Difference between revisions of "Part:BBa K3182000"
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[[File:T--Linkoping_Sweden--pinkbandageandnopink.png|400px|thumb|right|<b>Figure 7.</b> Lysate (via sonication) from BL21 E. coli was incubated with Epiprotect (microbial cellulose bandage) for 1h and washed thrice with 70% ethanol. To the right is Epiprotect that has not been incubated with AsPink-CBD lysate.]] | [[File:T--Linkoping_Sweden--pinkbandageandnopink.png|400px|thumb|right|<b>Figure 7.</b> Lysate (via sonication) from BL21 E. coli was incubated with Epiprotect (microbial cellulose bandage) for 1h and washed thrice with 70% ethanol. To the right is Epiprotect that has not been incubated with AsPink-CBD lysate.]] | ||
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<h3>AsPink bound to cellulose bandage</h3> | <h3>AsPink bound to cellulose bandage</h3> | ||
Epiprotect (microbial cellulose bandage, S2Medical) was incubated in AsPink lysate from BL21 (DE3) Gold for 30 min. Epiprotect was later washed with 70% ethanol three times. To the left is AsPink incubated Epiprotect and to the right is plain Epiprotect. | Epiprotect (microbial cellulose bandage, S2Medical) was incubated in AsPink lysate from BL21 (DE3) Gold for 30 min. Epiprotect was later washed with 70% ethanol three times. To the left is AsPink incubated Epiprotect and to the right is plain Epiprotect. |
Revision as of 15:37, 4 October 2019
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 580
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Introduction
No part name specified with partinfo tag.
This part consists of a carbohydrate binding domain (CBD) from Clostridium thermocellum (C. thermocellum) cellulose scaffolding protein (CipA) and is a central part Clostridium thermocellum's cellusome. The CBD-fusion were fused using a flexible GS-linker (-GGGGSGGGGS-). A thrombin cleavage site (-LVPRGS-) was added to the end of the linker and its breakage will leave a glycine and serine attached to the N-terminal of the fusion protein.
Protease site and use
The thrombin site was added to enable the ability to release the fusion protein down into skin wounds. Because of our integrated human practice we learned that infection span much deeper into wounds that we thought. Simply attaching the CBD-fusion protein to a carbohydrate material wouldn't make the fusion protein reach far into the wound. The thrombin site was also chosen because of thrombins endogenous occurrence in humans.
Assembly compabilities
An internal BamHI recognition sequence (RS) has been added to enable changeable fusion proteins. BamHI was chosen because its RS codes for glycine and serine, fitting it to the end of the thrombin site. It is also a cost-effective enzyme and is unaffected by methylated DNA.
CBDcipA crystal structure
Important molecular faces
CBDcipA is composed of a nine-stranded beta sandwich with a jelly roll topology and binds a calcium ion. It further contains conserved residues exposed on the surface which map into two clear surfaces on each side of the molecule. One of faces mainly contains planar strips of aromatic and polar residues which may be the carbohydrate binding part. Further aspect are unknown and unique with this CBD such as the other conserved residues which are contained in a groove.
Carbohydrate binding domain specificity
Since the CBD is from the cellusome of C. thermocellum some researches call it a cellulose binding domain. However, iGEM19 Linköping noticed that this domain could also bind to different sources of polysaccaride materials. This serves as a domain for iGEM19 Linköpings modular bandage, where the polysaccaride material can be changed for anything and not exclusively cellulose.
The choice of carbohydrate binding domain
iGEM Linköping 2019 choose CBDcipA due to many other iGEM teams exploring the possibilities of this domain. Our basic design was influenced by [http://2014.igem.org/Team:Imperial iGEM14 Imperial], [http://2015.igem.org/Team:edinburgh iGEM15 Edinburgh] and [http://2018.igem.org/Team:ecuador iGEM18 Ecuador]. Purification and where to place the fusion protein (N- or C-terminal) was determined by studying the former projects. CBDcipA also originates from a thermophilic bacteria which further increases the domains applications.
Expression system
The part has a strong expression with a T7-RNA-polymerase promotor (BBa_I719005) as well as a 5'-UTR (BBa_K1758100) region which has been shown to further increase expression in E. coli (BBa_K1758106), ([http://www.ncbi.nlm.nih.gov/pubmed/2676996 Olins et al. 1989]), ([http://www.ncbi.nlm.nih.gov/pubmed/23927491 Takahashi et al. 2013]).
Usage and Biology
A pink colony from an agar plate was selected and cultured in 10 ml LB-miller for 16h at 37°C, 180rpm. This culture was afterwards added to 1L of LB-miller which was grown for 16h, 80rpm at 16°C. This result can be observed in figure 4. Approximately 45ml of the culture was poured to a 50ml Falcon tube and sonicated which is displayed in figure 5. This tube was later centrifuged and a pink pellet was formed (seen in figure 6).
AsPink bound to cellulose bandage
Epiprotect (microbial cellulose bandage, S2Medical) was incubated in AsPink lysate from BL21 (DE3) Gold for 30 min. Epiprotect was later washed with 70% ethanol three times. To the left is AsPink incubated Epiprotect and to the right is plain Epiprotect.
Thrombin Cleavage
In figure 9 bacterial lysate of sonicated E. coli BL21 was incubated with cellulose (Whatman CF-11 Fibrous Medium Cellulose Powder) in 1h on an end-to-end rotator at R.T. The cellulose was saturated with AsPink lysate. The samples were afterwards centriuged at 3000 g in 1 min and the supernatant was removed. In all samples cleavage buffer (20 mM Tris-HCl pH 8.4, 150 mM NaCl and 2.5 mM CaCl₂) was added and three different dilutions of thrombin (Novagen, #69671) was added. The dilutions were 0,02, 9,94 and 0,08 units (U). One unit refers to the amount of thrombin needed to cleave 1 mg of protein for 16h at R.T in a 200 µl reaction containing the cleavage buffer. The samples were put on an end-to-end rotator over-night (ca 16h) and left at R.T. The control with only cleavage buffer has a transparent supernatant and a pink pellet of cellulose, the most diluted (0,02 U) has a slightly pinker supernatant and less pink pellet however the 0,04 U and 0,08 U dilutions has a pinker supernatant and paler pellet with 0,08 U dilution being the sample with most cleaved AsPink due to the more intense supernatant.