Team:SNU India/Experiments
Cloning and characterization of RFP-TAL composite part
Cloning was done using the bio-brick DNA parts from the iGEM kit. We used T7 with RBS (BBa_K525998), TAL coding sequence (BBa_K1033000) and RFP coding sequence (BBa_E1010). First we used 1 µl of plasmid DNA to transform competent E. coli DH5α cells which
were selected on chloramphenicol as all the 3 parts are in pSB1C3 vector which confers chloramphenicol resistance. The positive colonies were then used for plasmid extraction (Figure 1).
The extracted plasmids were then digested using restriction enzymes. For this RFP was linearized using Spe1 and Pst1 whereas TAL was digested with two restriction enzymes- Xba-I and Pst-I HF (Figure 2).
Subsequently the 1.5 kb band of TAL digestion product and lineraised RFP vector (2.7 kb) was used for ligation. The ligation reaction was transformed in E.coli DH5α cells and plasmid was isolated from positive colonies.
Simultaneously plasmid containing T7+RBS was linearized by sequentially digesting with Spe-I (Figure 4) and then Pst-I (Figure 5, second lane).
The released fragment of TAL+RFP (2.2 kb) was ligated with linearized plasmid containing T7-RBS. The ligation product was then transformed into competent cells. The positive colonies were screened followed by plasmid extraction and restriction digestion with Xba1 and Pst1 (Figure 5).
Simultaneously plasmid containing T7+RBS was linearized by sequentially digesting with Spe-I (Figure 4) and then Pst-I (Figure 5, second lane).
The released fragment of TAL+RFP (2.2 kb) was ligated with linearized plasmid containing T7-RBS. The ligation product was then transformed into competent cells. The positive colonies were screened followed by plasmid extraction and restriction digestion with Xba1 and Pst1 (Figure 5).
We, then moved ahead for sub-cloning of our entire construct of T7+RFP+TAL into pSB3C5 (a low copy plasmid, BBa_J04450). T7-RBS-RFP-TAL part was released from pSB1C3 using the enzymes Xba1 and Pst1 and ligated with the plasmid pSC3C5 was digested with
Xba1 and Pst1 (Figure 6). The positive clones were confirmed by mobility shift of the plasmid with respect to pSB3C5 (Figure 7).
Following the cloning, we checked for the expression of both RFP and p-coumaric acid in E.coli BL-21. RFP was visualized by fluorescence microscopy (in Leica DM IL LED)following IPTG (0.5mM) induction for 4 hrs (Figure 8, upper panel represents microscopic images of bacteria and lower panel is quantification of microscopy data) and p-coumaric acid (Figure 9a-d)
production was probed in filtered culture supernatant by mass spectrometry (Agilent Technologies 6540 UHD-Accurate Mass Q-TOF LC/MS in negative mode using methanol as solvent)
Characterization of Bacterial Laccase
Bacterial laccase construct – BBa_K863000 (iGEM kit) was used to transform competent BL21 (DE3) cells. The transformants were selected from LB agar plates containing chloramphenicol (50µg/ml).Since BPUL is under the T7 RNA promoter, 0.5 mM IPTG was used
to induce the bpul gene. The duration of induction was for 8 hours and the level of protein induced is seen to increase with increase in induction duration at the expected size (around 58 kDa similar to what has been seen for Part:BBa_K2043007,
http://parts.igem.org/Part:BBa_K2043007) as seen in Figure 10.
The same has also been confirmed by western blotting using anti-his antibody (as the laccase protein will have his-tag) which shows increase in band intensity at expected molecular weight in induced samples as opposed to uninduced samples (Figure 11).
After confirmation of protein induction we checked the localization of Bpu laccase in supernatant or cell pellet. Uninduced and induced cells were pelleted and the supernatant was collected for the induced sample. The pellets for both the samples was
resuspended in 1X PBS followed by SDS-PAGE to check if laccase protein secreted out of the bacterial cell into the supernatant (Figure 12). However we were not able to see induced protein band in supernatant by both coomassie and western
blotting. The reason this observation may be weak induction by IPTG as the amount of induced laccase protein is not significantly higher in induced samples as compared to uninduced samples in the pellet as well.
Another way of laccase secretion was evaluated was by plating the transformants on bromophenol LB plates with 0.5 mM IPTG. This was based on a protocol derived from Tekere et al. 2001. The secreted enzyme was expected to create clear zones around the
colonies that would grow. However, no clear zones were observed around the streaked colonies even after 48 hours of incubation. These observation rule out the secretion of laccase from bacterial cell under the conditions used.
Development of growth curves of algae and bacteria.
Growth curve of Algae in TAP media
Chlamydomonas reinhardtii was cultured in TAP media (recipe from [3]) at 25°C at 200 rpm. The growth curves were generated using OD measured at 750 nm [4],[5]. The growth curve was developed in duplicates and doubling time of Chlamydomonas reinhardtii
was calculated using the growthcurver package in R [4]. The estimated doubling time for first replicate (A1) was found to be 5.92 hours whereas the doubling time for second replicate (A2) was found to be 6.07 hours(Figure 14 and 15 where, black line represents actual data points and green lines represents fitted curves). Therefore, on average the doubling time for algae was calculated as 5.995 hours under the given growth conditions.
Growth curve of E.coli BL-21(DE3) in TAP media
E.coli BL-21(DE3) was cultured in TAP media at 25°C at 120 rpm. The growth curves were generated using OD measured at 600 nm [4],[5]. The growth curve was developed in duplicates calculated using the growthcurver package in R [4],[5].
