -weak RBS-sfGFP flank was SOEed with sfGFp-terminator-suffix
The construct was digested using EcoR1 and pst1
6-9-19
The construct was ligated with pSB1C3
The ligated plasmid was transformed into DH5alpha
7-9-19
Colony PCR was done and positive colonies were obtained
The culture was setup for plasmid isolation
8-9-19
Plasmid containing prefix-O1-P-O2 -weak RBS-sfGFP flank was SOEed with sfGFp-terminator-suffix was isolated.Insert release and linearisation were done and bands of required length were obtained
9-9-19
The ordered FimE knockout strain arrived
The FimE knockout strains were plated on LB agar plates
10-9-19
Prefix -O1-P O2 -strong RBS-sfGFP flank was SOEed with sfGFP-terminator-suffix
The construct was digested with EcoR1 and Pst1
Culture for the preparation of FimE knockout competent cell was set up
11-9-19
The digested construct was ligated with digested pSB1C3 plasmid
The ligated plasmid was transformed into DH5 alpha
FimE knockout competent cells were prepared
12-9-19
Colony PCR was done for the colonies transformed with plasmids having Prefix -O1-P-O2-strong RBS-sfGFP-terminator-suffix and positive colonies were obtained
Culture for the isolation of plasmid was set up
13-9-19
Plasmid was isolated from the positive colonies.Insert release and linearisation were done and bands of required length were obtained
The plasmid was transformed into BL21
14-9-19
FimHRPMrel was SOEed with prefix-strong Promoter-strong RBS-FimHRPMrel flank and fimHRPMrel flank-term-suffix
The construct obtained was digested with EcoR1 and Pst1
15-9-19
The digested plasmid was ligated with digested 1C3 plasmid
The plasmid was transformed in DH5alpha
16-9-19
Colony PCR was done but positive colonies were not obtained
17-9-19
Prefix-constitutive promoter-weak RBS-sfGFP flank was SOEed with sfGFP-terminator-suffix
The SOEed construct was digested with EcoR1 and Pst1
18-9-19
The digested construct was ligated with digested pSB1C3 plasmid
The ligated product was transformed into DH5alpha
19-9-19
Colony PCR was done and positive colonies were obtained
Culture for the isolation of plasmid was set up
20-9-19
The plasmid was isolated.insertrelease and linearisation were done and bands of expected length were obtained
The isolated plasmid and pSB3K3 plasmid were co-transformed into BL21
21-9-19 to 30-9-19
Mid semester exams
October
1-10-19
IL12 was SOEed with O1-P-O2-strong promoter-strong RBS-IL12 flank and IL12 flank-terminator-suffix,but the end construct obtained was not of the required size
2-10-19
IL12 was again SOEed with O1-P-O2-strong promoter-strong RBS-IL12 flank and Il12 flank-terminator-suffix
The construct obtained was digested with EcoR1 and Pst1
3-10-19
The digested construct was ligated with digested pSB1C3
The plasmid was transformed into DH5alpha
4-10-19
Colony PCR was done and positive colonies were obtained
Culture for the isolation of the positive plasmids was set up
5-10-19
The plasmids were isolated.insert release and linearisation were done and bands of expected length were obtained. The plasmid was transformed into BL21
6-9-19
Medium promoter -Strong RBS -lldD -terminator-suffix arrived from twist bioscience
The above construct was digested and ligated with pSB3K3
7-10-19
The construct was transformed into DH5alpha
8-10-19
The plamids were isolated from the colonies appeared. Insert release and linearisation were done and bands of expected length were obtained
The plasmid was transformed into BL21
6-10-19 to 17-10-19
Characterization of the constructs and western blot to detect IL12 was done
19-10-19
The lab was cleaned
"We play with bacteria and sometimes they play us"
Contact us
Email: igemiisertirupati@gmail.com
Address:
Indian Institute of Science Education and Research, Tirupati
C/o Sree Rama Engineering College (Transit Campus),
Rami Reddy Nagar, Karakambadi Road,
Mangalam (P.O.) Tirupati -517507.
Andhra Pradesh, INDIA
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