Difference between revisions of "Part:BBa K2008005"

Latest revision as of 23:43, 19 October 2016

pVeg->sec-TD1-KSCI-BBI

This part contains an active nonamer from the Bowman Birk Protease Inhibitor (BBI) with the addition of lysine, serine, cysteine, and isoleucine (KSCI) tag on the N-terminal end and a phenylalanine (F) tag on the C-terminal end to increase BBI solubility. A B. subtilis secretory signal peptide sequence is included to allow for secretion of the peptide directly into surrounding media. A transdermal tag, TD1, (https://parts.igem.org/Part:BBa_K1074000) is fused to the N-terminus of BBI to allow for diffusion of the peptide across the skin.

This coding sequence is under the control of the constitutive B. subtilis promoter pVeg (https://parts.igem.org/Part:BBa_K143012) and a strong B. subtilis RBS (https://parts.igem.org/Part:BBa_K780001).

Note: This part is a composite part, not a basic part, but it could not be added as composite at the time of entry.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Usage and Biology

Using HCT116 cells and a clonogenic viability assay, we were able to test the effectiveness of BBI's radioprotection on cancer cell lines. There was no trend observed in the data, and we concluded that further studies were needed to fully characterize this part. There are a variety of reasons as to why this experiment did not work, and foremost among them may be that the presence of TP53 (a critical component of the DBA damage response in cells) may not be enough for BBI to act as a radio-protector, as other proteins intrinsic in BBI's function could be non-functional.

Fig 1. Surviving fraction of cells when irradiated at 5 Gy and treated with a peptide construct or a DMSO and DTT control

Fig 2. The immunofluorescence assay performed on primary fibroblast cells (1BR4) using primary mouse H2AX and rabbit 53BP1 antibody. The secondary antibody used was Alexa 488, Cy3 and DAPI. The cells were fixed at regular intervals. Only 1 replicate is shown.

We further studied BBI by using 1BR3 Human Fibroblasts to assay whether BBI had an effect on the formation of double strand DNA breaks. Interestingly, this was found to be effective, and BBI was able to lower the amount of DNA double strand breaks in a modest fashion as is shown in Fig. 3. This is the most important point of data obtained in the functioning of BBI, and as such is valuable to any future iGEM teams wishing to pursue a project in dealing with this part.

Fig 3. y-H2AX data of 1BR3 Human Fibroblasts displaying the amount of y-H2AX foci over a 24-hour period when subjected to incubation with the Bowman-Birk Protease Inhibitor

We acquired mass spectra for a synthetic BBI standard [tagged with TD1 and KSCI]. The characterization of a BBI standard will be of value to future experiments when detection of BBI is necessary. MS spectrum of intact BBI in multiple charge states is shown in Figure 4; fragmentation of the 3+ and 4+ charge states, via CID, is shown in figures 5 and 6, respectively.

Fig 4. Standard mass spectra with states denoted.

T--UofC_Calgary--Spectrum%2B4.png
Fig 5. Mass spectra of BBI in a +3 state

Fig 6. Mass spectra of BBI in a +4 state

@@ Line 21: / Line 21: @@
 https://static.igem.org/mediawiki/2016/5/51/T--UofC_Calgary--k2008005img2.png<br>
-Fig 2. The Histology assay performed on primary fibroblast cells (1BR4) using primary mouse H2AX and rabbit 53BP1 antibody. The secondary antibody used was Alexa 488, Cy3 and DAPI. The cells were fixed at regular intervals. Only 1 replicate is shown.
+Fig 2. The immunofluorescence assay performed on primary fibroblast cells (1BR4) using primary mouse H2AX and rabbit 53BP1 antibody. The secondary antibody used was Alexa 488, Cy3 and DAPI. The cells were fixed at regular intervals. Only 1 replicate is shown.
 We further studied BBI by using 1BR3 Human Fibroblasts to assay whether BBI had an effect on the formation of double strand DNA breaks. Interestingly, this was found to be effective, and BBI was able to lower the amount of DNA double strand breaks in a modest fashion as is shown in Fig. 3. This is the most important point of data obtained in the functioning of BBI, and as such is valuable to any future iGEM teams wishing to pursue a project in dealing with this part.<br><br>
-https://static.igem.org/mediawiki/2016/2/2c/BTRES.png<br>
+https://static.igem.org/mediawiki/2016/c/c7/T--UofC_Calgary--k2008005img3.png<br>
 Fig 3. y-H2AX data of 1BR3 Human Fibroblasts displaying the amount of y-H2AX foci over a 24-hour period when subjected to incubation with the Bowman-Birk Protease Inhibitor<br><br>
-We were able to use mass spectroscopy to determine the spectra for BBI. These data are shown in Fig. 4 and Fig. 5 below. Fig 6 is a control standard spectra to compare this to the variable BBI spectra.<br><br>
+We acquired mass spectra for a synthetic BBI standard [tagged with TD1 and KSCI]. The characterization of a BBI standard will be of value to future experiments when detection of BBI is necessary. MS spectrum of intact BBI in multiple charge states is shown in Figure 4; fragmentation of the 3+ and 4+ charge states, via CID, is shown in figures 5 and 6, respectively.<br><br>
+https://static.igem.org/mediawiki/2016/5/52/T--UofC_Calgary--StandardMSSpec.png<br>
+Fig 4. Standard mass spectra with states denoted.<br><br>
 https://static.igem.org/mediawiki/2016/9/96/T--UofC_Calgary--Spectrum%2B4.png<br>
-Fig 4. Mass spectra of BBI in a +3 state<br><br>
+Fig 5. Mass spectra of BBI in a +3 state<br><br>
 https://static.igem.org/mediawiki/2016/9/9e/T--UofC_Calgary--Spectrum2.png<br>
-Fig 5. Mass spectra of BBI in a +4 state<br><br>
+Fig 6. Mass spectra of BBI in a +4 state<br><br>
-https://static.igem.org/mediawiki/2016/5/52/T--UofC_Calgary--StandardMSSpec.png<br>
-Fig 6. Standard mass spectra with states denoted.<br><br>