In our project, we challenged ourselves by attempting to express rather difficult biopolymers in Pichia pastoris. While we successfully managed to produce plasmids for the yeast, we also designed plasmids that are compatible with Synechocystis PCC 6803 for the option of expressing said polymers in the same organism we use for biomineralization. However, several parts of our work with Synechocystis have turned out to be not compatible with RFC10 and Type IIS assembly standards, thus are not represented here.
Below you may find a list of our coding sequences, promoters, terminators and secretion factors.
All of the parts listed are RFC 10 or Type IIS compatible. Have a look!
| Number | Name | Description |
|---|---|---|
| BBa_K4423001 | MaSp | Coding Sequence for main strain spider silk. |
| BBa_K4423002 | Sec. F. (P.) | Secretion factor for Pichia pastoris. |
| BBa_K4423003 | Sec. F. (S) | Secretion factor for Synechocystis. |
| BBa_K4423004 | PHA_A | Coding sequence for protein A for PHB. |
| BBa_K4423005 | PHA_B | Coding sequence for protein B for PHB. |
| BBa_K4423006 | PHA_C | Coding sequence for protein C for PHB. |
| BBa_K4423007 | PHA_E | Coding sequence for protein E for PHB. |
| BBa_K4423008 | Gelatin | Coding sequence for gelatin. |
| BBa_K4423009 | pGAP | pGAP promoter for Pichia pastoris. |
| BBa_K4423010 | pTEF | pTEF promoter for Pichia pastoris. |
| BBa_K4423011 | RPL2Att | RPL2Att terminator for Pichia Pastoris |
| BBa_K4423012 | RPP1Btt | RPP1Btt terminator for Pichia pastoris |
| BBa_K4423013 | RPS25Att | RPS25Att terminator for Pichia pastoris |
With all the coding sequences we created, we encountered several challenges with assembly compatibility in our composite parts. In this table, you may find the only composite part that we generated that were RFC10 or Type IIS compatible.
| Number | Name | Description | Promoter | Insert | Terminator |
|---|---|---|---|---|---|
| BBa_K4423021 | pTEF_MaSp_RPL2Att | Expression cassette for main strain spider silk including the secretion factor for Pichia pastoris. | pTEF | Sec. F. (P) and MaSp | RPL2Att |