We observe from the graphs that both cultures display almost similar trend of growth. The doubling time for first replicate (B1) was found to be 2.24 hours, whereas the doubling time for second replicate (B2) was found to be 2.25 hours (Figure 16 and 17 where, black line represents actual data points and red lines represents fitted curves). Therefore, on average the doubling time for bacteria was calculated as 2.245 hours under the tested conditions.
These growth curves will help us establish the consortium between algae and bacteria, as they provide us the doubling time of each organism which can then be used to decide the initial density of each organism that needs to be standardized for consortium establishment.
Improving a previous iGEM part
Estrogen sensitive T7 RNA polymerase
For our bacterial system, we planned to use the estrogen sensitive T7 RNA polymerase originally built by CMU iGEM team in 2014-15. Unfortunately, the part registry page (BBa_K1732015) showed that the estrogen sensitive T7 polymerase has an incomplete
or erroneous sequence. According to information on the original part page, it displayed a total size of 2301bp, which is much smaller than the expected functional gene, given that just the gene for T7 RNA polymerase with the ER-LBD
(estrogen responsive-ligand binding domain) is greater than 3kb. The sequence reads tab for the sample submission mentioned that the sequence read is good but inconsistent with part sequence. Furthermore, the listed sub-parts include
J23115 promoter, RBS and BBa_K1491022, which is titled as ‘N-Terminus of T7 RNA Polymerase’, which is not the entire estrogen sensitive T7 RNA polymerase. We could not find any other part in the registry in which the ER-LBD had been
fused with the wild type T7 RNAP.
Hence, we attempted to re-design the amino-acid sequence of this protein as described by McLachlan et.al; 2011. We fused the ER-LBD between positions 179 and 180 aa residues of the T7 RNAP. These domains of the T7 RNAP were described by Shis DL and Benett MR, 2013.
Unfortunately, the resulting sequence size exceeded the maximum gBlocks size of 2.3 kb. So, our strategy was to synthesize the whole sequence in two fragments: a 1.4kb N-terminal fragment featuring Promoter, RBS, T7RP positions 1-179 and ER-LBD, and a 2.1 kb fragment with the remaining T7RP positions 180-883. Our strategy was to assembled by Overlap-Extension Polymerase Chain Reaction.
Hence, we attempted to re-design the amino-acid sequence of this protein as described by McLachlan et.al; 2011. We fused the ER-LBD between positions 179 and 180 aa residues of the T7 RNAP. These domains of the T7 RNAP were described by Shis DL and Benett MR, 2013.
Unfortunately, the resulting sequence size exceeded the maximum gBlocks size of 2.3 kb. So, our strategy was to synthesize the whole sequence in two fragments: a 1.4kb N-terminal fragment featuring Promoter, RBS, T7RP positions 1-179 and ER-LBD, and a 2.1 kb fragment with the remaining T7RP positions 180-883. Our strategy was to assembled by Overlap-Extension Polymerase Chain Reaction.
The peptide sequences are as follows:
-
T7_pol_Nterminal (1-179)
MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYK -
ER_LBD_HERA (312-595)
ADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEAEGFPATV -
T7_Pol_Cterminal (179-883)
KAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA -
Complete protein sequence of Estrogen_Sensitive_T7_polymerase (1170AA) (~3.5kb)
MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYK ADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEAEGFPATV MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEAEGFPATVKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA
These fragments (1.4 kb from NT-ESRP PCR and 2.1 kb from CT_ESRP) with complimentary overhangs were gel-purified were then mixed in equimolar ratio and subjected to a touch-Down PCR in which the overlaps would anneal and form the 3.5 kb fragment. This
touch-down product had to be used as template for a final PCR reaction with the forward primer of the first fragment and reverse primer of the second fragment. However, even after multiple rounds of PCR troubleshooting, the final
product always yielded a product which appeared as a smear on agarose gel electrophoresis (Figure 20).
Finally, we used a commercial T/A Cloning kit (RBC) to clone the 2.1 kb CT-ESRP gBlock fragment obtained from IDT into TA vector. Isolated plasmids from transformed colonies showing mobility shift were used for PCR amplification, resulting in clear amplification
of 2.1 kb fragment with no non-specific amplification (Figure 21). This 2.1 kb fragment will be utilized for overlap extension PCR to obtain full length clone of T7_ESRP.
References
- Schindelin, J.; Arganda-Carreras, I. & Frise, E. et al. (2012), "Fiji: an open-source platform for biological-image analysis", Nature methods 9(7): 676-682, PMID 22743772, doi:10.1038/nmeth.2019
- https://www.chlamycollection.org/methods/media-recipes/tap-and-tris-minimal/
- R Core Team (2018). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/
- Sprouffske K, Wagner A. Growthcurver: An R package for obtaining interpretable metrics from microbial growth curves. BMC Bioinformatics. 2016 Apr 19;17:172. doi: 10.1186/s12859-016-1016-7.
- McLachlan, Michael J et al. “A new fluorescence complementation biosensor for detection of estrogenic compounds.” Biotechnology and bioengineering vol. 108,12 (2011): 2794-803. doi:10.1002/bit.23254
- Shis DL, Bennett MR. Library of synthetic transcriptional AND gates built with split T7 RNA polymerase mutants. Proc Natl Acad Sci U S A. 2013, 110(13):5028-33.
- www.parts.igem.org/Part:BBa_K1732015