Part:BBa_K2958013
LacP with RFP + Proinsulin Gene Block
This composite part contains RFP regulated by a Lac promoter (BBa_K2958002), and native Proinsulin gene block that contains an extended RBS (iGEM17_Sydney_Australia), ecotin tag(BBa_K2958015), a 6GGS-6His tag-6GGS linker (BBa_K2958003), TEV tag (BBa_K2958000), human proinsulin sequence coding sequence from iGEM17_Sydney_Australia (BBa_K2417006), and double terminator (B0015).
Description
The LacP + RFP portion of this composite part functions as the reporter gene used to confirm the expression of a circuit. RFP is easily visible, and the cells will glow red if it is properly expressed. This circuit is regulated by the Lac Promoter. The promoter will act as a constitutive promoter because there is no Lac repressor in this construct. However, the promoter still contains the binding site for the lac repressor native to E. coli, this construct can be treated with IPTG as a precaution to ensure expression.
This proinsulin gene block in this construct is the negative control for our experimental circuits.This gene block will allow us to compare the structure and function of our single chain insulins (single chain native proinsulin, fast acting single chain insulin, and long lasting insulin) to wildtype insulin. The proinsulin gene sequence is based on the contains 3 different tags for purification--an ecotin tag that is meant to send our Insulin to the periplasm of the cell for proper disulfide bond formation, a 6GGS-6 His tag-6GGS tag that is meant to aid in the first step of our insulin purification via nickel beads, and a TEV tag used for the site-specific cleavage of the his tag. It was important for the ecotin tag to be at the end of the protein in order for the proinsulin disulfide bonds to properly form in the periplasm, so the 6 his tag fell between multiple tags. To ensure the his tag is still functional, we added 2 6GGS spacers based on the iGEM17_Sydney_Australia design to increase flexibility of the his tag and allow for purification.
Figure 3. (A) Gel electrophoresis ran at 110V for 30 minutes. Lane 1 is APEX 500 bp ladder, lane 2 is PCR purified LacP+RFP at the correct base pair length (924 bp), lane 3 is PCR purified proinsulin at the correct base pair length (1068 bp), NL 5.50 Single Chain Proinsulin at the correct base pair length (not used for this project; 1002 bp), and lane 4 is long lasting single chain insulin at the correct base pair length (1002 bp). This gel electrophoresis result supports that the RFP gene block and the Proinsulin gene block with tags for purification was properly PCR'd. (B) Gel electrophoresis ran at 110V for 30 minutes. Lane 1 is APEX 500 bp ladder, lane 2 is PCR purified psB1C3 plasmid at the expected base pairs (2070 bp). This psB1C3 plasmid was used as the plasmid backbone for this LacP with RFP + Proinsulin gene block with tags for purification composite part.
Figure 4. Transformation of RFP + Proinsulin Gibson Assembly into DH5-Alpha (E.coli) cells exhibits colony growth.
Figure 5.Colony PCR of RFP+Proinsulin construct. This colony PCR isolates our construct form the plasmid, and is used as a confirmation test of our gibson assembly. The RFP + Proinsulin gene blocks result in a complete construct that is 1992 base pairs long. (RFP is 924 bp, and Proinsulin is 1068 bp; 924 bp + 1068 BP = 1992 bp). These colony screening results indicate a construct at ~2000 bp, as the band is right beneath the 2 kb band. From these results, we conclude that the Gibson Assembly was successful.
">Figure 6. EcoRI and PstI Restriction Digest of RFP + Proinsulin (left) versus Predicted Results of Proinsulin Restriction Digest. According to the virtual restriction digest conducted on serial cloner, a restriction digest of the RFP + Proinsulin construct will result in 3 fragments: one 2,033 bp fragment, one 1,028 bp fragment, and one 1,001 bp fragment. The two fragments at about 1,000 base pairs in length will appear as one band due to their similarity in length. Based on the results of the restriction digest, our bands appear to be at the expected fragment length. Through this digest, we were able to locate an illegal pstI site in our proinsulin fragment. However, analysis on serial cloner confirms that this pstI site does not affect the protein sequence, and this composite part is still RFC[1000]/Type IIS compatible. According to this data, the gibson assembly reaction of the RFP + Proinsulin gene block composite part was successful.
Figure 7. EcoRI Digest of RFP + Proinsulin (left) versus Predicted Results of Proinsulin Restriction Digest. This digest was performed in order to confirm the entire construct including the psB1C3 plasmid is present to further confirm successful Gibson Assembly. According to the virtual restriction digest conducted on serial cloner, a restriction digest of the RFP + Proinsulin construct will result in 1 fragment at 4,062 base pairs.Through this digest, we were able to locate an illegal pstI site in our proinsulin fragment. However, analysis on serial cloner confirms that this pstI site does not affect the protein sequence, and this composite part is still RFC[1000]/Type IIS compatible. According to this data, the gibson assembly reaction of the RFP + Proinsulin gene block composite part was successful.
Figure 8.Protein gel of the RFP + Proinsulin construct, courtesy of iGEM Team_Moscow 2019. According to this protein gel, our construct appears to have worked as expected, as seen by the bands at the expected proinsulin fusion construct band and expected RFP band in the (+) lysate of the protein gel (indicated by the red arrow). The expected molecular mass of the proinsulin is 31.75 kda, and the expected molecular mass of the RFP is 25.38 kda.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 996
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 996
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 996
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 996
Illegal AgeI site found at 781
Illegal AgeI site found at 893 - 1000COMPATIBLE WITH RFC[1000]
| None |