Overview

First Cycle

Design #1

We started documentation for the properties of alkB1, the main gene of interest. Another iGEM team(TzuChiU_Formosa) created a part containg alkB1 using RBS BBa_B0034, however we chose to use RBS BBa_B0032 instead because it has higher strength which results in higher protein synthesis. The idea then was to insert alkB1 with BBa_B0032 to have a continuous expression and degradation of present alkanes. As for the plasmid backbone we decided to use plasmid pSB1C5A from the distribution kit.
As only cloning and observing the permanent expression of alkB1 would not have been very efficient and might have led to an unsatisfactory result, we decided to use alkS as it is the transcription factor for palkB, which will activate the expression of alkB1, being the inducible promoter for this gene.

Figure 1: alkane degradation genes mechanism

The strategy was to take a plasmid with a constitutive promoter from the distribution kit and clone alkS in that plasmid. Then, using another plasmid from the distribution kit, clone palkB and alkB1 into it, with palkB placed upstream of alkB1, and then do a three-point ligation to insert both genes into the open plasmid. After that, the plan was to transform both plasmids into the same E. coli cells, so the alkS-palkB-alkB1 complex works. We chose to use the J23119 promoter for the alkS gene and clone both of them into pSB1C30 from the distribution kit (cut pSB1C30 and plasmid containing J23119 with BsaI and then ligate them, cut alkS with XbaI and PstI, cut pSB1C30-J23119 with SpeI and PstI and ligate alkS into open pSB1C30-J23119). For palkB and alkB1, we chose to use the pJUMP28-1A (sfGFP) containing BBa_J428353 as backbone (Kanamycin resistance) and clone both genes into the plasmid.

Build and Test #1

After looking into the BsaI enzyme, we decided it is not correct to use it as it cuts 4 bases away from the recognition sequence, therefore not creating compatible ends. This restriction enzyme is an IIS-type enzyme, not the IIP-type as the other ones, which means it cuts the DNA after the palindromic region that the enzyme recognizes, not in the recognition site. Thus, if we cut with this enzyme, we would obtain from the pSB1C30 plasmid the sequences found in the images below.

Figures 2: BsaI restriction enzyme cutting for plasmid pSB1C30

From the plasmid containing the J23119 promoter we obtained the sequences found in the images below:

Figures 3: BsaI restriction enzyme cutting for promoter J20119

Although there is nothing wrong with the cutting process itself, a problem arises when the piece of DNA containing the promoter is inserted into the pSB1C30 plasmid, because the overhangs that remain are not compatible with each other. For example, 5' GCTT 3' in figure 2b should be compatible with 3' GGAG 5' in figure 3b, which is not true. A solution to solve this problem would be to use PCR, with primers that amplify the promoter and add additional base pairs to be cut with BsaI so that ends are compatible.

Learn #1

The solution we chose however is to cut the pSB1C30 backbone with EcorI and PstI, order the J23119 promoter sequence separately and cut it with EcorI and SpeI, and then do a three-point ligation with the open plasmid, the cut J23119, and the alkS cut with XbaI and PstI.

Second Cycle

Design #2

Identified problem: Both the pSB1C30 and the pJUMP28-1A have the pMB1 origin, therefore making them incompatible to transform in the same cell. We also added “ggc” overhangs at the ends of the sequences which used enzymes that cut at the end of the sequence in order to make sure we have stable compatible ends.
pJUMP28-1A was replaced with pJUMP26-1A, which has a p15A origin with a medium copy number, therefore making the plasmids compatible. We decided to place the J23119-alkS complex in pJUMP26-1A and the palkB-alkB1 complex in pSB1C30, as pSB1C30 has a high copy number, hence we thought it would be better to use it to ensure the noticeable expression of alkB1.

Build and Test #2

The palkB sequence we were planning to order was incorrect, as the sequence we were using was supposed to be cloned upstream of alkB1, so we reversed the sequence, without changing the direction of the prefix and the suffix, so the promoter would transcribe in the right direction. We also replaced the EcorI recognition sites from the alkB1 sequence to prevent alteration of open reading frames by modifying codon nucleotides, without changing the corresponding amino acid sequence.
We realized it would be less complicated and we would have a higher chance of success if we cloned all 4 sequences of interest (J23119 promoter, alkS, palkB promoter and alkB1) into the same plasmid (pSB1C30, as it has a high copy number), with the J23119-alkS complex cloned upstream of the palkB-alkB1 complex, in order to minimize leakiness and problems derived from inefficient transcription termination or translation readthrough. After having both plasmids with their corresponding gene complexes inserted, we would cut J23119-alkS out of pJUMP26-1A using XbaI and PstI and ligate it with pSB1C30 cut with SpeI and PstI, therefore obtaining a bicistronic plasmid

Learn #2

As a result of all iterations, the final construct to be created was modeled in SnapGene.

Figure 4: plasmid construct with J23119, alkS, palkB and alkB1