Part:BBa_K1428001
Gene cluster for Expressing a purifiable Eut Bacterial Microcompartment
The following features of the gene cluster are listed from upstream to downstream: A T7 promoter, a T7 RBS, His6-tagged EutS, a Plac promoter, an RBS (B0034), EutM, an RBS (B0034), EutN, a Plac promoter, an RBS (B0034), EutL, an RBS (B0034), EutK. The whole gene cluster encodes five proteins, EutS, EutM, EutN, EutL, EutK, arranged in a specific order such that when induced with 0.2mM IPTG or allowed for leaky expression at 30C overnight, these 5 gene products will be expressed in a specific molar ratio so that they can assemble into the intact organelle of the bacterial microcompartment (BMC) of the Ethanolamine-utilization type (Eut). It was derived from BBa_K311004 and the pET28a vector in 2 cloning steps. If the whole part is put into a bacterial, one can readily express out a whole organelle in the form of BMC for compartmentalization, which, when combined with our second Biobrick BBa_K1428000, can target any coding Biobrick inside the BMC for both in vivo and in vitro applications, for example, metabolic engineering to avoid toxic intermediate damage to the cell, bioremediation for pollutant collection, drug delivery systems by storing nucleic acids, peptides and proteins for replacement therapy, or even virology model studies as the whole thing resembles a viral capsid.
Usage and Biology
Expression
The entire gene cluster can be expressed when compared with the original BBa_K311004, which is evidenced by the following gel picture:
- Pre-I: Pre-induction or expression; 3h Post-I: 3 hours post-induction with 0.2mM IPTG; ON-I: overnight induction with 0.2mM IPTG; ON-30°C: overnight incubation at 30°C without IPTG. Calculated sizes of proteins for reference: EutS 11.6kDa, His6-EutS 13.84kDa, EutM 9.8kDa, EutN 10.4kDa, EutL 22.7kDa, EutK 17.5kDa
There are several things to note about this piece of data. Firstly, the difference in size between EutS and the His6-tagged EutS is readily apparent in the strongest band, and it also shows that both EutS and His6-EutS are well-expressed. Smearing of the bands suspected to be EutS (11.6kDa), His6-EutS (13.8kDa) and EutM (9.8 kDa) showed some smearing that is typical of proteins rich in hydrophobic residues. The EutN (10.4kDa) band was difficult to discern from that of EutM in both cases, as in the literature. The band related to EutK (17.5kDa) was relatively vague in both pETE and BBa_K311004, but is also consistent with that observed in the literatures which showed similar results. EutL (22.7kDa) was also clearly expressed.
Interestingly, different conditions seems to have an impact on different system. Comparing pETE with BBa_K311004, the only differences are (1) EutS was under T7 promoter in pETE rather than under Plac in BBa_K311004, in both cases, the spacing regions between the promoter and putative transcriptional start site, and that between the consensus RBS and start codon are both optimal and the same. (2) EutS in pETE was His6-tagged together with a thrombin site before fusing to EutS, while that of BBa_K311004 is not. From our data, it seems that the expression for His6-EutS from pETE is better with 0.2mM IPTG room temperature overnight induction, while that of BBa_K311004 is better with overnight 30°C incubation. On the other hand, EutL is better expressed with overnight 30°C incubation than for using IPTG, although one could argue that these samples were normalized according to the cultures’ OD, so that the protein maybe under-represented in terms of percentage of global protein concentration and thus also for band intensity, such conclusion was invalid as the result from BBa_K311004 clearly shows such under-representation was absent. Furthermore, the regulation of the T7 promoter was rather tight, as indicated by the fact that very little His6-EutS was produced if IPTG was not added in 30°C incubation. Intriguingly, there is one ~26-28kDa band visible in both cases that does not match with any calculated sizes of proteins expressed by the plasmids compared with the controls, indicating that the presence of BMC might affect the intrinsic protein expression of BL21(DE3) cells.
Purification
Purification can be carried out using standard protein purification procedures. The detailed procedure was described below:
BL21(DE3) cells carrying BBa_K1428001 was inoculated and induced overnight using 0.2mM IPTG in a liter scale. Sonication was carried in sonication buffer* with 2s bursts-8s rest for 3 minutes for each intervals on ice, with all procedures post-sonication carried out at 4°C, and all solutions in contact with the sample and column was pre-chilled to 4°C. After sonication, the sample was clarified by centrifugation at 9,520g for 10min. The Supernatant containing the soluble protein fraction was collected. An adjustment solution* was added slowly with shaking to convert the composition of the sonication buffer into that of Ni-Binding buffer. The clarified, adjusted samples were filtered through a 0.45μm-pore membrane, and subsequently loaded separately onto 1ml HiTrap Chelating columns (GE Health Sciences). The column, bathed in 20% ethanol, was prepared by (1) washing with 5-columns of MiliQ water, (2) washing with 5-columns of Ni-Binding buffer, (3) loaded with 100mM NiSO4 in Ni-Binding buffer, and finally (4) equilibrated with Ni-Binding buffer just before sample loading. After sample loading, the column was washed with 5-columns of Ni-Binding buffer, and elution was carried out in gradients of 30%, 50% and 100% Ni-Elution buffer* in Ni-Binding buffer, and finally stripped with Strip buffer*. To preserve the column for future use, the column was stripped with 5-columns of Strip buffer noting that the column of the column bed should be pure white, and washed with 5-columns of MiliQ water and re-bathed in 20% ethanol. The BMC would most likely be on the 30% Eluent fraction.
- 2X Adjustment solution composition: 1M NaCl, 40mM Imidazole
Ni-Binding buffer composition: 25mM Tris-HCl, 500mM NaCl, 20mM Imidazole, pH 8.0 Ni-Elution buffer composition: 25mM Tris-HCl, 500mM NaCl, 500mM Imidazole, pH 8.0 Strip buffer composition: 25mM Tris-HCl, 500mM NaCl, 50mM EDTA, pH 7.4
Due to the freezed upload function of the iGEM Wiki, the gel picture can only be found in our team's wiki: http://2014.igem.org/Team:Hong_Kong_HKU
Cargo protein localization
You can localize any coding Biobricks into the BMC by fusing a signal peptide to your proteins' N-terminus, which is provided in BBa_K1428000. For more information please visit that page and our Wiki.
BMC-Cargo co-purification
Here, we have an example of localizing mCherry into the BMC and purifying that using the Ni-NTA affinity column. You would expect the column to look like this after loading all your clarified and filtered sample:
- Note the upper part of the column is colored red due to the trapped mCherry together with the BMC. The lower part is blue due to the color of the Ni2+-NTA-imidazole complex.
The procedure of purifying the BMC is the same as described above. One can do a Gel Filtration analysis to confirm the existence of a large protein complex of >2,000kDa. Note that the collected fraction has to be concentrated prior to loading on the gel filtration column as the whole process will dilute the sample very much. Use Ni-Binding buffer as the carrying buffer.
Our gel filtration results was as follows:
- Calculated sizes of molecular complexes for the four peaks from large to small: Peak 1 >2,000kDa, Peak 2 450kDa, Peak 3 154kDa, Peak 4 46kDa
Of course, one can carry out further characterizations by collecting the fractions for native PAGE, SDS-PAGE, and do a denaturation prior to loading on the gel filtration column using 6M Urea and 8M Guanidinium chloride. But we are unable to do all these within the timeframe of iGEM.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 484
Illegal NotI site found at 1394 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 697
Illegal BglII site found at 1607
Illegal XhoI site found at 492
Illegal XhoI site found at 1402 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1321
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 909
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