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Team:Buenos Aires/Results/Bb1
From 2012.igem.org
Contents |
BioBricks Design for Synthetic Ecology
In order for the cross-feeding scheme to work, we need the strains to export the amino acid they can produce and the other strains can’t. We are going to use yeast expression plasmids with inducible or constitutive promoters. The structure of the synthetic gene is represented in Figure 1.
Figure 1: Schematic representation of the synthetic gene for the cross-feeding design. RS: Restriction Sites, Prefix, Suffix: BioBrick standard, Signal: Secretion signal peptide. The ORF is highlighted in violet.
The synthetic gene has the following elements:
• Prefix of the BioBrick standard.
• Kozak consensus sequence for initiation of translation.
• Signal peptide that targets the product of the gene for secretion.
• Trojan peptide, to increase internalization in target cell.
• Payload: this is the exported amino acid rich domain of the protein.
• Suffix of the BioBrick standard.
Beside, the gene will have convenient restriction sites for directional cloning (RS1 and RS3). RS2 will allow to easily remove the sequence coding for the trojan peptide, by restriction and re-ligation.
BioBrick Prefix and Suffix Because the entire ORF is contained within the prefix and suffix, no care for in-frame assembly has to be taken. We can use the original RFC10 BioBrick standard.
Figure2: RFC10 BioBrick standard
Kozak Sequence
The Kozak sequence is analogous to the bacterial RBS, it is required for proficient initiation of translation. There is only one yeast Kozak sequence in the registry (part BBa_J63003, distributed in the 2012 kit). Note that this sequence codes for a glutamic acid (E) after the start codon. Alternatively we could use the sequence of the 5’UTR of the MF-alpha1 gene of yeast (see Signal Peptide).
| Name | DNA Sequence |
| BBa_J63003 | cccgccgccaccatggag |
| MF-alpha1[-12,6] | acgattaaaagaatgaga |
Table1: DNA sequences for Kozak consensus
Signal Peptide
The signal peptide directs the secretion of the produced protein, and therefore allows for the exportation of the payload. This peptides are cleaved once the protein is in the lumen of the ER.
We have several options for yeast secretion signal peptides
1) MRFPSIFTAVLFAASSALA
2) MKVLIVLLAIFAALPLALAQPVISTTVGSAAEGSLDKR
Number 1 is the signal peptide for the yeast alpha-mating factor [Water et al 1987]. Because its a part of a yeast gene, we don’t need to optimize it for yeast. Number 2 is part BBa_K416003 from the registry (not included in the kit), and was designed for yeast [Clements 1991].
The DNA sequence are shown in Table 2
| Name | DNA Sequence |
| 1: MF-alpha1 signal | atgagatttccttcaatttttactgcagttttattcgcagcatcctccgcattagct |
| 2: BBa_K416003 | atgaaagttttgattgttttgttggctattttcgctgctttgccattggctttggctcaaccagttatttctactactgttggttctgctgctgaaggttcactagataaaaga |
Table 2: DNA sequences for the signal peptides, atg codon in bold
Trojan peptide
Trojan peptides are short (15aa) sequences that penetrate through the plasma membrane inside the cell without the need of any receptor or endocitosis process [Derossi 1998]. We want to use them to increase the efficiency with witch the payload enters the target cell. Ideally they should not contain Trp or His, as those are the relevant amino acids for exportation. Two good candidates are the penetratin from the HIV TAT protein, and polyarginine [Jones et al 2005].
| Penetratin | Residue sequence |
| TAT | YGRKKRRQRRR |
| polyarginine | RRRRRRRRRRR |
Table 3: Primary structure for penetratins
This proteins are not from yeast, so we need to retro-translate them. Using the conventions in table 4, we get the DNA sequence in Table 5.
| R | Purine (A or G) |
| Y | Pyrimidine (C or T) |
| N | Any nucleotide |
| W | Weak (A or T) |
| S | Strong (G or C) |
| M | Amino (A or C) |
| K | Keto (G or T) |
| B | Not A (G or C or T) |
| H | Not G (A or C or T) |
| D | Not C (A or G or T) |
| V | Not T (A or G or C) |
Table 4: Nucleotide convention for retro-translated sequences
| Penetratin | DNA Sequence |
| TAT | TAYGGNMGNAARAARMGNMGNCARMGNMGN |
| polyarginine | MGNMGNMGNMGNMGNMGNMGNMGNMGNMGN |
Table 5: DNA sequence for penetratins. MGN=CGN or AGR
Next we need to codon optimize for yeast, and if posible avoid mRNA secondary structures.
Payloads
The payloads are the elements of the synthetic gene that code for the “amino acid rich” region of the secreted protein. By “a.a. rich” we mean, rich in the amino acid we want to export, Trp or His in our case. This domain should be soluble enough not to cause precipitation of the protein, and should be relatively stable not to be degraded before they are actually secreted from the cell.
| Payload | Residue sequence |
| PolyHa | HNHNHNHNHNHN |
| TrpZipper2 | SWTWENGKWTWK |
| PolyHb | HGDHDGHGKHKG HGDHDGHGKHKG HGDHDGHGKHKG |
| PolyWb | WGDWDGWGKWKG WGDWDGWGKWKG WGDWDGWGKWKG |
Table 6: Payloads protein sequences
PolyHa is Clontech’s His tag sequence. TrpZipper is a small peptide that folds into a beta-hairpin secondary structure. The indole rings of the Trp form a hydrophobic core. The protein is water soluble and monomer [Cochran 2001].
PolyHb and PolyWb were desing taking into acount the following consideration; 1) avoided repeating the same residue in tandem to minimize local tRNA depleation, 2) avoided Trp in tandem because of therir low solubility, 3) we included Gly to avoid the formation of stable structures and 4) included acidic and basic amino acids to increase solubility. Retro-translating using the conventions of table 4, we obtained the sequences in Table 7.
