Best Part Collection

Best Part Collection LTF Our project aims as using engineered E.coli to produce human lactoferrin(LTF). Human LTF has four known isoforms, from isoform1 to isoform4. Based on the description on NCBI databank, we find that isoform1 and isoform3 are preprotein, and isoform2 may lack a vital segment. Consequently, we choose isoform 4 as the gene we use. However, to help more people with the future production of LTF, we modified four isoforms to accurate format and up load them: BBa_K49710013 for isoform4, the isoform we use; BBa_K4971008, BBa_K4971009, BBa_K4971010 is for isoform1, isoform2, and isoform3 respectively. Besides, we upload the plasmid we used in part BBa_K4971015. We believe our work will inspire and benefit those who want to conduct further research in producing LTF in bacteria, lowering its market price, and benefitting infants and those patients with anemia. CRISPR for CRISPR In this part, I don’t mean that we use gene drive in our program, which is highly suspected and has potential dangerous. I mean that we try to use CRISPR/spCas9 to silence CRISPR system in the target bacteria. In fact, LTF is a protein that has bacteriostasis, so that the plasmid we insert is detrimental to the bacteria. We doubt that CRISPR system of our target bacteria damages the plasmid, which causes experimental failure for many times. Based on the result of CRISPR-Cas adaptation in Escherichia coli, by Damjan Mitić et al. we know that there are 7 functional Cas protein: Cas1, Cas2, Cas5, Cas6, Cas7, CasA, and CasB. Consequently, we design 7 pairs of CRISPR sequence, containing the transcripted result of gRNA alongside scaffold RNA. Since E.coli doesn’t have Cas9 protein, we use spCas9 as the bases, and use PAM “NGG” to find proper sequence.

We design 2 sequence for each protein, which part codes range from BBa_K4971001 to BBa_K4971007. This design also show our consideration about safty, since the bacteria with knocked CRISPR is very sensitive to the attack by bacteriaphage in the environment. Without considering the strict rules in our lab, potential leakage is about impossible.