Payload DNA Sequence
| PolyHa | CAYAAYCAYAAYCAYAAYCAYAAYCAYAAYCAY |
| TrpZipper2 | WSNTGGACNTGGGARAAYGGNAARTGGACNTGG |
| PolyHb | CAYGGNGAYCAYGAYGGNCAYGGNAARCAYAARCAYGGNGAYCAYGAYGGNCAYGGNAARCAYAARCAYGGNGAYCAYGAYGGNCAYGGNAARCAYAAR |
| PolyWb | TGGGGNGAYTGGGAYGGNTGGGGNAARTGGAARTGGGGNGAYTGGGAYGGNTGGGGNAARTGGAARTGGGGNGAYTGGGAYGGNTGGGGNAARTGGAAR |
Table 7: Payload’s DNA sequence. WSN=TCN or AGY
ORFs Arrangements
We are planning to synthesize 4 genes with different combinations of signal peptides, trojans and payloads. We want these genes to be immediately functional (no cloning necessary), but we also want o to have the possibility to recombine the parts in different arrangements. The 4 ORFs we will synthesize are described in Table 8.
| ORF# | Kozak | Signal | Trojan | Payload |
| 1 | MF1[-12,6] | 1: MF1 signal | TAT | PoliHa |
| 2 | MF1[-12,6] | 1: MF1 signal | TAT | TrpZipper2 |
| 3 | BBa_J63003 | BBa_K416003 | polyarginine | PoliHb |
| 4 | BBa_J63003 | BBa_K416003 | polyarginine | PoliWb |
Table 8: Arrangement of the synthesized ORFs
This arrangement would allow to compare ORF1 with ORF2, and ORF3 with ORF4.
Expression Plasmids
To determine which restriction sites to use for cloning (RS1 and RS3 in Figure 1), we need to know the MCS of the expression plasmids we are going to use.
One of these plasmids will probably of the pCM180-5 series, which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996].
The Multiple Cloning Site of these vectors is shown in Figure 3.
Figure 3. Multiple Cloning Site (MCS) of the pCM180 series plasmids.
The other plasmid we might use is pEG202, with a 2 ori, HIS3 marker and a constitutive promoter (PADH1).
Figure 4: pEG202 MCS sequence and restriction sites.
Comparing Figure 3 and Figure 4, BamHI and NotI appear in both MCS in the same order, so they are good candidates for RS1 and RS3 (Figure 1) respectively.
We will probably need to clone the construct in a general purpose plasmid for manipulation. For instance, to remove the trojan we need to clone the construct into a plasmid, cut it with the RE of RS2, precipitate the DNA (to get rid of the trojan fragment), and religate. For this to work we have to make sure that there is no RS2 in the vector. A common vector for this is pBluescript, wich has the MCS shown in Figure 5.
Figure 5: pBluescript Multiple Cloning Site
Restriction Enzymes
The restriction sites used for RS1-3 have to be different from the ones used in the BioBricks standard. The standard RE for BioBricks are EcoRI, NotI, XbaI, SpeI and PstI.
Unfortunately, NotI was our candidate for RS3, so we have a problem here. There are different solutions. We can either use two restriction sites instead of RS3, one for each plasmid, or we can change the BioBrick standard to something like RFC[21] (Berkeley standard) that has no NotI restriction site.
An other option would be to use the restriction sites in the prefix and suffix to clone the construct into the expression plasmid. This is appealing because we don´t need new REs. Anyhow we would need to include a RS in the 5’ end to be able to directionally clone into pCM180. The new design would look something like Figure 6.
Figure 6: Alternative scheme for the restriction sites
If we make RS4 a BamHI site, we can directionally clone the construct into both plasmids (pCM180 and pEG202 ) by cutting with BamHI and NotI. In this design we would not need RS1 and RS3, but we can include them just in case we need to clone them into an other vector.
Regarding RS2 we need a restriction enzyme that produces cohesive ends, codes for acceptable amino acids, is easily available and not used in an other part of the construct. Some candidate RE are listed in Table 9.
| R. Enzyme | R. Site sequence | Overhang | Codes for |
| HindIII | A/AGCTT | AGCT | Lys-Leu (KL) |
| XhoI | C/TCGAG | TCGA | Leu-Glu (LE) |
Table 9. Candidate restriction enzymes for RS2
Probably any of them will work, but the trojan peptide needs to be basic so the HindIII site looks better suited. If we want to remove the trojan, we will have to clone the construct into a vector with no HindIII site. One way to do this is to clone it into pBluescript in such a way that the HindIII restriction site of the MCS is removed.
Looking at Figure 5 we can see that if we cut pBluescript with XhoI and PstI, the HindIII site is removed. If we make RS1 -> XhoI (which is easily available) we can cut the construct with these same enzymes and directionally clone it into pBluescript.
Most likely we wont use RS3, but we can assign it a restriction site just in case. For example NcoI could be used instead of NotI to do the directional cloning into pEG202.
| RS# | R. Enzyme | R. Site Sequence |
| RS1 | XhoI | C/TCGAG |
| RS2 | HindIII | A/AGCTT |
| RS3 | NcoI | C/CATGG |
| RS4 | BamHI | G/GATCC |
Table 10. Assignation of restriction enzymes to the different restriction sites.
When we order the construct we will probably have to specify in which vector we want it shipped. RS3 (and perhaps RS1) could be changed depending on this vector. For example we could order the gene in pBluescript, in which case it might be convenient to make RS3 -> SacI. For other plasmids other RS might be needed, but if possible it would be convenient to retain RS1 -> XhoI that allows us to clone the gene into pBluescript as described above.
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